Microfluid platform and uses thereof in cell-based assays and precision medicine

Abstract

The present disclosure relates to integrated microfluidic platforms and systems that automate cell¬ based assays, including cell-cell interaction assays, with applications in precision medicine. Requiring only a minimal number of cells, the disclosed devices and systems enable the customization of treatment options by evaluating the functional responses of a patient's cells to a defined drug panel. This approach preserves the heterogeneity of the original sample, thereby enhancing the accuracy of personalized treatment regimens.

Description

[0001] MICROFLUID PLATFORM AND USES THEREOF IN CELL-BASED ASSAYS AND PRECISION MEDICINE

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to microfluidic platforms. More specifically, the present disclosure relates to devices and systems and uses thereof in cell-based, and cell-cell interaction assays, specifically for precision medicine.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. McDonald, J. C. et al. Fabrication of microfluidic systems in poly (dimethylsiloxane). ELECTROPHORESIS: An International Journal 21, 27-40 (2000).

[0007] 2. Unger, M. A., Chou, H.-P., Thorsen, T., Scherer, A. & Quake, S. R. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science (1979) 288, 113-116 (2000).

[0008] 3. Kipper, S. et al. Control and automation of multilayered integrated microfluidic device fabrication. Lab Chip 17, 557-566 (2017).

[0009] 4. Pak, C. et al. MicroC3: an ex vivo microfluidic cis-coculture assay to test chemosensitivity and resistance of patient multiple myeloma cells. Integrative Biology 7 , 643-654 (2015).

[0010] 5. Pradhan, S. et al. A microvascularized tumor-mimetic platform for assessing anti-cancer drug efficacy. Sci Rep 8, 3171 (2018).

[0011] 6. Zeng, S. et al. A cascade Fermat spiral microfluidic mixer chip for accurate detection and logic discrimination of cancer cells. Analyst 147, 3424-3433 (2022).

[0012] 7. Watson, C. et al. Multiplexed microfluidic chip for cell co-culture. Analyst 147, 5409-5418 (2022).

[0013] 8. Jang, M., Koh, I., Lee, S. J., Cheong, J. H. & Kim, P. Droplet-based microtumor model to assess cell- ECM interactions and drug resistance of gastric cancer cells. Sci Rep 7, (2017).

[0014] 9. Desyatnik, I. et al. An Integrated Microfluidics Approach for Personalized Cancer Drug Sensitivity and Resistance Assay. Adv Biosyst 1900001. Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0015] BACKGROUND

[0016] Precision medicine has emerged as a revolutionary paradigm in cancer treatment with the promise of enhanced patient outcomes [Hoeben, A., et al. Cancers (Basel) 13, 242 (2021); Schilsky, R. L. Discovery vol. 9 363-366 Preprint at https: / / doi.org / 10.1038 / nrd3181 (2010)]. This personalized approach became feasible with the advent of targeted therapies, which are continually refined to match ongoing advances in digital and medical sequencing and proteomics technologies [Suwinski, P. et al. Sequencing and Big Data Analytics. Front Genet 10, (2019); Frohlich, H. et al. 16, 150 (2018); Potti, A. et al. New England Journal of Medicine 355, 570-580 (2006); Verma, M. J. Pers. Med. J. Pers. Med 2, 1-14 (2010)]. The rapid development of increased therapeutic options brings about fresh challenges: the greater the number of choices, the harder it becomes to identify the optimal therapy for individual patients, significantly impacting their prognosis by treating them with harmful and / or ineffective treatments.

[0017] While research efforts have indeed shifted towards applications of personalized cancer treatment, they are still mostly based on molecular profiling and the identification of unique peripheral biomarkers in the tumor [Schilsky, R. L. Discovery vol. 9 363-366 Preprint at https: / / doi.org / 10.1038 / nrd3181 (2010); Meyer, J. M. & Ginsburg, G. S. Curr Opin Chem Biol 6, 434-438 (2002); Drilon, A. et al. New England Journal of Medicine 378, 731-739 (2018)]. This in turn raises another challenge, namely, that the response of similar tumor types and even genetic profile patients to identical treatment may be different in character and degree. Many theories have been proposed as to why this heterogenic response occurs, with leading theories relating it to the progression of genomic evolution within tumors themselves, as well as between primary tumors and metastases [Gerlinger, M. et al. New England journal of medicine 366, 883-892 (2012); Cyll, K. et al. Br J Cancer 117, 367 (2017); Dagogo-Jack, I. & Shaw, A. T. Nat Rev Clin Oncol 15, 81 (2018); Longo, D. L. New England Journal of Medicine 366, 956-957 (2012)]. New sequencing and bioinformatics technologies have been developed to combat these diagnostic challenges; however, these too are limited advantages for personalizing the treatments [Lloyd, K. L., et al. BMC Cancer 15, 117 (2015); Bartlett, R. et al. Transl Oncol 7, 657-664 (2014)].

[0018] To circumvent this issue, the use of chemosensitivity and resistance assay (CSRA) models on animal or tissue samples has also gained traction [Hidalgo, M. et al. Mol Cancer Ther 10, 1311-1316 (2011); Samson, D. J., et al. Journal of clinical oncology 22, 3618-3630 (2004); Burstein, H. J. et al. J Clin Oncol 29, 3328-3330 (2011)]. Today, animal models are practically the only clinical tool available for testing patient response to drugs before treatment. However, it takes too long (2-3 months at best to obtain results), these models are difficult to scale up and are a poor functional predictor of efficacy and cytotoxicity in humans [Atkins, J. T. et al. Br J Cancer 123, 1496-1501 (2020); Mak, I. W., et al. Am J Transl Res 6, 114-8 (2014)]. Likewise, Animal research in the European Union (EU) is tightly regulated (e.g. Directive 2010 / 63 / EU on the protection of animals used for scientific purposes), with dedicated body (the EURL ECVAM) set up towards the final aim of replacing all animal research with nonanimal alternatives, such as in-vitro cell-based models or through in-silico computer simulations. In addition, EU-wide support and initiatives are growing to promote the development of non-animal approaches to testing and research.

[0019] Alternative techniques such as the 3D structure of organoids have also arisen, which incorporate not only the genomic data but also the tumor microenvironment, thus allowing nucleotide profiling of the tumor and its protein composition and orientation [Zhao, Z. et al. Nature Reviews Methods Primers 2, 94 (2022); Silva-Pedrosa, R., et al. Cells 12, 930 (2023); Rosenwald, A. et al. New England Journal of Medicine 346, 1937-1947 (2002)].

[0020] The common problem preventing all these in vitro functional assays from reaching their maximal potential is the sensitivity of the sample and the need to deliver it to a central laboratory. The solution is to bring the test to the sample at the point of care [Hayes, B., et al. Diagnostics 8, 39 (2018)]. To that end, several ideas have been proposed, the most promising of which is the integration of a CSRA model within a microfluidic platform.

[0021] Microfluidics is the science of manipulating small volumes of fluids ranging from picolitres to microliters within channels ranging from one micron to one millimeter in size [Hong, J. W. & Quake, S. R. Nat Biotechnol 21, 1179-1183 (2003); Whitesides, G. M. Nature 442, 368-373 (2006)]. This field has been increasingly expanding since inception and is now incorporated into a plethora of regularly used molecular biology techniques [Klasner, S. A. et al. Anal Bioanal Chem 397, 1821— 1829 (2010); Glick, Y., et al. Journal of Visualized Experiments (2012) doi: 10.3791 / 3849]. The most common production of microfluidic devices is by soft-lithography of poly-dimethyl siloxane (PDMS), a clear, permeable to gas, and bio-compatible polymer, thus making it ideal for biological studies [1]. The additional incorporation of pneumatic micro-valves into the device, known as “integrated microfluidics,” allows for precise control over concentrations and combinations of drugs in small volumes, thereby enabling the conservation of reagents via a more tightly controlled high throughput approach [2, 3].

[0022] While microfluidic applications have improved the efficiency and reliability of these techniques immensely, applying microfluidics to primary patient samples such as tumor or tissue biopsies is a much less established technology. Several groups have attempted this undertaking, notable among them is Pak et al. who developed a microfluidic platform to study drug resistance in cells derived from bone marrow in patients with myeloma [4]. Another group, Pradhan et al. attempted to recompile tumor structure and then evaluate its’ drug response in vitro using a microfluidic device [5]. Several other groundbreaking microfluidic approaches have also been attempted [6-8]. The inventors have previously presented a microfluidic platform that displays capabilities for a high-throughput drug screening on cancer cells [9]. The device utilized a printed drug microarray at various concentrations and combinations to conduct a wide range of CSRA experiments simultaneously.

[0023] Still further, co-culturing different cell types is essential for studying cell heterogeneity, cell interactions, signaling pathways, and disease models. However, achieving exact and reproducible cell ratios remains challenging, particularly in microfluidic systems. Current microfluidic devices lack precise control over cell counting and collection, limiting their use in applications with limited samples size or requiring defined ratios, such as co-culture assays, therapeutic cell development, and molecular analyses.

[0024] GENERAL DESCRIPTION

[0025] Tools that can translate microfluidic functional assays into practical clinical applications are needed. Toward this goal, the inventors introduce the OncoFlow microfluidic device (also referred to throughout the present disclosure as the microfluidic platform and system). This innovative system features controllable pneumatic valves for complex process control and distinct cell chambers. It is initially designed to perform functional assays on primary cells obtained from patients and to measure their responses to various potential treatments. The inventors offer a-proof-of-concept for a highly promising point-of-care solution that meets the critical demand for personalized decision support, with potential for clinical settings. By screening a range of therapeutic agents on patient-derived cells and analyzing the outcomes, this device may help physicians identify ineffective treatments. This capability not only conserves time and resources but also enhances patient outcomes by facilitating the selection of optimal therapeutic strategies from a refined list of options, excluding any drugs unlikely to be effective.

[0026] Still further, co-culturing of different cell types is a fundamental technique in biological research, widely employed to study cell-cell interactions, signaling pathways, and disease models. A persistent challenge, however, lies in establishing co-cultures with exact and reproducible ratios of different cell types, a difficulty that is particularly pronounced in the constrained environment of microfluidic systems. Existing microfluidic devices for cell handling often lack the ability to precisely count and control the number of cells collected from a defined population. This limitation restricts their utility in applications where accurate cell ratios are critical, including co-culture assays, therapeutic cell development, and downstream molecular analyses. Accordingly, there remains a need for methods that enable the controlled collection and combination of different cell types at known concentrations within a microfluidic device, thereby ensuring reproducible and reliable experimental outcomes.

[0027] Therefore, according to a first aspect of the presently disclosed subject matter, there is provided a microfluidic platform, comprising: a primary block of substrate material, the primary block defining a first plurality of cell cultivation units, and a control system for enabling control of fluid flows with respect to the first plurality of cell cultivation units; wherein the cell cultivation units are fluidly isolated from one another; wherein each said cell cultivation unit comprises a medium channel, a cell channel and a second plurality of filter pathways, wherein: the medium channel is laterally spaced with respect to the cell channel; the second plurality of filter pathways laterally interconnects the medium channel with the cell channel; the cell channel defines a second plurality of serially adjacent cell cultivation zones, wherein each cell cultivation zone is in selective fluid communication with the medium channel via a respective said filter pathway; wherein for each said cell cultivation unit, the control system is configured for selectively blocking fluid communication via the cell channel between adjacent cell cultivation zones to thereby provide respective cell cultivation chambers, wherein each said cell cultivation chamber is in selective fluid communication with the medium channel exclusively via the respective said filter pathway; wherein each said cell cultivation unit comprises a dedicated input / output line providing selective exclusively fluid communication between an outside of the device and each one of the cell channel and the medium channel of the respective cell cultivation unit; wherein for each cell cultivation unit: the medium channel is configured for enabling fluids to be received therein from the dedicated input / output line or to be delivered therefrom to the dedicated input / output line; the cell channel is configured for enabling cells to be received therein from the dedicated input / output line or to be delivered therefrom to the dedicated input / output line; the filter pathways are configured for selectively providing fluid communication between the medium channel and the cell channel, while concurrently preventing transit of cells therethrough from the cell channel to the medium channel.

[0028] For example, the control system comprises a plurality of microfluidic valves, each said microfluidic valve being configured for selectively allowing or preventing flow therethrough under the control of the control system.

[0029] Additionally or alternatively, for example, each said cell cultivation unit extends between a first unit end and a second unit end. For example, each said dedicated input / output line is operatively coupled to the first unit end of the respective cell cultivation unit. For example, each said dedicated input / output line is operatively and separately coupled to the respective cell channel and the respective medium channel at a first cell channel port and a first medium channel port, respectively, at the first unit end of the respective cell cultivation unit. For example, the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective first cell channel address valve overlying the respective first cell channel port, wherein the control system is operable to selectively open or close the first cell channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said cell channel and the respective said dedicated input / output line. For example, the control system is configured for concurrently operating all the first cell channel address valves to thereby concurrently selectively open or close all the corresponding first cell channel ports. Additionally or alternatively, for example, the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective first medium channel address valve overlying the respective first medium channel port, wherein the control system is operable to selectively open or close the first medium channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said medium channel and the respective said dedicated input / output line. For example, the control system is configured for concurrently operating all the first medium channel address valves to thereby concurrently selectively open or close all the corresponding first medium channel ports.

[0030] Additionally or alternatively, for example, for each said cell cultivation unit the respective said medium channel extends longitudinally between the first unit end and the second unit end, and the respective cell channel extends longitudinally between the first unit end and the second unit end.

[0031] Additionally or alternatively, for example, each said filter pathway comprises a third plurality of lateral channels in selective fluid communication between the respective said medium channel and the respective said cell channel, each said lateral channel having a flow cross-section so dimensioned as to prevent transit of the cells therethrough from the cell channel to the medium channel.

[0032] Additionally or alternatively, for example, the control system comprises a said microfluidic valve in the form of a respective horizontal segmentation valve (neck valve) overlying each said filter pathway of each said cell cultivation unit, wherein the control system is operable to selectively open or close each said horizontal segmentation valve (neck valve) to thereby allow or prevent fluid communication, respectively, between the respective said cell cultivation chamber and the respective medium channel. For example, the control system is configured for concurrently operating all the horizontal segmentation valve (neck valves) to thereby concurrently selectively open or close all the horizontal segmentation valve (neck valve).

[0033] Additionally or alternatively, for example, the control system comprises a pair of said microfluidic valve in the form of a respective pair of longitudinally spaced horizontal segmentation valve (sandwich valves) longitudinally enclosing each said cell cultivation zone of each said cell cultivation unit, wherein the control system is operable to selectively close each said pair of horizontal segmentation valve (sandwich valves) to thereby provide the respective said cell cultivation chambers. For example, the control system is configured for concurrently operating all the horizontal segmentation valve (sandwich valves) of each said cell cultivation unit to thereby concurrently selectively close all the corresponding horizontal segmentation valve (sandwich valves) of the respective cell cultivation unit, thereby concurrently providing all the cell cultivation chambers of the respective cell cultivation unit. For example, the control system is configured for concurrently operating all the horizontal segmentation valve (sandwich valves) of said first plurality of cell cultivation unit to thereby concurrently selectively close all the corresponding horizontal segmentation valve (sandwich valves) of said first plurality of cell cultivation units, thereby concurrently providing all the cell cultivation chambers of said first plurality of cell cultivation units.

[0034] Additionally or alternatively, for example, the microfluidic platform further comprises a fluid inlet system operatively coupled to the respective second unit ends of the cell cultivation units. For example, the fluid inlet system comprises a manifold arrangement, the manifold arrangement having at least one inlet coupled to a first plurality of outlets via a succession of bifurcating flow paths defining flow path branches, wherein each said outlet is operatively coupled to a respective said second unit end of a different said cell cultivation unit. For example, the control system comprises a plurality of said microfluidic valves in the form of a respective plurality of manifold address valves, each overlying a respective said branch, wherein the control system is operable to selectively open or close each said manifold valve to thereby allow or prevent fluid communication, respectively, through the respective branch. For example, the control system is operative to selectively concurrently open or close all the manifold address valves to thereby enable or prevent, respectively, fluid communication between the at least one inlet and the first plurality of outlets. Additionally or alternatively, for example, the control system is operative to selectively concurrently open or close selected said manifold address valves to thereby enable fluid communication between the at least one inlet and only a selected one said outlet of said first plurality of outlets.

[0035] Additionally or alternatively, for example, each said outlet is operatively and separately coupled to the respective cell channel and the respective medium channel at a second cell channel port and a second medium channel port, respectively, at the second unit end of the respective cell cultivation unit. For example, the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective second cell channel address valve overlying the respective second cell channel port, wherein the control system is operable to selectively open or close the second cell channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said cell channel and the respective said outlet. For example, the control system is configured for concurrently operating all the second cell channel address valves to thereby concurrently selectively open or close all the corresponding second cell channel ports.

[0036] Additionally or alternatively, for example, the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective second medium channel address valve overlying the respective second medium channel port, wherein the control system is operable to selectively open or close the second medium channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said medium channel and the respective said outlet. For example, the control system is configured for concurrently operating all the second medium channel address valves to thereby concurrently selectively open or close all the corresponding second medium channel ports.

[0037] Additionally or alternatively, for example, the control system comprises, for each said cell cultivation unit, a plurality of said microfluidic valves in the form of a respective plurality of surface activation valves overlying a portion of the respective medium channel, wherein the control system is operable to selectively open or close each surface activation valve, and wherein when closed each respective said surface activation valve abutting seals against a respective contact zone on a surface of the medium channel, while concurrently permitting fluid communication through the medium channel. In at least some examples, the control system is operable to selectively open or close all surface activation valves together, wherein when closed each respective said surface activation valve abutting seals against a respective contact zone on a surface of the medium channel, while concurrently permitting fluid communication through the medium channel.

[0038] For example, each contact zone is capable of having applied thereto at least one affinity moiety, when the respective surface activation value is open, and wherein responsive to subsequently closing the respective activation value, the respective at least one affinity moiety is fluidly isolated from the respective medium channel. For example, application of the at least one affinity moiety on the respective contact zone is performed by the steps of:

[0039] (a) with the surface activation valve open, applying surface activating material to said surface of the medium channel;

[0040] (b) subsequently closing the surface activation valve;

[0041] (c) with the surface activation valve closed, applying a surface blocking material to said surface of the medium channel, excluding the now isolated contact zone;

[0042] (d) subsequently opening the surface activation valve;

[0043] (e) with the surface activation valve open, applying said at least one affinity moiety to the now exposed contact zone; thereby immobilizing the affinity moiety thereat (i.e., at the activated surface) to create a reaction zone at the contact zone.

[0044] Additionally or alternatively, for example, the control system is configured for concurrently operating the surface activation valves of all the cell cultivation units to thereby concurrently selectively open or close all the corresponding surface activation valves. Additionally or alternatively, for example, the microfluidic platform further comprises a gas channel configured for delivering a gas optionally including at least one of carbon dioxide (CO2), oxygen (O2) and nitrogen (N) in close proximity to the medium channels, and wherein the gas channel is laterally spaced from all the cell channels. For example, the gas channel comprises a channel inlet, a channel outlet, a plurality of longitudinal channel segments and a plurality of lateral channel segments, and wherein the longitudinal channel segments are in overlying relationship with respect to the medium channels.

[0045] Additionally or alternatively, for example, the cell cultivation units are in adjacent laterally spaced relationship with respect to one another within the primary block. For example, each pair of laterally adjacent said cell cultivation units are laterally separated by a solid wall of said substrate material that provides fluid isolation between the cell cultivation units of the respective pair.

[0046] Additionally or alternatively, for example, said control system comprises a plurality of microfluidic control lines, each said microfluidic control line configured for controlling operation of one or more said microfluidic valves associated with the respective said microfluidic control line.

[0047] Additionally or alternatively, for example, the microfluidic platform includes one of the following: wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension; wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a material transparent to electromagnetic radiation at least in the visible spectrum; wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a material transparent to electromagnetic radiation at least in the visible spectrum, and, wherein said material is or comprises at least one of polydimethylsiloxane, polycarbonate, polystyrene; wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a first block layer in overlying abutting relationship with a second block layer, wherein the second block layer comprises said control system, and said first block layer comprises said first plurality of cell cultivation units, and said dedicated input / output lines; wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a first block layer in overlying abutting relationship with a second block layer, wherein the second block layer comprises said control system, and said first block layer comprises said first plurality of cell cultivation units, the fluid inlet system, and said dedicated input / output lines.

[0048] Additionally or alternatively, for example, said first plurality of said cell cultivation units are arranged in an array with respect to the primary block of said substrate material.

[0049] Additionally or alternatively, for example, said first plurality is within a first integer range, the first integer range being between 2 and 20.

[0050] Additionally or alternatively, for example, said first plurality is 16.

[0051] Additionally or alternatively, for example, said second plurality is within a second integer range, the second integer range being between 2 and 20.

[0052] Additionally or alternatively, for example, said second plurality is 11.

[0053] According to a second aspect of the presently disclosed subject matter there is provided a microfluidic system comprising at least one microfluidic platform as defined herein regarding the first aspect of the presently disclosed subject matter, and an accessory module configured for being fluidly coupled with respect to said at least one microfluidic platform.

[0054] For example, the accessory module is in the form of a cell mixing module, wherein the mixing module comprises a secondary block, defining a cell mixing chamber, an inlet port array, a medium input port, an output port, a pump system, and an air purging system, wherein: the inlet port array comprising a plurality of inlet ports selectively connectable to the dedicated input / output lines of said cell cultivation units, such that when thus connected each such connected inlet port and dedicated input / output line are in fluid communication with one another, and wherein the inlet ports are in open fluid communication with the cell mixing chamber; the cell mixing chamber defining a mixing volume in open fluid communication with the medium input port, the output port and the air purging system; the pump system comprising at least one microfluidic pump operative to selectively mix contents of the cell mixing chamber when present; the output port configured for enabling outputting therefrom the contents of the mixing volume; the input port configured for enabling inputting desired media into the mixing volume; the air purging system configured for enabling selectively purging air that may be present in the mixing volume.

[0055] For example, the pump system comprises at least two spaced microfluidic valves operative to periodically open and close one said microfluidic valve while concurrently closing and opening the other microfluidic valve to thereby induce a pumping action within the mixing volume. Additionally or alternatively, for example, the microfluidic system comprises one said microfluidic platform operatively coupled with the cell mixing module, wherein a respective said dedicated input / output lines of at least one said cell cultivation unit is fluidly coupled to a corresponding said inlet port of the cell mixing module.

[0056] For example, the microfluidic system is for use in preparation and / or extraction of protein extract / material and / or nucleic acid extract / material, for use in at least one protein-based assay and / or nuclei acid-based assay.

[0057] Additionally or alternatively, for example, the microfluidic system comprises a first said microfluidic platform and a second microfluidic platform, wherein the first microfluidic platform is operatively coupled with the cell mixing module such that a respective said dedicated input / output lines of at least one said cell cultivation unit of the first said microfluidic platform is fluidly coupled to a corresponding said inlet port of the cell mixing module, and wherein the cell mixing module is coupled to the second said microfluidic platform such that the output port is coupled to the at least one inlet of the second said microfluidic platform. For example, cells mixed in the cell mixing module are for use in carrying out at least one cell-cell interaction assay.

[0058] For example, the accessory module is in the form of a sensor module and comprises at least one sensor configured for being selectively operatively coupled to at least one said dedicated input / output line; wherein the at least one sensor is configured for detecting and / or quantifying at least one parameter and / or at least one target molecule of a fluid present in said dedicated input / output line.

[0059] For example, said microfluidic platform comprises the gas channel as defined herein regarding the first aspect of the presently disclosed subject matter, and further comprising a controller and a gas source including at least one of CO2, O2 and N, wherein the controller is operatively coupled with the said at least one sensor and is configured to operate the gas source to control the flow of said at least one of CO2, O2 and N, to the platform, to provide a desired level of said at least one parameter. For example, said at least one parameter comprises pH of the fluid present in said dedicated input / output line.

[0060] For example, said sensor is configured to detect and / or quantify at least one target molecule in the fluid present in said dedicated input / output line, wherein said sensor comprises at least one affinity moiety specific for said target molecule.

[0061] According to a third aspect of the presently disclosed subject matter there is provided a cell mixing module, comprising a block defining a cell mixing chamber, an inlet port array, a medium input port, an output port, a pump system, and an air purging system, wherein: the inlet port array comprising a plurality of inlet ports in open fluid communication with the cell mixing chamber; the cell mixing chamber defining a mixing volume in open fluid communication with the medium input port, the output port and the air purging system; the pump system comprising at least one microfluidic pump operative to selectively mix contents of the cell mixing chamber when present; the output port configured for enabling outputting therefrom the contents of the mixing volume; the input port configured for enabling inputting desired media into the mixing volume; the air purging system configured for enabling selectively purging air that may be present in the mixing volume. A further aspect of the present disclosure provides a method for performing a cell-based assay. In some embodiments, the cell-based assay may be any assay performed on a population of cells, for example, cells of at least one type. In some embodiments, the cell-based assay may be performed in cells of at least one sample. More specifically, the method comprises the steps of:

[0062] In step (a), applying at least one sample comprising at least one type of cells into a microfluidic platform as defined by the present disclosure. It should be noted that the cells are applied into at least one of the cell cultivation units, via the cell channel, such that each of the cell cultivation chambers in each of the cell cultivation units are seeded with the same at least one type of cells. In step (b), providing at least one cell assay reagent / s and / or cell assay material / s via said medium channel into, or from each of the cell cultivation chambers in each of the cell cultivation units. More specifically, in one option (i), the cell assay reagent / s are applied on cells grown in each of the cell cultivation chambers, via the medium channel. Alternatively, or additionally (ii), the cell assay material / s are collected from and / or produced by, the cells in each of the cell cultivation chambers, and transferred via the medium channel of each of the cell cultivation units to the active zone / s formed by the surface activation valves, as defined by the present disclosure, in each of the medium channel of each of the cell cultivation unit. In step (b), determining at least one test parameter in each the cell cultivation unit: Either on cells of each cell cultivation chambers of option (i); or on cell assay material / s that were collected from and / or produced by, the cells on each cell cultivation chambers of (ii), and reacted with the reaction zone / s.

[0063] A further aspect of the present disclosure provides a method for performing a cell-cell interaction assay. The method comprising the steps of:

[0064] In step (a), applying at least one sample comprising at least two types of cells into a first microfluidic platform of a microfluidic system as defined in the present disclosure. The cells are applied into at least one of the cell cultivation unit, via the cell channel. Each of the cell cultivation units is applied with one type of cells, such that the cell cultivation chambers in each of the cell cultivation unit is seeded with the same one type of cells. Next in step (b), counting the number of cells in each of the cell cultivation chambers in each of the cell cultivation unit. Step (c), involves transferring cells from at least two cell cultivation units, each in a desired cell number, to the cell mixing module via dedicated input / output lines of at least one of the cell cultivation units of the first microfluidic platform. It should be noted that at least two cell cultivation units comprise at least two different types of cells. Still further, a desired number of cells from each type of cells is transferred to the mixing module, each from one or more the cell cultivation unit comprising the cell type. Next in step (d), mixing at least two types of cells, each in a desired number to obtain a cell mixture comprising at least two types of cells in a predetermined ratio. Next in step (e), transferring the cell mixture obtained in step (d), from the cell mixing module via the output port that is coupled to the at least one inlet of the second the microfluidic platform of the system, such that each of the cell cultivation chambers in each of the cell cultivation units is applied with a mixture of at least two types of cells at a predetermined ratio. In step (f), performing at least one cell-cell interaction assay in at least one of the cell cultivation units of the second microfluidic platform of the microfluidic system.

[0065] A further aspect of the present disclosure provides a method for performing nucleic acid-based assay and / or a protein-based assay in a population of cells comprising at least one type of cells. The disclosed method comprises the steps of: First in step (a), applying at least one sample comprising at least one type of cells into a first microfluidic platform of a microfluidic system as defined by the present disclosure. The cells are applied into at least one of the cell cultivation unit, via the cell channel. Still further, each of the cell cultivation units is applied with at least one type of cells, such that each of the cell cultivation chambers in each of the cell cultivation units is seeded with the same at least one type of cells. Next in step (b), transferring cells from one or more cell cultivation units, to the cell mixing module via dedicated input / output lines of at least one the cell cultivation unit of the first microfluidic platform as defined above. In step (c), mixing the at least one type of cells, and subjecting the cell mixture to at least one protein preparation procedure and / or at least one nucleic acid preparation procedure. In step (d), applying the nucleic acid preparation and / or protein preparation obtained in step (c) on at least one protein-cased and / or nucleic acid-based assay.

[0066] A further aspect of the present disclosure relates to a method for detecting and / or quantifying at least one target molecule in at least one sample comprising at least one type of cells. The method comprising: (a), applying at least one sample comprising at least one type of cells into a first microfluidic platform of a microfluidic system as defined by the present disclosure. The system comprises at least one microfluidic platform and at least one sensor, and the cells are applied into at least one of the cell cultivation unit / s, via the cell channel. Each of the cell cultivation units is applied with at least one type of cells, such that the cell cultivation chambers in each of the cell cultivation units are seeded with the same at least one type of cells. Next in step (b), transferring cells from one or more cell cultivation units, to the sensor module via dedicated input / output lines of at least one the cell cultivation unit of the first microfluidic platform. The sensor is configured to detect and / or quantify at least one target molecule in the fluid present in the dedicated input / output line. It should be further noted that the sensor comprises at least one affinity moiety specific for said target molecule. In step (c), determining the presence and / or the quantity of the target, if an end-point indication that reflects recognition and / or binding of the target molecule to the affinity moiety, is detected.

[0067] BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0069] Figure 1. is an isometric view of a microfluidic platform according to an example of the presently disclosed subject matter.

[0070] Figure 2. is a schematic plan view of the fluid inlet system, cell cultivation units, dedicated input / output lines, and the control system of the example of Fig. 1.

[0071] Figure 3. is a schematic plan view of the fluid inlet system, cell cultivation units, and dedicated input / output lines of the example of Fig. 1.

[0072] Figure 4. is a schematic plan view of the control system of the example of Fig. 1.

[0073] Figure 5A-5B. is a schematic plan view of a single cell cultivation unit and its dedicated input / output line, and associated part of the control system, of the example of Fig. 1; Fig. 5B is a schematic plan view of the single cell cultivation unit and its dedicated input / output line only of the example of Fig. 5A; Fig. 5C is a schematic plan view of the associated part of the control system only of the example of Fig. 5A.

[0074] Figure 6-6A. is a plan view of a part of a single cell cultivation unit including a cell cultivation chamber thereof of the example of Fig. 1; Fig. 6A is a cross-sectional side view of the example of Fig. 6, taken along A-A.

[0075] Figure 7. is a cross-sectional side view of a portion of the example of Fig. 2, including a microfluidic control line.

[0076] Figure 8. is a schematic plan view of the fluid inlet system, cell cultivation units, dedicated input / output lines, and a first portion of the control system of the example of Fig. 1.

[0077] Figure 9. is a schematic plan view of the fluid inlet system, cell cultivation units, dedicated input / output lines, and a second portion of the control system of the example of Fig. 1. Figure 10. is a schematic plan view of the fluid inlet system, cell cultivation units, dedicated input / output lines, and a third portion of the control system of the example of Fig. 1.

[0078] Figure 11-11B. is a schematic plan view of the fluid inlet system, cell cultivation units, dedicated input / output lines, and a fourth portion of the control system of the example of Fig. 1 including surface activation valves thereof; Fig. 11A is a schematic plan view of a portion of a cell cultivation units and the respective part of the fourth portion of the control system of the example of Fig. 11; Fig.llB is a schematic cross-sectional side view of the portion of the cell cultivation units and the respective part of the fourth portion of the control system of the example of Fig. 11 A.

[0079] Figurel2. is a schematic plan view of the fluid inlet system, cell cultivation units, dedicated input / output lines, and a fifth portion of the control system of an alternative variation of the example of Fig. 1.

[0080] Figure 13. is an isometric view of a microfluidic platform according to the example of Fig. 12 Figure 14. is a schematic plan view of a microfluidic system according to an example of the presently disclosed subject matter, including one or more microfluidic platforms according to the example of Fig. 1 or Fig. 12, and at least one accessory module.

[0081] Figure 15. is a schematic plan view of an accessory module according to an example of the presently disclosed subject matter, in the form of a cell mixing module.

[0082] Figure 16. is a cross-sectional side view of the example of Fig. 15, taken along C-C.

[0083] Figure 17. is a schematic plan view of an application of the microfluidic system of the example of Fig. 14.

[0084] Figure 18. is a schematic plan view of another application of the microfluidic system of the example of Fig. 14.

[0085] Figure 19A-19B. Design of the OncoFlow device

[0086] Fig. 19A. The OncoFlow chip includes 16X16 chambers for cell cultivation and a set of valves controlling reagent flow. The lower valve (a) allows full separation between drugs application in parallel.

[0087] Fig. 19B. Zoom in presentation of two units within the OncoFlow device. The neck and sandwich valves create 256 individual cultivation chambers on activation. The filters allow continuous nutrition of the cultivated cells by diffusion.

[0088] Figure 20A-20B. Crizotinib and alectinib dose response of H2228 EML4-ALK cells

[0089] Fig. 20A. A clear dose response to crizotinib was observed 18 hours post exposure to the drug, with IC50 of 5pM and maximal effect in concentrations above lOuM crizotinib. Fig. 20B. For alectinib treatment dose dependency started 30 hours post drug exposure with 2.5-5 pM resulting in survival of 40% and maximal drug effect (10% survival) at concentrations above lOpM Alectinib.

[0090] Figure 21A-21B. Cultivation of cells isolated from pleural effusion of patient PE-1 within the OncoFlow device

[0091] Fig. 21A. The cells were cultivated inside the chip, with continuous supply of medium and imaging for 48 hours. A live / dead assay was applied for the evaluation of survival rate. Dead cells were stained with Propidium Iodide (Magenta) while live cells were stained with Cytopainter (Green).

[0092] Fig. 21B. Boxplot representation of the survival rate of PE-1 cells. The graph depicts the survival rate over a period of 48-hours. (n=20, 20 cell chambers were analyzed in each time point).

[0093] Figure 22A-22F. Patient cell samples response to alectinib and crizotinib (15pM) was quantified by live imaging

[0094] Fig. 22A, 22C, 22E. Live imaging of cell samples was performed within the OncoFlow device for 24 hours post drug exposure. Live cells are stained using Cytopainter (Green) and dead cells are stained with Propidium Iodide (Magenta).

[0095] Fig. 22B, 22D, 22F. Cells response to both drugs and Control (without drug) was quantified using survival rate analysis. Resistance to alectinib and sensitivity to crizotinib were observed in the samples from patients PE-1 and PE-11. While cells from patient PE-7 showed resistance to both drugs. (n=20, the number of analyzed chambers).

[0096] Figure 23. PE-4 response to Alectinib (15pM) and Crizotinib (15pM) following 24 hours of on chip exposure

[0097] Graphical presentation of cells’ response to both drugs within a period of 24 hours. Results indicate resistance to the Alectinib treatment (-90% survival), and moderate sensitivity to the Crizotinib treatment resulting in only -70% cell survival.

[0098] Figure 24. PE-6 response to Alectinib (15pM) and Crizotinib (15pM) following 24 hours of on chip exposure

[0099] Graphical presentation of PE-6Bcells’ response to both drugs within a period of 24 hours resulting in -90% survival with Alectinib treatment and only -40% survival following Crizotinib treatment.

[0100] Figure 25A-25B. Summary of patient response to Alectinib / Crizotinib (15pM) exposure

[0101] Fig. 25A. Exposure to Alectinib for 24 hours. All patients demonstrated resistance to alectinib, with about 90% survival rates. Fig. 25B. Exposure to crizotinib for 24 hours. Samples from PE-7 & PE-5 were not affected by the drug, while PE-1 & PE-4 presented mild crizotinib response and cells from patients PE-6 & PE- 11 presented high sensitivity.

[0102] Figure 26A-26B. PE-7 cells in-chip response to 20pM of Pemetrexed

[0103] Fig. 26A. PE-7 cells were grown in the chip and exposed to 20pM of Pemetrexed for 48 hours. Live cells are marked green with Cytopainter while dead cells are colored Magenta with Propidium Iodide. Fig. 26B. The survival rate of PE-7 boxplot depiction of PE-7 cells following no treatment (control) vs. exposure to 20pM of Pemetrexed. Survival rates after 48 hours were -85%, indicating a resistance to the treatment.

[0104] Figure 27A-27B. Characterization and Tyrosine kinase inhibitors cytotoxicity of pleural effusion cells from ALK+ NSCLC patient that progressed on first line Alectinib (PE-11)

[0105] Fig. 27A. Western blot analysis. The levels of ALK, in a sample from NSCLC patients (PE-11), were evaluated by western blot analysis. The patient sample showed high expression levels of ALK. H2228- positive control for ALK, H1975 - negative control for ALK, PEI 1 - the patient sample.

[0106] Fig. 27B. Cell viability following treatment with three different ALK inhibitors at three concentration (l-25pM). Cell viability was assessed using AlamarBlue following 24 hours of treatment. Cell viability decreased following treatment with Crizotinib at 25 pM. The other two ALK inhibitors Alectinib and Lorlatinib did not affect the survival rates as Crizotinib (mean±SD, n=6).

[0107] Figure 28A-28B. Cell counting and manipulations for cell-cell interactions assays

[0108] Fig. 28A. shows a fluorescent micrograph showing two columns, from each type of cell, within a microfluidic chip. Columns 1 and 2 present MM1R cells containing overall 125 cells before (1) and after cells removal (2). Columns 3 and 4 present Jurkat cells containing overall 492 cells before (3) and after cells removal (4).

[0109] Fig. 28B. is a magnified view of a single chamber containing cells, which are subsequently washed out, leaving an empty chamber.

[0110] Figure 29. Cell counting and manipulations for cell-cell interactions assays

[0111] Fluorescent images of different chambers within a microfluidic chip, each containing a co-culture of two cell types, Jurkat (blue) and MM1R (green), at various pre-determined ratios (1: 1, 1:2, and 1:3).

[0112] Figure 30. Detection of ALK-EML4 in the supernatant of cultivated cell lines

[0113] Three cell lines were cultivated in the chip on separate columns: H-2228, which secrets the ALK- EML4 protein and Hl 650 and HEK-293 that do not secret the protein and served as negative controls. The ELISA zone was prepared for protein capturing by surface chemistry procedures. The fused protein, within the medium was captured using a biotinylated anti-ALK antibody and detected using a fluorescently labeled anti-EML4 antibody. The lower pictures present the scanned results, showing protein capture in the H-2228 cells (circles) while no protein was detected in the Hl 650 and HEK-293 cells.

[0114] Figure 31. Connecting the microfluidic cell culture device with a downstream electrochemical biosensor

[0115] Fig 31A. Connected device with red dye flowing over the sensor at lul per minute. Inset shows the PDMS microchannel on top of a dime for scale.

[0116] Fig. 31B. Magnified top view showing the PDMS channel aligned over the sensing area.

[0117] DETAILED DESCRIPTION OF EMBODIMENTS

[0118] While biomarker-guided treatments and NGS-based approaches are refining precision medicine, they are not universally applicable. The gap between the genomic characterization of tumors and their functional behavior is becoming increasingly evident. There is an escalating demand for functional assays that can customize cancer treatments for individual patients and bridge this gap. The inventors have developed OncoFlow, an integrated microfluidic platform that automates viability assays. This platform customizes treatment options by assessing the functional responses of a patient's tumor cells to a specific drug panel. This study specifically addressed non-small cell lung adenocarcinoma (NSCLC) in patients presenting pleural effusion.

[0119] The NCI-H2228 adenocarcinoma cell line was used, which harbors the EML4-ALK fusion oncogene, to develop and fine-tuned the viability assay. Cells cultivated in microfluidic chambers were treated with various concentrations of the tyrosine kinase inhibitors alectinib and crizotinib, and the cytotoxic effects were measured. The results were consistent with those from conventional cell culture methods, thereby validating the assay's reliability. Next, pleural effusion samples from six NSCLC patients, four of them harboring the EML4-ALK rearrangement were tested with alectinib and crizotinib using the OncoFlow system. Monitoring and analysis of cell viability showed varied sensitivities to crizotinib, while all samples exhibited resistance to alectinib. These findings underscore OncoFlow' s potential to enhance physician decision-making and customize treatment plans, ultimately improving patient outcomes.

[0120] The platforms, systems, and methods disclosed herein enable the handling of small cell samples for assaying heterogeneous cancer tissues with high fidelity. In certain embodiments, such samples may be processed, assayed, and subsequently eluted for downstream genomic or proteomic analysis using the same mixing module and workflow. This streamlined approach differs markedly from conventional functional precision oncology (FPO) systems, such as patient-derived xenograft (PDX) models and organoid cultures, which suffer from significant limitations that hinder clinical applicability. These limitations include prolonged assay turnaround times, often extending over weeks to months and thereby exceeding clinically actionable timeframes; selection biases that enrich for highly proliferative clones at the expense of faithfully representing the diversity of the tumor; and the inability to adequately recapitulate the in vivo tumor microenvironment. Collectively, such drawbacks contribute to unreliable predictions of drug responsiveness, thereby diminishing the utility of conventional systems for real-time therapeutic decision-making. By contrast, the platforms, systems, and methods disclosed herein directly address and overcome these shortcomings, providing clinically relevant, timely, and representative assessments of tumor behavior.

[0121] According to an aspect of the presently disclosed subject matter, and referring to Fig. 1, a first example of a microfluidic platform, generally designated with reference numeral 10, is in the form of a primary block 11 of a substrate material, and comprises a block member 200 affixed in overlying relationship with a base member 300.

[0122] Herein, "microfluidic platform" is used interchangeably with any one of "platform", "microfluidic cell cultivation platform", "cell cultivation platform", "device", "microfluidic device", "microfluidic CSRA device", "CSRA device".

[0123] The block member 200 has a generally planar first block face 210 facing and in contact with a complementary first base face 310 of the base member 300. The block member 200 in at least this example is generally parallelepiped in form, and has a length dimension LI, width dimension Wl, and thickness dimension tl. For example, the length dimension LI is about 5cm to 6cm, the width dimension Wl is about 4cm to 5cm, and the thickness dimension tl is about 0.5cm to 0.7cm. It is to be noted that in alternative variations of this examples, and in some other examples, the block member can have any other suitable shape, regular or irregular, and any suitable size. The block member 200 can be made from any suitable bio-compatible material. In at least this example, the block member 200 is made from a material that is transparent to electromagnetic radiation, particularly in the visible spectrum and / or in the spectrum corresponding to fluorescence imaging.

[0124] Furthermore, in at least this example, the block member 200 is made from a substrate material that is gas-permeable, in particular permeable to gaseous carbon dioxide. For example, the block member 200 is made from one or more of: polydimethylsiloxane (PDMS), polycarbonate, polystyrene. In at least this example, the base member 300 has a length dimension L2, width dimension W2, and thickness dimension t2. For example, the length dimension L2 is about 5cm to 6cm, the width dimension W2 is about 4cm to 5cm, and the thickness dimension t2 is about 0.1cm.

[0125] The base member 300 can be made from any suitable bio-compatible material, for example glass, silicon or any other suitable material. In at least this example, the base member 300 is made from a material that is transparent to electromagnetic radiation, particularly in the visible spectrum.

[0126] The block member 200 has a second block face 220, facing a direction generally opposed to that of the first block face 210, and is spaced by the block thickness dimension tl from the second block face 220.

[0127] The base member 300 has a second base face 320, facing a direction generally opposed to that of the first base face 310, and is spaced by the base thickness dimension t2 from the second base face 320. Referring also to Figs. 2, 3 and 4, the block member 200 is configured with a first plurality of cell cultivation units 400 and a control system 700.

[0128] In at least this example, the block member 200 comprises two block layers: a first block layer 230 and a second block layer 260, which are affixed to one another is overlying relationship.

[0129] As best seen in Fig. 3 and Fig. 1, the first block layer 230 is configured with the first plurality of cell cultivation units 400, and further comprises the first block face 210, and a first inter-layer face 215 spaced from the first block face 210 by a first layer thickness tl'.

[0130] As best seen in Fig. 4 and Fig. 1 , the second block layer 260 is configured with the control system 700, and further comprises the second block face 220, and first inter-layer face 215 spaced from the second block face 220 by a second layer thickness tl".

[0131] As will become clearer herein, the control system 700 comprises a plurality of microfluidic valves MV, and a plurality of microfluidic control lines 790. Each microfluidic valve MV is configured for selectively allowing or preventing flow therethrough under the control of the control system 700. Each microfluidic control line 790 is configured for controlling operation of one or more microfluidic valves MV associated with the respective microfluidic control line 790, as will become clearer herein.

[0132] In at least this example, the cell cultivation units 400 are arranged in a parallel spaced arrangement, in the form of a plurality of two or more corresponding array columns. In at least this example, the array columns are arranged parallel to length dimension LI, and laterally spaced from one another along the width dimension Wl.

[0133] According to an aspect of the presently disclosed subject matter, the cell cultivation units 400 are fluidly isolated from one another. By "fluidly isolated from one another" is meant herein that there is an absence of any physical channels laterally coupling any pair of cell cultivation units 400 (at least along the longitudinal length of the cell cultivation units) that, if present, would otherwise provide full, partial or selective fluid communication laterally between the cell cultivation units.

[0134] It is to be noted that in alternative variations of this example, any suitable number of cell cultivation units 400 can be provided, in any desired arrangement.

[0135] In at least this example, the microfluidic platform 10 comprises 11 cell cultivation units 400. However, in alternative variations of this example, the respective microfluidic platform can have any integer number of cell cultivation units 400 greater than 1. For example, the number of cell cultivation units 400 can be any one of or greater than any one of the following: 2, 4, 8, 10, 16, 24, 32.

[0136] Referring in particular to Fig. 5A, Fig. 5B, Fig. 5C, and Fig. 3, in at least this example, each cell cultivation unit 400 comprises a respective medium channel 510, a cell channel 610, and a second plurality of filter pathways 810.

[0137] In each cultivation unit 400, the respective medium channel 510 is laterally spaced with respect to the respective cell channel 610, by a respective lateral spacing SP1.

[0138] Furthermore, and as will become clearer herein, in each cultivation unit 400, the respective second plurality of filter pathways 810 laterally interconnects the respective medium channel 510 with the respective cell channel 610.

[0139] Also as will become clearer herein, in each cultivation unit 400, the respective cell channel 610 defines a second plurality of serially adjacent cell cultivation zones 420. Each such cell cultivation zone 420 is in selective fluid communication with the medium channel 510 via a respective filter pathway 810. Without being bound to theory, inventor considers that such fluid communication is primarily via diffusion.

[0140] As will be disclosed in greater detail herein, for each said cell cultivation unit 400, the control system 700 is configured for selectively blocking fluid communication via the cell channel 610 between adjacent cell cultivation zones 420 to thereby provide respective cell cultivation chambers 450. Each said cell cultivation chamber 450, once formed, is in selective fluid communication with the medium channel 510 exclusively via the respective filter pathway 810.

[0141] Also as will become clearer herein, in each cultivation unit 400 comprises a dedicated input / output line 580 providing selective exclusively fluid communication between an outside of the device 10 and each one of the cell channel 610 and the medium channel 510 of the respective cell cultivation unit 400. As will become clearer herein, the medium channel 510 is configured for enabling fluids to be received in the medium channel 510 from the dedicated input / output line 580 and / or for enabling fluids to be delivered from the medium channel 510 to the dedicated input / output line 580.

[0142] Such fluids include any fluids suitable for sustaining cells in vitro, including culture media such as DMEM, RPMI-1640, EMEM, or IMDM, optionally supplemented with serum, serum-free formulations, or chemically defined media. Such fluids may further include specialized media, balanced salt solutions such as PBS or HBSS, washing fluids, and supplements including antibiotics, glutamine, cytokines, or growth factors, alone or in combination, to support cell viability, proliferation, and function.

[0143] Furthermore, each medium channel 510, juxtaposed and laterally spaced from a respective cell channel 610, operates essentially as a bus.

[0144] Also as will become clearer herein, the cell channel 610 is configured for enabling cell samples CS to be received therein from the dedicated input / output line 580 or to be delivered therefrom to the dedicated input / output line 580. Thus, each cell channel 610 is configured for selectively delivering cell samples CS in suitable media, one or more source agents, for example nutrients for cell growth, culture medium, and / or dyes to the respective cell cultivation units 400, under the action of the control system 700.

[0145] Referring again to to Fig. 5A, Fig. 5B, Fig. 5C, and Fig. 3, each cell cultivation unit 400 extends between a first unit end 401 and a second unit end 402. Furthermore, in at least this example, for each cell cultivation unit 400 the respective medium channel 510 extends longitudinally (i.e., in a direction parallel to the length dimension LI) between the first unit end 401 and the second unit end 402, and the respective cell channel 610 also extends longitudinally between the first unit end 401 and the second unit end 402.

[0146] Each dedicated input / output line 580 is operatively coupled to the first unit end 401 of the respective cell cultivation unit 400.

[0147] Each dedicated input / output line 580 is operatively and separately coupled to the respective cell channel 610 at a first cell channel port 620, at the first unit end 401 of the respective cell cultivation unit 400.

[0148] Furthermore, each dedicated input / output line 580 is at the same time also operatively and separately coupled to the respective medium channel 510 at a first medium channel port 520, also at the first unit end 401 of the respective cell cultivation unit 400. In at least this example, the control system 700 comprises, for each cell cultivation unit 400, a microfluidic valve MV in the form of a respective first cell channel address valve 710 overlying the respective first cell channel port 620. The control system 700 is operable to selectively open or close the first cell channel address valve 710 to thereby allow or prevent fluid communication, respectively, between the respective cell channel 610 and the respective dedicated input / output line 580. The control system 700 is also configured for selectively concurrently operating all the first cell channel address valves 710 to thereby concurrently selectively open or close all the corresponding first cell channel ports 620 of the respective cell cultivation units 400. Thus, when desired, the control system 700 can be operated to concurrently open (or concurrently close) all the first cell channel address valves 710, thereby concurrently opening (or closing) all the corresponding first cell channel ports 620 of the respective cell cultivation units 400. Alternatively, the control system 700 can be operated to concurrently open (or concurrently close) a selected number of the first cell channel address valves 710, thereby concurrently opening (or closing) the selected first cell channel ports 620 of the respective cell cultivation units 400.

[0149] The control system 700 also comprises, for each cell cultivation unit 400, a microfluidic valve MV in the form of a respective first medium channel address valve 720 overlying the respective first medium channel port 520. The control system 700 is operable to selectively open or close the first medium channel address valve 720 to thereby allow or prevent fluid communication, respectively, between the respective medium channel 510 and the respective dedicated input / output line 580. The control system 700 is also configured for selectively concurrently operating all the first medium channel address valves 720 to thereby concurrently selectively open or close all the corresponding first medium channel ports 520 of the respective cell cultivation units 400. Thus, when desired, the control system 700 can be operated to concurrently open (or concurrently close) all the first medium channel address valves 720, thereby concurrently opening (or closing) all the corresponding first cell medium ports 520 of the respective cell cultivation units 400. Alternatively, the control system 700 can be operated to concurrently open (or concurrently close) a selected number of the first medium channel address valves 720, thereby concurrently opening (or closing) the selected first medium channel ports 520 of the respective cell cultivation units 400.

[0150] For each cell cultivation unit 400, the respective filter pathways 810 are configured for selectively providing fluid communication between the respective medium channel 510 and the cell channel 610, while concurrently preventing transit of cells through the respective filter pathways 810 from the cell channel 610 to the medium channel 510. Referring also to Fig. 6, each filter pathway 810 comprises a plurality of lateral channels 830 in selective fluid communication between the respective medium channel 510 and the respective cell channel 610. In particular, each lateral channel 830 has a flow cross-section so dimensioned as to prevent transit of the cells CS therethrough from the cell channel 610 to the medium channel 510. Thus, each lateral channel 830 has a respective cross-sectional area, in particular a cross-sectional profile, such as to allow flow of liquids therethrough, but not of cells CS that may be accommodated in the cell channel 610. For example, each lateral channel 830 can have a width of about 5 micron, while respective medium channel 510 and / or the respective cell channel 610 can each have a width of between about 100pm and about 200pm. In this manner, each group 605 of seeding channels 610 operates as a filter and blocks passage of cells (of the cell sample) therethrough.

[0151] The lateral channels 830 of each filter pathway 810 are, at least in this example, generally rectilinear, and run parallel to the width direction Wl. For each cell cultivation unit 400 each adjacent pair of respective filter pathway 810 is longitudinally spaced from one another by a longitudinal spacing parallel to the length dimension LI.

[0152] The control system 700 also comprises a microfluidic valve MV in the form of a respective horizontal segmentation valve 730 (also interchangeably referred to herein as neck valves) overlying each filter pathway 810 of each cell cultivation unit 400. The control system 700 is operable to selectively open or close each horizontal segmentation valve 730 to thereby allow or prevent fluid communication, respectively, between the respective cell cultivation chamber 450 (when formed) and the respective medium channel 510. The control system 700 is configured for selectively concurrently operating all the horizontal segmentation valves 730 to thereby concurrently selectively open or close all the horizontal segmentation valves 730.

[0153] As disclosed above, for each said cell cultivation unit 400, the control system 700 is configured for selectively blocking fluid communication via the cell channel 610 between adjacent cell cultivation zones 420 to thereby provide respective cell cultivation chambers 450; furthermore, once each cell cultivation chamber 450 is formed, the cell cultivation chamber 450 is in selective fluid communication with the medium channel 510 exclusively via the respective filter pathway 810.

[0154] In this connection, the control system 700 comprises a pair of microfluidic valves MV in the form of a respective pair of longitudinally spaced vertical segmentation valves 740 (also interchangeably referred to herein as sandwich valves). The vertical segmentation valves 740 of each such pair are longitudinally spaced by a longitudinal spacing LS, and longitudinally enclose a respective cell cultivation zone 420 of the respective cell cultivation unit 400. The control system 700 is operable to selectively close each such pair of vertical segmentation valves 740 to thereby provide the respective cell cultivation chamber 450.

[0155] In at least this example, for each cell cultivation unit 400, all the respective vertical segmentation valves 740 thereof are fluidly interconnected via a common fluid passage 745 (Fig. 6) that operates essentially as a bus.

[0156] The control system 700 is further configured for selectively concurrently operating all the vertical segmentation valves 740 of one cell cultivation unit 400 to thereby concurrently selectively close all the corresponding vertical segmentation valves 740 of the respective cell cultivation unit 400, thereby concurrently providing all the cell cultivation chambers 450 of the respective cell cultivation unit 400. Thus, the control system 700 can be selectively operated to form all the cell cultivation chambers 450 of one cell cultivation unit 400 by selectively closing all the vertical segmentation valves 740 of the respective cell cultivation unit 400.

[0157] The control system 700 is further configured for selectively concurrently operating all the vertical segmentation valves 740 of all the cell cultivation units 400 to thereby concurrently selectively close all the corresponding vertical segmentation valves 740 of all the cell cultivation units 400, thereby concurrently providing all the cell cultivation chambers 450 of all the cell cultivation units 400. Thus, the control system 700 can be selectively operated to form all the cell cultivation chambers 450 of all the cell cultivation units 400 by selectively closing all the vertical segmentation valves 740 of all the cell cultivation unit 400.

[0158] Referring in particular to Fig. 3, the microfluidic platform 10 further comprises a fluid inlet system 800 operatively coupled to the respective second unit ends 402 of the cell cultivation units 400.

[0159] In at least this example, the fluid inlet system 800 comprises an inlet manifold arrangement 835. The inlet manifold arrangement 835 has an inlet arrangement comprising one or a plurality of inlets 820 coupled to a plurality of outlets 890 via a succession of bifurcating flow paths defining flow path branches, wherein each outlet 890 is operatively coupled to a respective second unit end 402 of a different cell cultivation unit 400.

[0160] The inlet manifold arrangement 835 is configured for distributing and controlling fluid flow from a main feeding inlet 834 to each of the cell cultivation units 400, via a plurality of a microfluidic valves MV, in the form of a respective plurality of manifold address valves 750, which are comprised in and are under the control of control system 700. The main feeding inlet 834 comprises a manifold address valve 750 in the form of primary manifold address valve 750A configured for selectively allowing or preventing fluid flow therethrough from the inlet manifold arrangement 835.

[0161] The main feeding inlet 834 bifurcates, downstream of the primary manifold address valve 750A, into two first branches 831, each said first branch 831 comprising a manifold address valves 750 in the form of respective secondary manifold address valves 750B configured for selectively allowing or preventing fluid flow therethrough from just downstream of the primary manifold address valves 750A of the main feeding inlet 834.

[0162] Each first branch 831 bifurcates, downstream thereof into two second branches 832, each said second branch 832 comprising a manifold address valves 750 in the form of respective tertiary manifold address valves 750C configured for selectively allowing or preventing fluid flow therethrough from just downstream of the respective secondary manifold address valves 750B of the respective said first branch 831.

[0163] Each second branch 832 bifurcates, downstream thereof into two third branches 833, each third branch 833 comprising a manifold address valves 750 in the form of respective quaternary manifold address valves 750D configured for selectively allowing or preventing fluid flow therethrough from just downstream of the respective tertiary manifold address valves 750C of the respective said second branch 832.

[0164] Each third branch 833 bifurcates, downstream thereof into two fourth branches 834, each fourth branch 834 comprising a manifold address valves 750 in the form of respective quinary manifold address valves 750E configured for selectively allowing or preventing fluid flow therethrough from just downstream of the respective quaternary manifold address valves 750D of the respective said third branch 833.

[0165] Thus, in at least this example, there are 16 fourth branches 834.

[0166] Each fourth branch 834 comprises a respective outlet 890 connected to, and in selective fluid communication with a respective cell cultivation unit 400 via the respective quinary manifold address valves 750E and the respective second end 402.

[0167] It is to be noted that in alternative variations of this example, some of the manifold address valves 750 of the inlet manifold arrangement 835 can be omitted while retaining other manifold address valves 750, to thereby alter the level of control of flow through the various branches of the inlet manifold arrangement 835. The inlet manifold arrangement 835 is configured for selectively delivering each one of a plurality of fluids to the main seeding inlet 834 from a corresponding plurality of sources, under the control of control system 700. For example, such fluids can each include any one or more of: suitable media for cell samples CS; one or more source agents, for example nutrients for cell growth; culture medium; dyes.

[0168] As disclosed above, the control system 700 further comprises a plurality of microfluidic valves MV in the form of a respective plurality of manifold address valves 750. Each manifold address valve 750 is in overlying relationship with a respective branch, and the control system 700 is operable to selectively open or close each manifold valve 750 to thereby allow or prevent fluid communication, respectively, through the respective branch.

[0169] The control system 700 is operative to selectively concurrently open or close all the manifold address valves 750 to thereby enable or prevent, respectively, fluid communication between the at least one inlet 820 and all the outlets.

[0170] The control system 700 is operative to selectively concurrently open or close selected manifold address valves 750 to thereby enable fluid communication between the at least one inlet and only a selected one said outlet of said first plurality of outlets 890.

[0171] Each said outlet is operatively and separately coupled to the respective cell channel 610 and the respective medium channel 510 at a second cell channel port 640 and a second medium channel port 540, respectively, at the second unit end of the respective cell cultivation unit 400.

[0172] The control system 700 comprises, for each cell cultivation unit 400, a microfluidic valve MV in the form of a respective second cell channel address valve 760 overlying the respective second cell channel port 640. The control system 700 is operable to selectively open or close the second cell channel address valve 760 to thereby allow or prevent fluid communication, respectively, between the respective cell channel 610 and the respective outlet 890.

[0173] The control system 700 is configured for concurrently operating all the second cell channel address valves 760 to thereby concurrently selectively open or close all the corresponding second cell channel ports 640.

[0174] The control system 700 comprises, for each cell cultivation unit 400, a microfluidic valve MV in the form of a respective second medium channel address valve 770 overlying the respective second medium channel port 540. The control system 700 is operable to selectively open or close the second medium channel address valve 770 to thereby allow or prevent fluid communication, respectively, between the respective medium channel 510 and the respective said outlet.

[0175] The control system 700 is configured for concurrently operating all the second medium channel address valves 770 to thereby concurrently selectively open or close all the corresponding second medium channel ports.

[0176] In at least this example, the inlet arrangement has a plurality of inlets 820, and each inlet 820 can optionally be used to introduce a different fluid into the cell cultivation units 400 via the inlet manifold arrangement 835.

[0177] The control system 700 further comprises a plurality of microfluidic valves MV, each in the form of a respective inlet valve 780 overlying each inlet 820. The control system 700 is operable to selectively open or close the inlet valves 780 to thereby allow or prevent fluid communication, respectively, between the respective inlet 810 and the inlet manifold arrangement 835. The control system 700 is also configured for selectively concurrently operating all the inlet valves 780 to thereby concurrently selectively open or close all the corresponding inlets 820. Thus, when desired, the control system 700 can be operated to concurrently open (or concurrently close) all the inlet valves 780, thereby concurrently opening (or closing) all the corresponding inlets 820. Alternatively, the control system 700 can be operated to concurrently open (or concurrently close) a selected number of the inlet valves 780, thereby concurrently opening (or closing) the selected inlets 820 of the inlet manifold arrangement 835.

[0178] Referring to Fig. 11, Fig. 11A and Fig. 11B, in at least this example, the control system 700 further compromises a plurality of microfluidic valve MV, each in the form of a respective surface activation valve 765 (also interchangeably referred to herein as button valves). In at least this example, three such surface activation valves 765 are serially overlying a portion of each respective medium channel 510 between the respective second medium channel ports 540 and the respective filter pathways 810. It is to be noted that in at least some alternative variations of this example, some or all of the surface activation valves can be omitted from the respective microfluidic platform.

[0179] The control system 700 is operable to selectively open or close the surface activation valves 765 of each respective cell cultivation unit 400. Referring to Fig. 11 A and Fig. 1 IB in particular, it is to be noted that in the closed position, the respective surface activation valves 765 do not fully block flow of fluid within the respective medium channel 510.

[0180] Rather, the lateral dimension of each surface activation valve 765 is less than the lateral width of the medium channel 510 thereby allowing fluid to flow in-between the sides of the surface activation valve 765 and the side walls of the medium channel 510 in either longitudinal direction, as marked at Al and A2 in Fig. 11 A. Thus, even with the surface activation valves 765 in the closed position, there is still fluid communication between the respective first medium channel port 520 and the respective second medium channel port 540.

[0181] Referring in particular to Fig. 1 IB, it is to be noted that in the closed position, the respective surface activation valves 765 are in abutting sealing contact with the bottom surface 510B of the respective medium channel 510 at a contact zone CZ. In at least this example the bottom surface 510B is provided by corresponding portions of the first base face 310 of the base member 300.

[0182] Such sealing contact at the contact zone CZ between the respective surface activation valves 765 and the bottom surface 510B essentially fluidly isolates the contact zone CZ from the remainder of the internal volume of the respective medium channel 510, and thus from any fluids that can be present therein.

[0183] After being in the closed position, the respective surface activation valves 765 can be opened to thereby expose the contact zones CZ to the internal volume of the respective medium channel 510, and thus to any fluids that can be present therein.

[0184] As with the other types of microfluidic valves MV disclosed herein, each surface activation valve 765 comprises a diaphragm member that is deformable, under the action of the control system 700.

[0185] In at least this example, the control system 700 is also configured for selectively concurrently operating all the surface activation valves 765 to thereby concurrently selectively open or close all the corresponding surface activation valves 765. Thus, when desired, the control system 700 can be operated to concurrently open (or concurrently close) all the surface activation valves 765, thereby concurrently sealing abutting (or exposing) all the corresponding contact zones CZ. Alternatively, the control system 700 can be configured to be operated to concurrently open (or concurrently close) a selected number of the surface activation valves 765, independently of the other surface activation valves 765, thereby concurrently sealing abutting (or exposing) the respective selected contact zones CZ.

[0186] Each such contact zone CZ is capable of having applied thereto at least one affinity moiety, when the respective surface activation value 765 is open, and, responsive to subsequently closing the respective activation value 765, the respective at least one affinity moiety is fluidly isolated from the respective medium channel 510.

[0187] The aforesaid application of the at least one affinity moiety on the respective contact zone CZ can be performed by the steps of: (a) with the surface activation valve 765 open, applying surface activating material to the bottom surface of the medium channel 510;

[0188] (b) subsequently closing the surface activation valve 765;

[0189] (c) with the surface activation valve 765 closed, applying a surface blocking material to the bottom surface of the medium channel 510, excluding the now-isolated contact zones CZ;

[0190] (d) subsequently opening the surface activation valves 765;

[0191] (e) with the surface activation valves 765 open, applying the at least one affinity moiety to the now-exposed contact zones CZ; thereby immobilizing the affinity moiety thereat (the activated surface) to create a reaction zone at the contact zone CZ.

[0192] More specifically, additionally or alternatively, for example, the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective second medium channel address valve overlying the respective second medium channel port, wherein the control system is operable to selectively open or close the second medium channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said medium channel and the respective said outlet. For example, the control system is configured for concurrently operating all the second medium channel address valves to thereby concurrently selectively open or close all the corresponding second medium channel ports.

[0193] Additionally or alternatively, for example, the control system comprises, for each said cell cultivation unit, a plurality of said microfluidic valves in the form of a respective plurality of surface activation valves overlying a portion of the respective medium channel, wherein the control system is operable to selectively open or close each surface activation valve , and wherein when closed each respective said surface activation valve abutting seals against a respective contact zone on a surface of the medium channel, while concurrently permitting fluid communication through the medium channel, flowing around the contact zone. For example, each contact zone is capable of having applied thereto at least one affinity moiety, when the respective surface activation value is open, and wherein responsive to subsequently closing the respective activation value, the respective at least one affinity moiety is fluidly isolated from the respective medium channel.

[0194] In the exemplary device described herein, activation of the valve regulates fluid flow collectively for all of the cultivation units within one half of the microfluidic chip, rather than for each individual cultivation unit. As indicated above, in some embodiments, the application of the at least one affinity moiety on the respective contact zone in the microfluidic platform of the present disclosure, is performed by the following steps of:

[0195] In step (a), with the surface activation valve open, applying surface activating material to the surface of the medium channel. Upon activation of the surface of the entire channel, in step (b), subsequently closing the surface activation valve. Next in (c), with the surface activation valve closed, applying a surface blocking material to the surface of the medium channel, excluding the now isolated contact zone. Next, after blocking the entire channel while maintaining the contact zone protected by the valve, activated, the affinity moiety is next applied on the activated contact zone. Thus step (d) involves subsequently opening the surface activation valve. Next in step (e), with the surface activation valve open, applying the at least one affinity moiety to the now exposed contact zone (that remains activated). Opening the valve exposes the contact zone, such that the affinity moiety applied on the medium channel is attached thereto; thereby immobilizing the affinity moiety thereat. The activated surface that now contains attached thereto the affinity mobilities forms a reaction zone at the contact zone. As all the surface of the medium channel is blocked, the affinity moiety will be immobilized and attached to the activated surface only at the contact zone, to create the reaction zone.

[0196] In some embodiments, a surface activating material used in step (a) herein, may be any reactive group that may allow activation of the surface of the medium channel for immobilizing materials needed for attaching the affinity moiety, either directly or indirectly to the surface of the medium channel at the specified location (the contact zone) as discussed herein after.

[0197] To enable immobilization of an affinity moiety such as antibody, or any other protein-based affinity moiety, the channel walls first need to be functionalized. In the case of silicon, the surface can be silanized with functional silanes to introduce reactive groups such as amines, thiols, or carboxyls. These groups can then be coupled to at least one protein-based affinity moiety (e.g., antibodies), using chemistries like EDC / NHS for carboxyl-to-amine coupling or maleimide-thiol chemistry for sitespecific attachment. Epoxy-silanes are also widely used to provide a reactive surface inside channels. For PDMS, plasma oxidation may be performed first to introduce hydroxyl groups, which can then be further derivatized with silanes carrying reactive groups for crosslinking. Alternatively, channels can be coated with polymers like poly-L-lysine or polydopamine to create a reactive surface. Still further, in some non-limiting embodiments, the activating material may be biotinylated BSA, as described herein after. Once functional groups are introduced, antibodies or any other affinity moiety may be immobilized directly to the activated surface of the channels. Subsequently, following activation of the channel surface, the valve is closed protecting the contact zone, to ensure that the entire surface of the channel is blocked, while the protected contact zone remains activated. Thus, the next step (c) involves blocking step, by applying a surface blocking material at the channel to block the entire surface, except for the contact zone. In some embodiments, where the activating material is biotinylated BSA, the blocking material may comprise avidin, for example, Neutravidin, as discussed herein after. In some alternative embodiments, other blocking materials may be applicable. Common blocking materials include bovine serum albumin (BSA), casein, or fish gelatin, which adsorbs unreacted hydrophobic or charged sites and provide a protein cushion that reduces background binding. Polyethylene glycol (PEG) and its derivatives are frequently used in microfluidic systems, either as passivating coatings on silanized surfaces or grafted polymers, because they are highly effective at minimizing nonspecific protein interactions. Another approach is using synthetic polymers such as polyvinyl alcohol or pluronic F127 to coat the walls, creating a hydrophilic barrier that discourages unwanted binding. The choice of blockers depends on the channel material, the immobilization chemistry, and the downstream assay; for example, BSA or casein are simple and effective for most immunoassays, while PEGylation is preferred for highly sensitive microfluidic biosensors where background noise must be minimized. In yet some further embodiments, if the activated surface is activated with biotin (e.g., biotinylated BSA), blocking may be performed by applying avidin on the surface, specifically, avidin, while the contact zone is protected and remains unblocked. After blocking, the valve is opened and the contact zone that is still activated, is exposed to the affinity moiety that is then immobilized such that the contact zone creates the active zone having affinity moieties attached therein, ready for specific binding of the target molecule that is produced by the cells in the culturing chamber. In some alternative embodiments, specifically where the activating material is applied in more than one layer, biotin (e.g., biotinylated BSA) can be used as a first activating material, followed by avidin (Neutravidin), used herein as the activating material that will directly bind and immobilize the affinity moiety (e.g. biotinylated antibody) to the surface of the contact zone. The blocking material is biotinylated PEG. Accordingly, in some embodiments, the contact zone that creates the reaction zone in each of the cell cultivation units, may be configured to perform an ELISA-like assay. In some specific and non-limiting embodiments, that are also presented by Example 6, the ELISA area within the reaction zones is prepared by the following surface chemistry. Biotinylated BSA is flowed through the medium channel for 30 min, binding the BSA to the surface of the medium channel. As a second layer of activating material, specifically, on top of the biotinylated BSA, 0.5 pg / pL Neutravidin (Pierce) is added for 30 min. This creates a medium channel having an activated surface comprises avidin. The surface activation valves are then closed, and biotinylated PEG (1 pg / pL; Nanocs), used herein as the blocking material is flowed through the medium channel for 30 min, passivating, specifically blocking the rest of the device. Only the contact zones were protected from blockage and included activated surface. Then, the surface activation valves are released, and a specific anchoring biotinylated affinity moiety, specifically, biotinylated antibody is applied, for 20 minutes, via the corresponding addressed valves, at a pressure of 3psi. The antibody bound to the exposed Neutravidin, specifically to the area under the surface activation valves, creating a “trap”, that is the reaction zone, for the target molecule of interest. The surface activation valves are closed (20psi) and medium (e.g., RPMI) is applied to remove unbonded materials. ELISA assay specific conditions are conducted based on the biological question. For antigen trapping, the neck valve is open and so are the input valves of the medium channel side (left for each pair of columns). This set up allows lateral diffusion of molecules from the cell cultivation chamber into the medium stream, and the accumulation of the molecule of interest on the activated surface. Now the valve is closed, protecting the captured molecule, and the reported fluorescent antibody is flowed for 5 minutes allowing binding thereof to the immobilized target molecule.

[0198] Referring also to Fig. 6A, the plurality of cell cultivation units 400, control system 700, and inlet manifold arrangement 835, including all the respective microfluidic valves MV, can be provided by forming suitably shaped and sized recesses projecting inwards from the first block face 210 to a suitable respective depth, relative to the thickness dimension tl ' of the first block layer 230.

[0199] For example, each cell cultivation chamber 450 can be formed as a square-shaped recess, of sides 250 micron in each direction parallel to the length dimension and the width dimension. Such a squareshaped recess can have, for example, a depth of about 20micron from the first block face 210. This arrangement leaves a residual thickness RT sufficient to maintain the mechanical integrity (and thus internal volume) of the cell cultivation chamber 450 essentially unchanged when the control system 700 is being operated.

[0200] Similarly, for example, each cell channel 610 can be formed as a rectangular-shaped recess, of sides 250 micron in each direction parallel to the length dimension and the width dimension, and spanning the spacing between the first unit end 401 and the second unit end 402. This arrangement leaves a residual thickness RT sufficient to maintain the mechanical integrity (and thus internal volume) of the cell channel 610 essentially unchanged when the control system 700 is being operated.

[0201] Similarly, for example, each medium channel 510, and for example each branch of the inlet manifold arrangement 835, and the dedicated input / output lines 580, can also be provided as a recess having a width of about 220micron and depth of about 20 micron, running along the entire length of each medium channel 510, each branch of the inlet manifold arrangement 835, and each dedicated input / output line 580, except for at the locations of the respective microfluidic valves MV thereof. This arrangement leaves a residual thickness RT sufficient to maintain the mechanical integrity (and thus internal volume) of each medium channel 510, each branch of the inlet manifold arrangement 835, and each dedicated input / output line 580 essentially unchanged when the control system 700 is being operated.

[0202] In at least this example, each of the microfluidic valves MV of the platform 10, has a normally open configuration, and a closed configuration in response to selective actuation of the control system 700. In particular, each such microfluidic valve MV is caused to adopt the respective closed configuration responsive to a threshold pressure being applied thereto via the control system 700 and the respective microfluidic control lines 790.

[0203] Referring for example to Fig. 6A, one such microfluidic valve MV, in this case a horizontal segmentation valve 730, comprises a respective valve channel 248 and a respective valve diaphragm 245. The respective valve channel 248 defines a respective valve flow area 247 that is normally open defining the respective open configuration of the respective microfluidic valve MV.

[0204] In at least this example, the respective valve channel 248 can be formed as a first recess 248A projecting inwardly from the first block face 210, and further comprising a second recess 248B extending further inwardly from the first recess 248A. the second recess 248B has a curved generally concave cross-section facing in a direction towards the first block face 210, and thereby defining the respective valve diaphragm 245.

[0205] The valve diaphragm 245 thus has an inner valve surface 245A facing towards the first block face 210, and an outer valve surface 245B facing in a direction away from the first block face 210. The inner valve surface 245A and the outer valve surface 245B are spaced by a valve diaphragm thickness VT. The valve diaphragm thickness VT is significantly smaller than the residual thickness RT, and does not maintain its mechanical integrity when the control system 700 is being operated; rather, when the control system 700 is operated to selectively provide a threshold pressure on the outer valve surface 245B via the respective microfluidic control line 790, the valve diaphragm 245 essentially deforms or otherwise displaces into abutting contact with the first base face 310 in a manner blocking fluid communication via the respective valve flow area, as illustrated by the phantom line 245C in Fig. 6A. Referring again to Fig. 4 and Fig. 7, the control system 700 comprises a plurality of microfluidic control lines 790, each microfluidic control line 790 configured for controlling operation of one or more microfluidic valves MV associated with the respective microfluidic control line 790. In at least this example, the plurality of microfluidic control lines 790 are provided in the second block layer 260.

[0206] Each microfluidic control line 790 has an open first end 752, a closed second end 754, and a lumen 756 extending between the first end 752 and the second end 754. The lumen 756 comprises one or more lumen stations 758, each of which is in overlying relationship with a respective microfluidic valve MV associated with the respective microfluidic control line 790. In particular, in the block member 200, each lumen station 758 is in overlying relationship with the respective diaphragm member 245 of the respective microfluidic valve MV associated with the respective microfluidic control line 790.

[0207] The control system 700, in particular the plurality of microfluidic control lines 790, can be provided by forming suitably shaped and sized recesses projecting inwards from the second interlayer face 225 to a suitable respective depth, relative to the thickness dimension tl" of the second block layer 260. For example, at least a portion of the lumen 756, including all the respective lumen stations 758 of the respective microfluidic control line 790, in at least this example, can be formed as a rectangular-shaped recess, of suitable width and depth, and spanning the spacing between the respective first end 752, and the respective closed second end 754. This arrangement leaves a residual thickness RTT with respect to the second block face 220, sufficient to maintain the mechanical integrity of the respective lumen 756 essentially unchanged when the control system 700 is being operated.

[0208] Each such recess of the lumen 756, in particular of the respective lumen stations 758, has an open end 759 opposite to the respective residual thickness RTT. These open ends 759 are essentially closed by the first interlayer face 215 of the first block layer 230, when the first block layer 230 and the second block layer 260 are affixed to one another in overlying relationship to provide the block member 200. Thus, the respective external valve surface 245B, of the respective diaphragm members 245 of the respective microfluidic valves MV associated with the respective microfluidic control line 790, are exposed to the respective lumen 756 of the respective microfluidic control line 790, at the respective lumen station 758. In operation of the control system 700, each microfluidic control line 790 is operatively and selectively coupled to a pressure source (nor shown), such that a threshold pressure can be selectively applied to the respective lumen 756 via the pressure source. When such a threshold pressure is applied to a particular microfluidic control line 790, the respective diaphragm members 245 of the respective microfluidic valves MV associated with the respective microfluidic control line 790 are correspondingly exposed to the threshold pressure, and thus deform to the respective closed potion of the respective microfluidic valve MV. In this manner, all the microfluidic valves MV associated with the respective microfluidic control line 790 are concurrently closed when the threshold pressure is applied to the microfluidic control line 790. Conversely, when threshold pressure is eliminated from the microfluidic control line 790, all the microfluidic valves MV associated with the respective microfluidic control line 790 are concurrently opened to the respective open configurations.

[0209] For example, each microfluidic control line 790 can be connected to a pressure source, for example in the form of a pneumatic pressure source or in the form of a hydraulic pressure source, to selectively provide the required threshold pressure when desired.

[0210] Referring again to Fig. 4, the control system 700 comprises a plurality of said microfluidic control lines 790, each such microfluidic control line being referred to specially as a first microfluidic control line and designated with reference numeral 790A. The first microfluidic control lines 790A are configured for controlling operation of some of the inlets 820, in particular of the six centrally located inlets 820 that are directly coupled to the main feeding inlet 834.

[0211] Each first microfluidic control line 790A is configured for controlling operation of a different one of these inlets 820.

[0212] Each first microfluidic control line 790A has a respective lumen station 758 overlying the respective inlet valve 780 of these respective inlets 820.

[0213] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to one or more of the first microfluidic control lines 790A results in the respective inlet valve 780 to be allowed to remain open, or in the respective inlet valve 780 to be closed, to thereby allow or prevent respective fluids at the respective inlet 820 to flow to the inlet manifold arrangement 835 (when the primary manifold address valve 750A is also open).

[0214] Referring also to Fig. 8, the control system 700 comprises a second said microfluidic control line 790, being designated with reference numeral 790B. The second microfluidic control line 790B is configured for controlling operation of the remaining two inlets 820.

[0215] The second microfluidic control line 790B has a respective lumen station 758 overlying each one of the two respective secondary manifold address valves 750B of these respective inlets 820.

[0216] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the second microfluidic control line 790B results in the two respective secondary manifold address valves 750B to be allowed to remain open, or in the two respective secondary manifold address valves 750B to be closed, to thereby allow or prevent respective fluids at the two respective inlets 820 to flow to the inlet manifold arrangement 835 via the two first branches 831.

[0217] In at least this example, the second microfluidic control line 790B controls operation of the remaining two inlets 820 concurrently; however, in least some alternative variations of this example, the respective device can instead comprise two separate second microfluidic control lines wherein each controls operation of one of the remaining two inlets 820, independently of the other.

[0218] Referring again to Fig. 4 and to Fig. 8, the control system 700 comprises two third said microfluidic control lines 790, being designated with reference numeral 790C.

[0219] The third microfluidic control lines 790C are configured for controlling operation of the flow through the second branches 832. In at least this example, the second branches 832 are divided into two groups - a first group including the second branches 832 that turn to the right in these figures, and a second group including the second branches 832 that turn to the right in these figures.

[0220] Each third microfluidic control line 790C is configured for controlling operation of a different one of the two groups of the second branches 832.

[0221] Each third microfluidic control line 790C has a respective lumen station 758 overlying the respective tertiary manifold address valves 750C of these second branches 832.

[0222] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to one or more of the third microfluidic control lines 790C results in the respective tertiary manifold address valves 750C to be allowed to remain open, or in the respective tertiary manifold address valves 750C to be closed, to thereby allow or prevent respective fluids at the respective first branches 831 to flow to the inlet manifold arrangement 835 via the second branches 832.

[0223] In at least this example, each third microfluidic control line 790C concurrently controls operation of all the tertiary manifold address valves 750C in the respective group; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate third microfluidic control lines wherein each controls operation of a respective tertiary manifold address valve 750C, independently of the others.

[0224] Similarly, the control system 700 comprises two fourth said microfluidic control lines 790, being designated with reference numeral 790D.

[0225] The fourth microfluidic control lines 790D are configured for controlling operation of the flow through the third branches 833. In at least this example, the third branches 833 are divided into two groups - a first group including the third branches 833 that turn to the right in these figures, and a second group including the third branches 833 that turn to the right in these figures.

[0226] Each fourth microfluidic control line 790D is configured for controlling operation of a different one of the two groups of the third branches 833.

[0227] Each fourth microfluidic control line 790D has a respective lumen station 758 overlying the respective quaternary manifold address valves 750D of these third branches 833.

[0228] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to one or more of the fourth microfluidic control lines 790D results in the respective quaternary manifold address valves 750D to be allowed to remain open, or in the respective quaternary manifold address valves 750D to be closed, to thereby allow or prevent respective fluids at the respective second branches 832 to flow to the inlet manifold arrangement 835 via the third branches 833.

[0229] In at least this example, each fourth microfluidic control line 790D concurrently controls operation of all the quaternary manifold address valves 750D in the respective group; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate fourth microfluidic control lines wherein each controls operation of a respective quaternary manifold address valve 750D, independently of the others.

[0230] Similarly, the control system 700 comprises two fifth said microfluidic control lines 790, being designated with reference numeral 790E.

[0231] The fifth microfluidic control lines 790E are configured for controlling operation of the flow through the fourth branches 834. In at least this example, the fourth branches 834 are divided into two groups - a first group including the fourth branches 834 that turn to the right in these figures, and a second group including the fourth branches 834 that turn to the right in these figures.

[0232] Each fifth microfluidic control line 790E is configured for controlling operation of a different one of the two groups of the fourth branches 834. Each fifth microfluidic control line 790E has a respective lumen station 758 overlying the respective quinary manifold address valves 750E of these fourth branches 834.

[0233] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to one or more of the fifth microfluidic control lines 790E results in the respective quinary manifold address valves 750E to be allowed to remain open, or in the respective quinary manifold address valves 750E to be closed, to thereby allow or prevent respective fluids at the respective third branches 833 to flow to the inlet manifold arrangement 835 via the fourth branches 834.

[0234] In at least this example, each fifth microfluidic control line 790E concurrently controls operation of all the quinary manifold address valves 750E in the respective group; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate fifth microfluidic control lines wherein each controls operation of a respective quinary manifold address valves 750E, independently of the others.

[0235] Similarly, the control system 700 comprises a sixth said microfluidic control line 790, being designated with reference numeral 790F.

[0236] The sixth microfluidic control line 790F is configured for controlling operation of the flow through the second medium channel ports 540 of the respective cell cultivation units 400.

[0237] Each sixth microfluidic control line 790F has a respective lumen station 758 overlying the respective second medium channel address valve 770.

[0238] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the sixth microfluidic control line 790F results in the respective second medium channel address valves 770 to be allowed to remain open, or in the respective second medium channel address valves 770 to be closed, to thereby allow or prevent respective fluids at the respective fourth branches 834 to flow to the respective medium channels 510 via the respective second medium channel address valves 770. In at least this example, each sixth microfluidic control line 790F concurrently controls operation of all the second medium channel address valves 770; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate sixth microfluidic control lines wherein each controls operation of a respective second medium channel address valve, independently of the others.

[0239] Similarly, the control system 700 comprises a seventh said microfluidic control line 790, being designated with reference numeral 790G.

[0240] The seventh microfluidic control line 790G is configured for controlling operation of the flow through the second cell channel ports 640 of the respective cell cultivation units 400. Each seventh microfluidic control line 790G has a respective lumen station 758 overlying the respective second cell channel address valve 760.

[0241] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the seventh microfluidic control line 790G results in the respective second cell channel address valves 760 to be allowed to remain open, or in the respective second cell channel address valves 760 to be closed, to thereby allow or prevent respective fluids at the respective fourth branches 834 to flow to the respective cell channels 610 via the respective second cell channel address valves 760.

[0242] In at least this example, each seventh microfluidic control line 790G concurrently controls operation of all the second cell channel address valves 760; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate seventh microfluidic control lines wherein each controls operation of a respective second cell channel address valve, independently of the others.

[0243] Similarly, the control system 700 comprises an eighth said microfluidic control line 790, being designated with reference numeral 790H.

[0244] The eighth microfluidic control line 790H is configured for controlling operation of the flow through the first cell channel ports 620 of the respective cell cultivation units 400.

[0245] Each eighth microfluidic control line 790H has a respective lumen station 758 overlying the respective first cell channel address valve 710.

[0246] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the eighth microfluidic control line 790H results in the respective first cell channel address valves 710 to be allowed to remain open, or in the respective first cell channel address valves 710 to be closed, to thereby allow or prevent respective fluids flowing between the respective cell channels 610 and the respective dedicated input / output lines 580 via the respective first cell channel ports 620 and respective first cell channel address valves 710.

[0247] In at least this example, each eighth microfluidic control line 790H concurrently controls operation of all the first cell channel address valves 710; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate eighth microfluidic control lines, wherein each controls operation of a respective first cell channel address valve, independently of the others.

[0248] Similarly, the control system 700 comprises a ninth said microfluidic control line 790, being designated with reference numeral 7901. The ninth microfluidic control line 7901 is configured for controlling operation of the flow through the first medium channel ports 520 of the respective cell cultivation units 400.

[0249] Each ninth microfluidic control line 7901 has a respective lumen station 758 overlying the respective first medium channel address valve 720.

[0250] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the ninth microfluidic control line 7901 results in the respective first medium channel address valves 720 to be allowed to remain open, or in the respective first medium channel address valves 720 to be closed, to thereby allow or prevent respective fluids flowing between the respective medium channels 510 and the respective dedicated input / output lines 580 via the respective first medium channel ports 520 and respective first medium channel address valves 720.

[0251] In at least this example, each ninth microfluidic control line 7901 concurrently controls operation of all the first medium channel address valves 720; however, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate ninth microfluidic control lines, wherein each controls operation of a respective first medium channel address valve, independently of the others.

[0252] Referring to Fig. 4 and Fig. 9, the control system 700 comprises a tenth said microfluidic control line 790, being designated with reference numeral 790J.

[0253] The tenth microfluidic control line 790J is configured for controlling operation of the flow through the filter pathways 810 of the respective cell cultivation units 400.

[0254] The tenth microfluidic control line 790J has a respective lumen station 758 overlying each respective horizontal segmentation valve 730.

[0255] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the tenth microfluidic control line 790J results in the respective horizontal segmentation valves 730 to be allowed to remain open, or in the respective horizontal segmentation valves 730 to be closed, to thereby allow or prevent respective fluid communication between the respective cell channels 610 and the respective medium channels 510 of each respective cell cultivation unit 400.

[0256] In at least this example, each tenth microfluidic control line 790J concurrently controls operation of all the filter pathways 810 of all the respective cell cultivation units 400. However, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate tenth microfluidic control lines, wherein each separate tenth microfluidic control line controls operation of all the filter pathways of a respective cell cultivation unit, independently of the other cell cultivation units. In least some other alternative variations of this example, the respective device can instead comprise a different plurality of separate tenth microfluidic control lines, wherein each separate tenth microfluidic control line controls operation of one or a group of filter pathways of a respective cell cultivation unit, independently of the other filter pathways of the same or other cell cultivation units.

[0257] Referring to Fig. 4 and Fig. 10, the control system 700 comprises an eleventh said microfluidic control line 790, being designated with reference numeral 790K.

[0258] The eleventh microfluidic control line 790K is configured for controlling operation of the flow through the cell channels 610 of the respective cell cultivation units 400, to thereby provide the distinct cell cultivation chambers 450.

[0259] The eleventh microfluidic control line 790K has a respective lumen station 758 overlying each respective vertical segmentation valve 740.

[0260] Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the eleventh microfluidic control line 790K results in the respective vertical segmentation valves 740 to be allowed to remain open, or in the respective vertical segmentation valves 740 to be closed, to thereby allow or prevent the formation of the respective cell cultivation chambers 450 of each respective cell cultivation unit 400.

[0261] In at least this example, each eleventh microfluidic control line 790K concurrently controls operation of all the vertical segmentation valves 740 of all the respective cell cultivation units 400. However, in least some alternative variations of this example, the respective device can instead comprise a plurality of separate eleventh microfluidic control lines, wherein each separate eleventh microfluidic control line controls operation of all the vertical segmentation valves of a respective cell cultivation unit, independently of the other cell cultivation units. In least some other alternative variations of this example, the respective device can instead comprise a different plurality of separate eleventh microfluidic control lines, wherein each separate eleventh microfluidic control line controls operation of one or a group of vertical segmentation valves of a respective cell cultivation unit, independently of the other vertical segmentation valves of the same or other cell cultivation units.

[0262] Referring to Fig. 4 and Fig. 11, in at least this example the control system 700 comprises a twelfth said microfluidic control line 790, being designated with reference numeral 790L.

[0263] The twelfth microfluidic control line 790L is configured for controlling operation of the surface activation valves 765.

[0264] Referring also to Fig. 1 IB, the twelfth microfluidic control line 790L has a respective lumen station 758 overlying each respective surface activation valve 765. Thus, operation of the control system 700 such as to selectively provide the threshold pressure to the twelfth microfluidic control line 790L results in the respective surface activation valves 765 to be allowed to remain open, or in the respective surface activation valves 765 to be closed, to thereby concurrently sealing abutting (or exposing) all the corresponding contact zones CZ.

[0265] The device 10 can be produced as follows, for example.

[0266] The first layer 230 can be provided as a block of suitable material, for example PDMS, of suitable thickness tl', length dimension LI and width dimension Wl. Depending on whether thickness tl' is relatively thick or relatively thin, this layer can be manufactured by a suitable casting process or a suitable spin coater process. Thereafter, the reaction units 400, the first network 500 of feeding channels 510, the second network 600 of seeding channels 610, each in the form of suitable recesses of varying depths, can be formed in the first layer via a suitable soft-lithography process.

[0267] Similarly, the second layer 260 can be provided as a second block of suitable material, for example PDMS, of suitable thickness tl", length dimension LI and width dimension Wl. Depending on whether thickness tl" is relatively thick or relatively thin, this layer can be manufactured by a suitable casting process or a suitable spin coater process. Thereafter, the control system 70, in the form of suitable recesses, can be formed in the second layer via a suitable soft-lithography process.

[0268] Thereafter, the first layer 230 and the second layer 260 are aligned such that the lumen stations 758 of each control line 750 overlies the respective microfluidic valve 240 of the first layer 230, and the two layers 230, 260 are fixed to one another. For example the two layers 230, 260 are aligned with respect to one another via manually with the aid of a stereoscope, by aligning each of the microfluidic valves 240 at their correct locations with the respective lumen stations 758. Alternatively, alignment can be performed with any automated method, such as using Microfluidic Device Assembly System (pDAS) (Gerber D. et al., Lab Chip, 2017,17, 557-566).

[0269] Once aligned the two layers 230, 260, in overlying and abutting relationship, are placed in a suitable oven for bonding. Alternatively, bonding between the two layers 230, 260 can be performed by exposing the two layers to oxygen plasma prior to the alignment, in particular by exposing the first interlayer face 215 and second interlayer face 225 to oxygen plasma prior to the alignment. Once aligned and in abutment, the two layers 230, 260 become bonded to one another.

[0270] It is to be noted that in general the block layer having the greatest thickness dimension is held in a fixed manner, while the thinner layer is moved into alignment therewith. For example, the first block layer 230 is held in a fixed manner while the second block layer 260 is moved into alignment therewith. In at least an alternative variation of this example, and referring to Fig. 12, the device 10 further comprises a gas channel 900. The gas channel 900 can be selectively operatively coupled to a gas source to thereby cause gas from the gas source to flow within the gas channel 900.

[0271] The gas channel 900 comprises a channel inlet 910, a channel outlet 920, a plurality of longitudinal channel segments 940 and a plurality of lateral channel segments 960.

[0272] The longitudinal channel segments 940 are superposed over the respective medium channels 510, and thus laterally displaced from the respective cell channels 610.

[0273] Each lateral channel segment 960 couples adjacent pairs of longitudinal channel segments 940 at one or the other longitudinal ends, in an alternating manner, to provide an undulating or serpentine configuration, as illustrated in Fig. 12.

[0274] The channel inlet 910 is coupled to one end of the first longitudinal channel segment 940, and the channel outlet 920 is coupled to an end of the last longitudinal channel segment 940.

[0275] The channel inlet 910, the longitudinal channel segments 940, the lateral channel segments 960, and the channel outlet 920, are thus coupled to one another to provide a contiguous flow path between the channel inlet 910 and the channel outlet 920.

[0276] The longitudinal lengths of the longitudinal channel segments 940 is such that no part of the lateral channel segments 960 overlie any of the cell cultivation chambers 450 or corresponding cell cultivation zones 420.

[0277] Nevertheless, and without being bound to theory, inventor considers that gas when present and / or flowing in the gas channel 900 can diffuse into the cell cultivation chambers 450, which in at least some examples can be spaced from the nearest parts of the gas channel 900 by a spacing of around 100 micron, for example.

[0278] In at least this example, and referring to Fig. 13, the block 11 can comprises an additional layer 270, of thickness tl'", superposed over second layer 260, with channels formed in the underside thereof corresponding to the gas channel 900.

[0279] The additional layer 270 has similar length dimension and width dimension as the second layer 260, for example.

[0280] The underside of the additional layer 270 is in sealing abutting contact with the second block face 220. In at least this example, the channel inlet 910 is connectable to a gas source, for example a pressurised bottle of gas, and the channel outlet 920 is open to the atmosphere. When thus connected, gas from the gas source can selectively flow within the gas channel 900, between the channel inlet 910 and the channel outlet 920, and at least some of this gas can diffuse into the various medium channels 510. In at least this example, the gas can include at least one of carbon dioxide, oxygen and nitrogen, or a mixture of two or three of these gases. In such a mixture, the relative proportions of each gas can be chosen. For example, a separate gas cannister can be provided for each gas, and each cannister is coupled to a gas mixing chamber via a respective valve, which can be fully opened, or fully closed, or adjusted to provide the required relative proportions of each gas to the gas mixing chamber. Alternatively, a canister including premixed proportions of each gas can be used.

[0281] For example, the presence of carbon dioxide in the gas channel 900 can affect the pH of the contents in the cell cultivation chambers 450.

[0282] The additional layer 270 is made from a substrate material that can be similar to that of at least the second layer 260. In particular, in at least this example, the additional layer 270 is transparent to electromagnetic energy, in particular corresponding to the ultraviolet range of wavelengths, the visible spectrum, and the infrared range of wavelengths, as described for example in Example 7.

[0283] According to another aspect of the presently disclosed subject matter, and referring to Fig. 14, a microfluidic system, generally designated with reference numeral 20, comprises one or more microfluidic platforms 10, and at least one accessory module 30 configured for being fluidly coupled to at least one such microfluidic platforms 20.

[0284] According to another aspect of the presently disclosed subject matter, and referring to Fig. 15, a first example of an accessary module is in the form of a cell mixing module, which is pe se novel, and is generally designated with reference numeral 1000. In at least this example, the cell mixing module 1000 comprises a block 1010 defining a cell mixing chamber 1200, an inlet port array 1400, a medium input port arrangement 1600, an output port arrangement 1700, a pump system 1800, and an air purging system 1900.

[0285] The inlet port array 1400 comprises a plurality of inlet ports 1410 in open fluid communication with the cell mixing chamber 1200.

[0286] The cell mixing chamber 1200 defines a mixing volume in open fluid communication with the medium input port arrangement 1600, the output port arrangement 1700, and the air purging system 1900.

[0287] In at least this example, the medium input port arrangement 1600 comprises a single input port, selectively connectable to a medium source (not shown). The medium input port arrangement 1600 configured for enabling inputting desired media into the mixing volume.

[0288] In at least this example, the output port arrangement 1700 comprises a single output port, selectively connectable to at least one of the following: cell-based assays, for example any affinity -based detection of target molecules (either proteinbased or nucleic acid-based target molecules); cell viability assays; cell phenotype assays, for example, cell culture module for modifying and labelling cell mixtures for imaging and / or quantification; downstream proteomic, transcriptomic or genomic analysis.

[0289] Cell-cell interaction assays, such as killing assays, activation assays;

[0290] All various cell-based assays and cell-cell interaction assays provided by the platform, system and methods of the present disclosure are described in more detail herein after.

[0291] The port arrangement 1700 is configured for enabling outputting therefrom the contents of the mixing volume.

[0292] In at least this example, the pump system 1800 comprises a microfluidic pump 1810 operative to selectively mix contents of the cell mixing chamber 1200 when such contents are present in the cell mixing chamber 1200.

[0293] The air purging system 1900 is configured for enabling selectively purging air that may be present in the mixing volume. Thus in operation of the cell mixing module 1000, air is displaced out of the mixing volume via the air purging system 1900 as fluids are inputted into the mixing volume via the medium input port arrangement 1600 and / or via the inlet port array 1400.

[0294] Referring also to Fig. 16, the microfluidic pump comprises a pair of spaced microfluidic valves 1830, operatively coupled to a module control system 1890.

[0295] The block 1010 comprises a block member 1020 affixed in overlying relationship with a base member 1300.

[0296] The block member 1020 in at least this example is generally parallelepiped in form, and has a corresponding length dimension, width dimension, and thickness dimension. For example, the length dimension is about 5cm to 6cm, the width dimension is about 4cm to 5cm, and the thickness dimension is about 0.5cm to 0.7cm. It is to be noted that in alternative variations of these examples, and in some other examples, the block member 1020 can have any other suitable shape, regular or irregular, and any suitable size.

[0297] The block member 1020 can be made from any suitable bio-compatible material. In at least this example, the block member 1020 is made from a material that is transparent to electromagnetic radiation, particularly in the visible spectrum and / or in the spectrum corresponding to fluorescence imaging. Furthermore, in at least this example, the block member 1020 is made from a substrate material that is gas-permeable, in particular permeable to gaseous carbon dioxide. For example, the block member 1020 is made from one or more of: polydimethylsiloxane (PDMS), polycarbonate, polystyrene.

[0298] In at least this example, the base member 1300 has a length dimension, width dimension, and thickness dimension; for example, the length dimension is about 5cm to 6cm, the width dimension is about 4cm to 5cm, and the thickness dimension is about 5m to about 6mm.

[0299] The base member 1300 can be made from any suitable bio-compatible material, for example glass, silicon or any other suitable material. In at least this example, the base member 1300 is made from a material that is transparent to electromagnetic radiation, particularly in the visible spectrum.

[0300] In at least this example, the block member 1020 comprises two block layers: a first block layer 1030 and a second block layer 1060, which are affixed to one another is overlying relationship.

[0301] The first block layer 1030 is configured with the cell mixing chamber 1200, the inlet port array 1400, the medium input port arrangement 1600, the output port arrangement 1700, the air purging system 1900, and the microfluidic valves 1830 of the pump system 1800.

[0302] The second block layer 1060 is configured with the module control system 1890, which comprises at least two microfluidic control lines 1790

[0303] Each microfluidic valve 1830 comprises a respective valve channel 1248 and a respective valve diaphragm 1245. The respective valve channel 1248 defines a respective valve flow area 247 that is normally open defining the respective open configuration of the respective microfluidic valve 1830.

[0304] In at least this example, the respective valve channel 1248 can be formed as a first recess 1249 projecting inwardly from the block face of the first block 1030, and further comprising a second recess 1247 extending further inwardly from the first recess 1249. The second recess 1247 has a curved generally concave cross-section facing in a direction towards the base member 1300, and thereby defining the respective valve diaphragm 1245.

[0305] The valve diaphragm 1245 thus has an inner valve surface 1245A facing towards the base member 1300, and an outer valve surface 1245B facing in a direction away from the base member 1300. The inner valve surface 1245A and the outer valve surface 245B are spaced by a valve diaphragm thickness VT'. The valve diaphragm thickness VT' is significantly smaller than the residual thickness RT', and does not maintain its mechanical integrity when the module control system 1890 is being operated; rather, when the module control system 1890 is operated to selectively provide a threshold pressure on the outer valve surface 1245B via the respective microfluidic control line 1790, the valve diaphragm 1245 essentially deforms or otherwise displaces towards, and optionally into abutting contact with, the base member 1300, as illustrated by the phantom line 1245C in Fig. 6A.

[0306] The module control system 1890 is configured for opening and closing the two microfluidic valves 1830 cyclically and in alternating manner. Thus, to provide a pumping action in the cell mixing chamber, two microfluidic valves 1830 are continuously opened and closed at a desired frequency, but out of phase, such that when one microfluidic valves 1830 is open, the other microfluidic valves 1830 is closed, and vice versa.

[0307] Referring to Fig. 17, in one application of the microfluidic system 20, one microfluidic platform 10 is coupled with one cell mixing module 1000, such that each dedicated input / output line 580 is coupled to a different inlet port 1410 of the inlet port array 1400.

[0308] In at least one example, the microfluidic platform 10 can be coupled with a cell mixing module 1000 by integrating the two components directly on a mold which includes a plurality of connecting channels, each having one end at the respective dedicated input / output line 580 and having another end at the respective inlet port 1410.

[0309] In at least one other example, the microfluidic platform 10 can be coupled with a cell mixing module 1000 (provided as a discreet module separate from the microfluidic platform) via a plurality of connecting tubes, each having one end at the respective dedicated input / output line 580 and having another end at the respective inlet port 1410.

[0310] The microfluidic system 20 of the example of Fig. 17 can be used for performing any cell-cell interaction assay or other cell-based assays, as described herein in connection with additional aspects of the present disclosure, a predetermined number of cells of a first type may be transferred from at least one cell cultivation unit of a first microfluidic platform into a cell mixing module and combined with a predetermined number of cells of at least one other type transferred from a separate cultivation unit of the same microfluidic platform. Within the mixing module, the cells are combined at a desired ratio and may thereafter be utilized directly in cell-based assays, including but not limited to proteomic, transcriptomic, or genomic analyses. In alternative configurations, such as the embodiment illustrated in Figure 18, the mixed cells may be transferred from the mixing module into a second microfluidic platform for use in cell-cell interaction assays, including, for example, killing assays and activation assays. Referring to Fig. 18, in another application of the microfluidic system 20, two microfluidic platforms 10 are coupled with one cell mixing module 1000.

[0311] A first microfluidic platform 10 is coupled with the cell mixing module 1000 such that each dedicated input / output line 580 is coupled to a different inlet port 1410 of the inlet port array 1400.

[0312] In at least one example, the microfluidic platform 10 can be coupled with a cell mixing module 1000 by integrating the two components directly on a mold which includes a plurality of connecting channels, each having one end at the respective dedicated input / output line 580 and having another end at the respective inlet port 1410.

[0313] In at least one other example, the microfluidic platform 10 can be coupled with a cell mixing module 1000 (provided as a discreet module separate from the microfluidic platform) via a plurality of connecting tubes, each having one end at the respective dedicated input / output line 580 and having another end at the respective inlet port 1410.

[0314] The cell mixing module 1000 is coupled to the second microfluidic platform 10 such that the output port arrangement 1700 is coupled to the inlet manifold arrangement 835 of the second microfluidic platform 10.

[0315] In at least one example, the cell mixing module 1000 can be coupled with the second microfluidic platform 10 by integrating the two components directly on a mold which includes a connecting channel, having one end at the output port arrangement 1700 and having another end at the respective inlet manifold arrangement 835.

[0316] In at least one other example, the cell mixing module 1000 can be coupled with the second microfluidic platform 10 (provided as a discreet module separate from the cell mixing module) via connecting tube, having one end at the output port arrangement 1700 and having another end at the respective inlet manifold arrangement 835.

[0317] The microfluidic system 20 of the example of Fig. 18 can be used for performing a cell-cell-interaction assay as discussed by the present disclosure.

[0318] According to another aspect of the presently disclosed subject matter, and referring again to Fig. 14, a second example of an accessary module is in the form of a sensor module, which is pe se novel, and is generally designated with reference numeral 2000. In at least this example, the sensor module 2000 comprises at least one sensor 2010 configured for being selectively operatively coupled to at least one dedicated input / output line 580 of the microfluidic platform 10. The one or more sensors 2010 are configured for detecting and / or quantifying at least one parameter and / or at least one target molecule of a fluid present in the dedicated input / output line 580.

[0319] For example, at least one such parameter comprises pH of the fluid present in dedicated input / output line 580.

[0320] Accordingly, the present disclosure provides a method for operating a microfluidic platform comprising an accessory module in the form of a sensor module, the sensor module comprising at least one sensor configured to be selectively and operatively coupled to at least one dedicated input / output line of the platform. The at least one sensor is configured to detect and / or quantify at least one parameter and / or at least one target molecule of a fluid present in the dedicated input / output line. In some embodiments, the microfluidic platform further comprises a gas channel, a controller, and a gas source comprising at least one of CO2, O2, and N2, wherein the controller is operatively coupled with the at least one sensor and is configured to operate the gas source to control the flow of said at least one of CO2, O2, and N2 to the platform so as to provide a desired level of the at least one parameter. In certain embodiments, the parameter comprises the pH of the fluid present in the dedicated input / output line, such that when the at least one sensor comprises a pH sensor, the method includes determining the pH of the medium using the sensor and manipulating the pH to a desired level through modulation of the gas flow via the gas channel.

[0321] At least one such sensor 2010 can be configured to detect and / or quantify at least one target molecule in the fluid present in dedicated input / output line 580. For example, the sensor comprises at least one affinity moiety specific for the target molecule. Non-limiting embodiments for such sensors are provided by Example 7, and Figure 31, and include various biosensors applicable for performing affinity assays, for example, electrochemical biosensors, which measure changes in current, potential, or impedance resulting from molecular interactions; optical biosensors, which exploit fluorescence, absorbance, or surface plasmon resonance to register binding events; and mass-sensitive biosensors, such as quartz crystal microbalance devices, which detect changes in resonance frequency caused by molecular adsorption.

[0322] It is to be noted that the sensor module 2000 can be configured with a number of such sensors 2020, each sensor being coupled to a different dedicated input / output line 580. Alternatively, the sensor module 2000 can comprise a chamber, coupled to all the dedicated input / output line 580 of interest, such that fluid present in all these dedicated input / output lines 580 can be caused to flow into the chamber. In such a case, one or more such sensors can be accommodated in the chamber for detecting and / or quantifying at least one parameter and / or at least one target molecule of a fluid present in the chamber.

[0323] It is also to be noted that the microfluidic platform 10 can be coupled at the same time to both a cell mixing module 1000 and to a sensor module 2000. For example, a first group of dedicated input / output lines 580 of the microfluidic platform 10 can be coupled to the cell mixing module 1000, while a second group of dedicated input / output lines 580 of the same microfluidic platform 10 can be coupled to the sensor module 2000.

[0324] In at least some examples, and referring again to Fig. 14, in which the microfluidic platform comprises the gas channel 900, for example as illustrated in Fig. 12, the respective microfluidic system 20 further comprises a controller 990 and a gas source 980. The gas source 900 includes at least one of, or a mixture of at least two of, carbon dioxide, oxygen or nitrogen.

[0325] The controller 990 is operatively coupled with the one or more sensors 2010 of the sensor module 2000, and is configured to operate the gas source 980 to control the flow of at least one of carbon dioxide, oxygen or nitrogen to the microfluidic platform 10, to provide a desired level of the parameter. For example, and with such a parameter being the pH of the fluids in the input / output lines 580 of the microfluidic platform 10, the sensor 2010 determines the pH and the controller compares the level of the measured pH against a datum value or range for a particular use of the microfluidic platform 10. According to whether the measured level of pH is above or below such a datum, the controller 990 can provide more or less carbon dioxide, respectively, to restore the level of pH to the datum.

[0326] Still further, according to this aspect of the presently disclosed subject matter there is provided a microfluidic system comprising at least one microfluidic platform as defined herein regarding the first aspect of the presently disclosed subject matter, and an accessory module configured for being fluidly coupled with respect to said at least one microfluidic platform.

[0327] For example, the accessory module is in the form of a cell mixing module, wherein the mixing module comprises a secondary block, defining a cell mixing chamber, an inlet port array, a medium input port, an output port, a pump system, and an air purging system, wherein:

[0328] The inlet port array comprising a plurality of inlet ports selectively connectable to the dedicated input / output lines of said cell cultivation units, such that when thus connected each such connected inlet port and dedicated input / output line are in fluid communication with one another, and wherein the inlet ports are in open fluid communication with the cell mixing chamber; the cell mixing chamber defining a mixing volume in open fluid communication with the medium input port, the output port and the air purging system; the pump system comprising at least one microfluidic pump operative to selectively mix contents of the cell mixing chamber when present; the output port configured for enabling outputting therefrom the contents of the mixing volume; the input port configured for enabling inputting desired media into the mixing volume; the air purging system configured for enabling selectively purging air that may be present in the mixing volume.

[0329] For example, the pump system comprises at least two spaced microfluidic valves operative to periodically open and close one said microfluidic valve while concurrently closing and opening the other microfluidic valve to thereby induce a pumping action within the mixing volume.

[0330] Additionally or alternatively, for example, the microfluidic system comprises one said microfluidic platform operatively coupled with the cell mixing module, wherein a respective said dedicated input / output lines of at least one said cell cultivation unit is fluidly coupled to a corresponding said inlet port of the cell mixing module.

[0331] For example, the microfluidic system is for use in preparation and / or extraction of protein extract / material and / or nucleic acid extract / material, for use in at least one protein-based assay and / or nuclei acid-based assay.

[0332] In some embodiments, the disclosed microfluidic system discussed herein, may be used for performing a protein-based assay, where at least one of: the target and / or the affinity moiety is composed of a proteaceous material. Affinity assay may include any affinity assay performed using any affinity pair, for example, immune-assays, that are based on antibody-target affinity. One non-limiting embodiments of such affinity assay is the ELISA-like assay described herein above in connection with the microfluidic platform that includes the reaction zone. Such affinity-assays are therefore applicable for any of the microfluidic systems disclosed by the present disclosure, provided that such system comprises at least one microfluidic platform that comprises the reaction zone / s as discussed above. In such case, either the affinity moiety or alternatively, the target molecule comprises antibody or any antigen-binding fragments thereof. Other affinity assays applicable in the present disclosure are based on affinity pairs such as aptamer-target, receptor-ligand, enzyme-substrate and the like.

[0333] In addition to the affinity assays discussed above, a system that further comprise as an accessory module, a mixing module, allows the use of such module not only for mixing cells obtained from one or more of the cell cultivation units of the microfluidic platform attached to the mixing module, but also for preparation of proteinaceous and / or nucleic acid material from the cells that may be used for performing assays such as proteomics, genomics and transcriptomic assays, or any protein-based and / or nucleic acid based assay.

[0334] More specifically, a protein-based assay applicable in the present disclosure may include proteomics, to analyze the complete set of proteins expressed in the cells in the cultivating chambers, or in each of the cell cultivation units. Proteomics typically begins with protein extraction from biological samples, e.g., from each of the cultivation chambers followed by separation, identification, and quantification. Mass spectrometry (MS) has become the central technology in this field, often combined with liquid chromatography (LC-MS / MS) for high-resolution separation and identification of peptides generated through enzymatic digestion (commonly with trypsin).

[0335] Still further, in some other embodiments, the disclosed platform and system may be applicable for performing any nucleic acid-based assay. Non-limiting examples for nucleic acid-based assays include sequencing, Next-generation sequencing (NGS) NGS, as well as RNA-based assays such as RT-PCT, and RNA sequencing, transcriptomic assay and genomics assay. More specifically, in some embodiments, transcriptomic assay measures the complete set of RNA transcripts expressed in a cell or tissue at a given time, providing insight into gene activity and cellular states. The main approaches include microarrays, which rely on hybridization to predesigned probes and are useful for profiling known genes; RNA sequencing (RNA-seq), now the gold standard, which uses high-throughput sequencing to quantify expression and detect novel transcripts and splice variants; and single-cell RNA sequencing (scRNA-seq), which captures expression patterns in individual cells to reveal heterogeneity within complex tissues. Genomic assays that may be applicable in the disclosed systems, applies technologies such as whole-genome sequencing, whole-exome sequencing, and targeted gene panels are commonly employed, each offering different levels of resolution and scope. According to such embodiments, nucleic acids are prepared from the cells in each cultivation chamber (using any nucleic acid extracting reagents that may be provided through the cell channel). The nucleic acid preparation is transferred to the reaction zones in each of the cell culture units and reacted with nucleic acid-based affinity molecules attached to the reaction zone. Upon specific recognition and binding, the target nucleic acids prepared from the cells are detected.

[0336] In some embodiments, the disclosed microfluidic system comprises a first microfluidic platform as disclosed by the present disclosure, and a second microfluidic platform. The first microfluidic platform is operatively coupled with the cell mixing module such that a respective dedicated input / output lines of at least one of the cell cultivation units of the first microfluidic platform is fluidly coupled to a corresponding the inlet port of the cell mixing module. Still further, the cell mixing module is coupled to the second microfluidic platform such that the output port is coupled to the at least one inlet of the second microfluidic platform.

[0337] In some embodiments of such configuration of the disclosed microfluidic system, the cells provided from the first microfluidic platform are mixed in the cell mixing module and then are applied into the second microfluidic platform ready for carrying out at least one cell-cell interaction assay.

[0338] As will be discussed in detail herein after, a cell-cell interaction assay may be any assay involving the interaction between at least two cells, specifically, the direct communication and physical contacts between at least two cells or two populations of cells, while the result of this interaction is detected and monitored by the disclosed systems. In some embodiments, a cell-cell interaction assay as used herein may include killing assay, and / or cell activation assay, and / or differentiation assay, and / or adhesion assay. More specifically, killing assays are used to evaluate the ability of effector cells such as cytotoxic T lymphocytes or natural killer cells to eliminate target cells, often by monitoring target cell lysis or death. Activation assays are designed to assess how one cell type stimulates another, for example when antigen-presenting cells activate T cells, leading to measurable outcomes such as cytokine secretion, upregulation of activation markers, or proliferation. Adhesion assays focus on the strength and specificity of binding between two cell populations, highlighting the role of adhesion molecules and receptor-ligand interactions in maintaining contact. Differentiation assays are employed in co-culture settings where one cell type directs the fate of another, as seen in stromal- stem cell interactions. In some embodiments, the accessory module of the disclosed microfluidic system is in the form of a sensor module and comprises at least one sensor configured for being selectively operatively coupled to at least one said dedicated input / output line. More specifically, the at least one sensor is configured for detecting and / or quantifying at least one parameter and / or at least one target molecule of a fluid present in the dedicated input / output line. A schematic presentation of this embodiment is provided by Figure 14.

[0339] More specifically, the microfluidic systems of the present disclosure integrate microfluidic platforms with two broad categories of sensors, acting herein as the sensor module. The first encompasses biosensors for detecting target molecules, which rely on different transduction principles to convert biological recognition events into measurable signals. These include electrochemical biosensors, which measure changes in current, potential, or impedance resulting from molecular interactions; optical biosensors, which exploit fluorescence, absorbance, or surface plasmon resonance to register binding events; and mass-sensitive biosensors, such as quartz crystal microbalance devices, which detect changes in resonance frequency caused by molecular adsorption. Such biosensors, that may be coupled each to each of the cell cultivation units of the disclosed microfluidic platform of the discussed systems, provide high specificity and sensitivity, making them well suited for real-time monitoring of diverse analytes in environmental, medical, and industrial contexts. The second category comprises sensors for physical parameters of the medium, including pH sensors (optical or electrochemical flow-through modules), conductivity-based salinity sensors, and temperature sensors such as thermistors or resistance temperature detectors. Additional devices, such as optical oxygen probes, can further extend monitoring to dissolved gases. Specific embodiments for such sensor modules are disclosed by Example 7, and Figure 31.

[0340] For example, the accessory module is in the form of a sensor module, and comprises at least one sensor configured for being selectively operatively coupled to at least one said dedicated input / output line; wherein the at least one sensor is configured for detecting and / or quantifying at least one parameter and / or at least one target molecule of a fluid present in said dedicated input / output line.

[0341] For example, said microfluidic platform comprises the gas channel as defined herein regarding the first aspect of the presently disclosed subject matter, and further comprising a controller and a gas source including at least one of CO2, O2 and N, wherein the controller is operatively coupled with the said at least one sensor and is configured to operate the gas source to control the flow of said at least one of CO2, O2 and N, to the platform, to provide a desired level of said at least one parameter. For example, said at least one parameter comprises pH of the fluid present in said dedicated input / output line. Thus, where the sensor attached to the microfluidic platform of the disclosed systems is a sensor for physical parameters of the medium, for example, pH sensors, upon determining the pH of the medium using the sensor, the manipulation and modulation of the pH to the desired levels may be achieved using the gas channel as defined herein.

[0342] In other embodiments, the sensor is configured to detect and / or quantify at least one target molecule in the fluid present in the dedicated input / output line, wherein said sensor comprises at least one affinity moiety specific for said target molecule. Such sensors may be any of the biosensors are as described above.

[0343] A further aspect of the present disclosure provides a method for performing a cell-based assay. In some embodiments, the cell-based assay may be any assay performed on a population of cells, for example, cells of at least one type. In some embodiments, the cell-based assay may be performed in cells of at least one sample. More specifically, the method comprises the following steps:

[0344] In step (a), applying at least one sample comprising at least one type of cells into a microfluidic platform as defined by the present disclosure. It should be noted that the cells are applied into at least one of the cell cultivation units, via the cell channel, such that each one of the cell cultivation chambers in each of the cell cultivation units is seeded with the same at least one type of cells. In step (b), providing at least one cell assay reagent / s and / or cell assay material / s via said medium channel into, or from each of the cell cultivation chambers in each of the cell cultivation units. More specifically, in one option (i), the cell assay reagent / s (e.g., therapeutic agent) are applied on cells grown in each of the cell cultivation chambers, via the medium channel. Alternatively, or additionally (ii), the cell assay material / s (protein-based or nucleic acid based material that are either secreted by the cells or produced from the cells) are collected from and / or produced by, the cells in each of the cell cultivation chambers, and transferred via the medium channel of each of the cell cultivation units to the active zone / s formed by the surface activation valves, as defined by the present disclosure, in each of the medium channel of each of the cell cultivation unit. In step (c), determining at least one test parameter in each of the cell cultivation unit. This step is performed either on cells of each cell cultivation chambers of option (i); or on cell assay material / s that were collected from and / or produced by, the cells on each cell cultivation chambers of (ii), and reacted with the reaction zone / s. More specifically, in some embodiments (i), the test reagents (e.g. the therapeutic agent or candidate compound) are provided via the medium channel of the cultivation units and contacted with the cells in each of the cultivation chambers. In such case, the test parameter (e.g., cell viability or cell morphology), is determined in each of the cell cultivation chambers. In other alternative or additional embodiments (ii), cell assay material / s, that may be any protein-based target or nucleic acid-based target (either produced by the cells and secreted to the medium) is collected from each of the cultivation chambers and via the medium channel is transferred to the reaction zones as described above. The test parameter (e.g., presence or quantity of at least one target molecule) is thus determined in each of the reaction zones.

[0345] As used herein, the term “cell-based assay” refers to any biological, research, or molecular assay that is performed on cells, cell parts, or on any material produced by cells and / or secreted by cells into a medium or environment, and / or on any material derived from cells by molecular or chemical methods. Such materials include, for example, proteins, nucleic acids, whole cells, cellular organelles, or cellular fractions. In a non-limiting example, a cell-based assay may comprise evaluating cell morphology, assessing cell viability, or analyzing target substances secreted by the cells or obtained from the cells, such as nucleic acids or proteins. In some particular and non-limiting embodiments, for preparation of material for a cell-based assay, the required reagents generally include three categories: cell culture components, assay-specific detection reagents, and appropriate controls. In addition, these reagents may comprise a test compound (e.g., therapeutic compound or any other candidate compound), may be also provided with the medium. Cell culture is maintained in a basal medium such as DMEM, RPMI, or MEM supplemented with serum, typically fetal bovine serum, and optionally antibiotics or antimycotics. Washing steps employ buffered saline solutions such as PBS or HBSS, and adherent cells may be detached using trypsin-EDTA or a non-enzymatic dissociation buffer. Cell number and viability are determined using standard dyes such as trypan blue. Assay readout determines the detection reagents employed in each case, as will be discussed for example for cell viability assay, herein below.

[0346] In some embodiments, cell assay material, as used herein, is any material (e.g., protein-based or nucleic acid based) collected from the cells in each cultivation chambers, maybe any material or compound present in the cell medium in each chamber. For example, any compound secreted by the cells or produced directly or indirectly by the cells. For example, any protein molecules secreted by the cells (cytokines, enzymes and the like). Alternatively, any protein or nucleic acid material that is produced, extracted and / or prepared from the cells, may be collected from each cultivation chamber and transferred via the medium channel to the reaction zone in each of the cell cultivation units. In the preparation of proteins or nucleic acids from cellular material, the process generally involves cell disruption, stabilization of the desired biomolecule, and removal of contaminants. Typically, cells are lysed using mechanical, enzymatic, or chemical means, often with buffers containing detergents such as SDS, Triton X-100, or CHAPS to solubilize membranes. For nucleic acids, nuclease inhibitors and chelating agents such as EDTA are included to prevent degradation, while proteolytic enzymes like proteinase K or chemical denaturants assist in liberating DNA or RNA from protein complexes. For protein extraction, protease inhibitors are incorporated to preserve protein integrity, and salts or chaotropic agents may be used to facilitate solubilization and stabilization. In such case, according to some embodiments, reagents for the preparation of this cell assay material are provided to each cultivation chamber via the cell cannel.

[0347] In more specific embodiments, the microfluidic test platform used in the disclosed methods comprises: a primary block of substrate material, the primary block defining a first plurality of cell cultivation units, and a control system for enabling control of fluid flows with respect to the first plurality of cell cultivation units. The cell cultivation units are fluidly isolated from one another. Still further, each said cell cultivation unit comprises a medium channel, a cell channel and a second plurality of filter pathways, wherein: the medium channel is laterally spaced with respect to the cell channel; the second plurality of filter pathways laterally interconnects the medium channel with the cell channel; the cell channel defines a second plurality of serially adjacent cell cultivation zones, wherein each cell cultivation zone is in selective fluid communication with the medium channel via a respective said filter pathway;

[0348] Still further, for each of the cell cultivation unit / s, the control system is configured for selectively blocking fluid communication via the cell channel between adjacent cell cultivation zones to thereby provide respective cell cultivation chambers. Each of the cell cultivation chamber / s is in selective fluid communication with the medium channel exclusively via the respective said filter pathway;

[0349] Still further, each of the cell cultivation unit comprises a dedicated input / output line providing selective exclusively fluid communication between an outside of the device and each one of the cell channel and the medium channel of the respective cell cultivation unit; wherein for each cell cultivation unit: the medium channel is configured for enabling fluids to be received therein from the dedicated input / output line or to be delivered therefrom to the dedicated input / output line; the cell channel is configured for enabling cells to be received therein from the dedicated input / output line or to be delivered therefrom to the dedicated input / output line; the filter pathways are configured for selectively providing fluid communication between the medium channel and the cell channel, while concurrently preventing transit of cells therethrough from the cell channel to the medium channel.

[0350] It should be understood that, in some non-limiting embodiments, the microfluidic platform applicable in the disclosed methods for performing any cell-based assay may correspond to any of the platforms illustrated in Figures 1 to 13, as well as to the systems illustrated in any one of Figures 14 to 18 and Figure 31. Furthermore, in certain embodiments, the disclosed methods may employ the microfluidic platform as designed in Example 1.

[0351] In some embodiments, the cell-based assay is a cell viability assay. It should be noted that cell viability assay is demonstrated by Examples 2 and 3. In some embodiments, the cell assay reagent / s comprise at least one candidate compound or therapeutic agent that may affect cell viability. Accordingly, the method comprising: (i), exposing cells applied in step (a), in each of the cell cultivation chambers, to at least one candidate compound or therapeutic agent (e.g., affecting cell viability). The compound or agent (also referred to herein as a test compound) is provided via the medium channel of each of the cell cultivation units. Next in (ii), determining cell viability and / or cell number, and / or cell phenotype in each of the cell cultivation chamber. Wherein the cells in all cell cultivation chambers of each of the cell cultivation unit / s are exposed to the same at least one candidate compound or therapeutic agent. A non-limiting embodiment for a cell viability assay is provided by Examples 2 and 3.

[0352] In some embodiments, cells were cultured within the microfluidic test platform for a suitable time period before being exposed and / or contacted with the test active agent. In some embodiments, cells may be cultured for lhr-48hr, specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48hrs, and more, 72, 96 hours and more.

[0353] In some embodiments, the cells may be exposed several time points to the candidate active agent specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 time points.

[0354] In some embodiments the additional time of exposure to the candidate active agent may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48hrs, and more, 72, 96 hours and more.

[0355] In some embodiments, the concentration of the cells grown in the microfluidic test platform may vary between 104cells mL1to 1010cells mL1, specifically 105cells mL1or 106cells ml / 1or 2T06cells mL'1or 3T06cells mL'1or 4- 106cells mL'1or 5T06cells mL'1or 6T06cells mL'1or 7T06cells mL1or 8- 106cells mL1or 107cells ml1or 15T06cells ml / 1or 108cells ml / 1or 109cells ml / 1or IO10cells mL1.

[0356] Still further, the average number of cells per chamber may vary between 0 to 1000 cells per chamber, specifically about 1, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 cells.

[0357] In some embodiments, the cells in all cell cultivation chambers of a cell cultivation unit are exposed to the same candidate compound or therapeutic agent or the same amount / dilution / concentration of the candidate compound or therapeutic agent.

[0358] In some embodiments, the cell viability is determined by using at least one cell-impairment DNA binding dyes and / or nuclear staining, apoptosis.

[0359] In more specific embodiments, cell viability is determined Viability and proliferation may be assessed using tetrazolium salts (MTT, XTT, WST-1), resazurin, or ATP-based luminescence kits. Cytotoxicity can be measured using LDH-release reagents or apoptosis markers such as Annexin V and propidium iodide. Reporter assays utilize substrates such as luciferin for luciferase or fluorescence readouts from GFP or RFP. Signal transduction assays may employ calcium-sensitive dyes (Fura-2, Fluo-4), cAMP detection reagents, or kinase substrates. Immunostaining methods require fixatives such as paraformaldehyde, permeabilizers including Triton X-100 or saponin, and suitable antibodies.

[0360] Controls are included to validate assay performance. A positive control compound with known biological activity relevant to the assay is applied, and a negative control consisting of vehicle (e.g., DMSO) or buffer is maintained. Reference standards are prepared when quantitative data are required. In some embodiments, the parameter determined in step (ii), may be cell phenotype upon exposure of the cells to the test compound or agent. As used herein, a cellular phenotype may be any detectable characteristic or property of a cell. In some embodiments, cell phenotype may refer to at least one of: cell activity, cell morphology, expression profile, functional response, enzymatic activity of enzymes secreted by the cells (metaloproteases), post translational modification profile, expression profile, of cell surface markers (e.g., TAA's), metastatic properties (invasiveness, cell motility, cell migration and cell adhesion), accumulation of metabolites (e.g., metabolite is any one of a nucleobase, nucleoside, nucleotide, amino acid residue / s, carbohydrate / s, fatty acid and ketone, sterols, calcium accumulation, porphyrin and haem, lipid, sphingolipid, phospholipid, and lipoprotein, neurotransmitters, vitamins and (non-protein) cofactors, pterin, trace elements, metals, metabolites associated with energy metabolism, metabolites associated with peroxisome functions, or any intermediate product, derivative or metabolite there). Thus, cell phenotype also refers to expression of different markers and receptors that characterize the cells.

[0361] Particular and additional methos applicable in the present aspect for determining cell viability and cell phenotype are disclosed in more detail herein after in connection with other aspects of the present disclosure.

[0362] Different types of cells may be suitable for the methods of the present disclosure, as well as for all microfluidic platforms, and systems disclosed by the present disclosure. Specifically in some embodiments, cells suitable for the methods of the disclosure are eukaryotic cells.

[0363] A eukaryote cell or eukaryotic cells as herein defined refer to cells within an organism that contain complex structures enclosed within membranes. All large complex organisms are eukaryotes, including animals, plants and fungi. Thus eukaryotic cells as herein defined may be derived from animals, plants and fungi, for example, but not limited to, insect cells, yeast cells or mammalian cells. There are several types of eukaryotic cells that may be used by the methods of the present disclosure. By way of example, eukaryotic cells may be, but are not limited to, stem cells, e.g. embryonic stem cells, totipotent stem cells, pluripotent stem cells or induced pluripotent stem cells, multipotent progenitor cells and plant cells.

[0364] Stem cells are generally known for their three unique characteristics: (i) they have the unique ability to renew themselves continuously; (ii) they have the ability to differentiate into somatic cell types; and (iii) they have the ability to limit their own population into a small number. In mammals, there are two broad types of stem cells, namely embryonic stem cells (ESCs), and adult stem cells. Stem cells may be autologous or heterologous to the subject. In order to avoid rejection of the cells by the subject’s immune system, autologous stem cells are usually preferred.

[0365] Thus, in some embodiments, the eukaryotic cells according to the disclosure may be embryonic stem cells, or human embryonic stem cells (hESCs), that were obtained from self-umbilical cord blood just after birth. Embryonic stem cells are pluripotent stem cells derived from the early embryo that are characterized by the ability to proliferate over prolonged periods of culture while remaining undifferentiated and maintaining a stable karyotype, with the potential to differentiate into derivatives of all three germ layers. hESCs may be also derived from the inner cell mass (ICM) of the blastocyst stage (100-200 cells) of embryos generated by in vitro fertilization. However, methods have been developed to derive hESCs from the late morula stage (30-40 cells) and, recently, from arrested embryos (16-24 cells incapable of further development) and single blastomeres isolated from 8-cell embryos. In some embodiments, the embryonic stem cells applicable by the disclosed methods may be ESCs of a non-human subject. Stil further, in some embodiments, the ESCs applicable herein may be of a non-primate subject. In yet some further embodiments, in case a human ESCs are used in the disclosed methods, these cells may be any ESCs with the proviso that they cannot form an entire organism. Still further, in some further embodiments, in case human ESCs are used in the disclosed methods, these cells may be any ESCs with the proviso that they were not obtained from embryos or living embryos distracted for the preparation of the cells.

[0366] In further embodiments, the eukaryotic cells according to the disclosure are totipotent stem cells. Totipotent stem cells are versatile stem cells and have the potential to give rise to any and all human cells, such as brain, liver, blood or heart cells or to an entire functional organism (e.g. the cell resulting from a fertilized egg). The first few cell divisions in embryonic development produce more totipotent cells. After four days of embryonic cell division, the cells begin to specialize into pluripotent stem cells. Embryonic stem cells may also be referred to as totipotent stem cells. In some embodiments, the totipotent stem cells applicable by the disclosed methods may be ESCs of a non-human subject. Stil further, in some embodiments, the totipotent stem cells applicable herein may be of a non-primate subject. In yet some further embodiments, in case a human totipotent stem cells are used in the disclosed methods, these cells may be any totipotent stem cells with the proviso that they cannot form an entire organism. Still further, in some further embodiments, in case human totipotent stem cells are used in the disclosed methods, these cells may be any totipotent stem cells with the proviso that they were not obtained from embryos or living embryos distracted for the preparation of the cells.

[0367] In further embodiments, the eukaryotic cells according to the disclosure are pluripotent stem cells. Similar to totipotent stem cells, a pluripotent stem cell refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). Pluripotent stem cells can give rise to any fetal or adult cell type. However, unlike totipotent stem cells, they cannot give rise to an entire organism. On the fourth day of development, the embryo forms into two layers, an outer layer which will become the placenta, and an inner mass which will form the tissues of the developing human body. These inner cells are referred to as pluripotent cells.

[0368] In still further embodiments, the eukaryotic cells, according to the disclosure are multipotent progenitor cells. Multipotent progenitor cells have the potential to give rise to a limited number of lineages. As a non-limiting example, a multipotent progenitor stem cell may be a hematopoietic cell, which is a blood stem cell that can develop into several types of blood cells, but cannot into other types of cells. Another example is the mesenchymal stem cell, which can differentiate into osteoblasts, chondrocytes, and adipocytes. Multipotent progenitor cells may be obtained by any method known to a person skilled in the art.

[0369] In yet further embodiments, the eukaryotic cells according to the disclosure are induced pluripotent stem cells. Induced pluripotent stem cells, commonly abbreviated as iPS cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, even a patient’s own. Such cells can be induced to become pluripotent stem cells with apparently all the properties of hESCs. Induction requires only the delivery of four transcription factors found in embryos to reverse years of life as an adult cell back to an embryo-like cell.

[0370] In some further embodiments, cells suitable for the methods of the disclosure may be cells from a cell line e.g. cells from Jurkat cel line, MM1R cell line (human multiple myeloma cell line), MCF7 cell lines or MCF7 / Dx cell line, or 293T cell line and others.

[0371] In yet some further embodiments, cells suitable for the disclosed methods, microfluidic platforms and systems, are cells derived from or present in at least one biological sample, specifically, cells obtained from a subject. It should be understood that suitable samples and cells derived therefrom are discussed herein after in connection with other aspects of the present disclosure and are applicable for the present aspect as well.

[0372] It should be understood that the cells, samples and tested parameters described herein above are applicable for each and every method of the present disclosure and in each and every aspect disclosed herein in the present disclosure.

[0373] In some embodiments, the cells are of a subject suffering from at least one pathologic disorder. In yet another alternative or additional embodiments, the pathologic disorder is any one of a malignant proliferative disorder, an inflammatory condition a metabolic condition, an infectious disease, an autoimmune disease, protein misfolding disorder or deposition disorder.

[0374] Still further, in some alternative or additional embodiments, the pathologic disorder is a malignant proliferative disorder. In yet some further embodiments, the cells are primary cancer cells of the subject. In some alternative or additional embodiments, the candidate active agent is at least one of a chemotherapeutic agent, a biological therapy agent, an immuno therapeutic agent, an hormonal therapy gent or any combination thereof. In more specific alternative or additional embodiments, the candidate active agent is at least one of Alectinib, Crizotinib, doxorubicin, docetaxel, paclitaxel, methotrexate, and any combinations thereof.

[0375] In some embodiments, the malignant proliferative disorder is any one of carcinoma, melanoma, lymphoma, leukemia, myeloma and sarcoma.

[0376] In some embodiments, the disclosed methods are particularly suitable for, and / or adapted for use in screening, and / or is used in screening for an active agent that affects cell viability and / or at least one cell phenotype. More specifically, the method comprising the steps of: in step (a), exposing cells in at least one cell chamber of at least one cell cultivation unit of a microfluidic test platform as defined by the present disclosure, to at least one candidate active agent, that is provided via the medium channel of each of the cell cultivation units. It should be understood that the cells in each of the cultivation chambers are exposed to the test compound either immediately after seeding or alternatively after growing for a suitable period of time. In some embodiments, the step of exposing the sample to the agent can be performed in several time points or for at least one time interval. In some embodiments, time intervals include but is not limited to every 5 minutes, 10 minutes, 30 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9., 10, 11, 12, 13, 14, 15, 16, 17, 18, 9, 0, 21, 22, 23, 24 , 48, 72, 96 hours or more, 2 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14 days or more. Step (b) of the disclosed method involves determining that the candidate is an agent that affects cell viability and / or phenotype if at least one of: cell viability and / or at least one cell phenotype is modulated as compared with the cell viability and / or at least one cell phenotype in the absence of the candidate active agent.

[0377] The disclosed screening method is thus for identifying an active agent that affects cell viability and / or at least one cell phenotype, specifically, morphology, activity, invasiveness, expression of various markers, functional response, and post-translational modifications.

[0378] In some embodiments, the methods of the present disclosure involve determination of cell viability, for example, after exposure to a candidate compound. In yet some further embodiments, the cell viability can be determined by using at least one cell-impermeant DNA-binding dyes (propidium iodide (PI, measuring dead cells), nuclear staining (Hoechst 33342) and XTT.

[0379] In another embodiments, living cells may also be stained with Calcein-AM.

[0380] More specifically, in some embodiments, Cell viability is a measure of the proportion of live, healthy cells within a population. As used herein, a cell viability assay refers to is an assay for determining the ability of cells to maintain or recover a state of survival. In some specific embodiments, cell viability may be determined using Propidium iodide, which dyes the nuclei of dead cells (magenta).

[0381] In some embodiments, assays for cell viability applicable in the present disclosure include but are not limited to fluorescent resazurin assay, MTT (tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, WST (water-soluble tetrazolium salts ) assay, ATP uptake assay and glucose uptake assay.

[0382] In some further embodiments, cell viability may be further determined by 2,3-bis-(2-methhoxy-4- nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) viability assay, PrestoBlue viability reagent, the fluorescent intercalator 7-aminoactinomycin D (7-AAD), LIVE / DEAD Viability Kits, cell growth by turbidity, for example at OD600, or by any means for cell counting.

[0383] In yet some further embodiments, cell viability may be determined by evaluating cell toxicity e.g. by measuring apoptosis of the cells. In some embodiments, apoptosis may be determined by at least one of DNA fragmentation (TUNNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling), caspase and / or PARP1 phosphorylation, annexin V and propidium iodide (PI) assay.

[0384] It should be noted that in some embodiments, other toxic phenotype that may be determined on the organism / cell may include, activation of cell stress pathways (e.g., heat shock response), poor fertility, destruction of organs and tissue damages, DNA mutagenesis, ER stress, cell energy status and ATP content, oxidative stress, mitochondrial dysfunction, mitochondrial damage, activation of autophagy and activation of necrosis.

[0385] In some embodiments, upon exposure to the candidate compound, a cell phenotype is determined. The cell phenotype may be determined according to the disclosure using different types of labelling methods, specifically by marker labeling.

[0386] Labels generally provide signals detectable by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. Examples of labels include haptens, enzymes, enzyme substrates, coenzymes, enzyme inhibitors, fluorophores, quenchers, chromophores, magnetic particles or beads, redox sensitive moieties (e.g., electrochemically active moieties), luminescent markers, radioisotopes (including radionucleotides), and members of binding pairs.

[0387] More specific examples include fluorescein, phycobiliprotein, tetraethyl rhodamine, and betagalactosidase. Binding pairs may include biotin / Strepavidin, biotin / avidin, biotin / neutravidin, biotin / captavidin, GST / glutathione, maltose binding protein / maltose, calmodulin binding protein / calmodulin, enzyme-enzyme substrate, receptor-ligand binding pairs, and analogs and mutants of the binding pairs.

[0388] Thus in some embodiments, specific markers of the cells may be labeled or tagged. In some embodiments, the term "labeled" or "tagged" may refer to direct labeling of a protein via, e.g., coupling (i.e., physically linking) or incorporating of a detectable substance, or a "separation substance", to the protein. Useful labels in the present disclosure may include but are not limited to include isotopes (e.g. 13C, 15N), or any other radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), magnetic beads (e.g. DYNABEADS), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

[0389] Furthermore, the cell phenotype may be determined by tagging specific proteins, such as surface receptors with antibodies.

[0390] More specifically, methods applicable in the present disclosure for determining the cell phenotype may include but are not limited to Live cell imaging, Immunofluorescence microscopy, Plasmon- enhanced fluorescence, Raman spectroscopy or Surface-enhanced Raman spectroscopy (SERS).

[0391] Immunofluorescence microscopy enables visualization of the phenotype of the cells. In some embodiments, cells are incubated with relevant first and secondary antibodies. The cells are then visualized using a confocal microscope (such as for example Zeiss LSM 700).

[0392] Live cell imaging of the cell phenotype consists in tagging the living cells with a fluorescent probe, thereby allowing in vivo detection via confocal fluorescence microscopy. For example, the cells may be tagged with any tag such as GFP, e.g. the P4, Rpn2, Rpn6.

[0393] Plasmon-enhanced fluorescence (PEF) that is also referred to as metal-enhanced fluorescence (MEF) represents an attractive method for shortening detection times and increasing sensitivity of various fluorescence -based analytical technologies. In PEF, fluorophore labels are coupled with the tightly confined field of surface plasmons — collective oscillation of charge density and associated electromagnetic field on a surface of metallic films and nanostructures. This interaction can be engineered to dramatically enhance emitted fluorescence light intensity which is desired for detecting minute amounts of analytes with improved limit of detection and shorten analysis time.

[0394] Raman spectroscopy is a spectroscopic technique that relies upon inelastic scattering of photons, known as Raman scattering. A source of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range is used, although X-rays can also be used. The laser light interacts with molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the vibrational modes in the system. Infrared spectroscopy typically yields similar yet complementary information.

[0395] Surface-enhanced Raman spectroscopy (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures such as plasmonic-magnetic silica nanotubes. The enhancement factor can be as much as IO10to 1011, which means the technique may detect single molecules.

[0396] In yet another embodiment, the cell phenotype may be determined by examining different metabolites of the cells, e.g. by examining metabolite aggregation or by performing metabolic profiling.

[0397] In yet some further embodiments, metabolite aggregation may be measured using at least one of Dye -binding specificity (for example, using thioflavin T (ThT) and congo red, or staining with Proteostat) microscopy, circular dichroism (CD) spectrometry, vibrational CD, Raman Spectroscopy, density functional theory (DFT) quantum mechanics methods, Fourier-transformed infrared spectroscopy dynamic light scattering (DLS), liquid chromatography and NMR. Microscopy, such as TEM (transmission electron microscope), confocal fluorescence microscopy, confocal Raman microscopy, indirect immunofluorescence.

[0398] More specifically, Transmission electron microscopy (TEM, also sometimes conventional transmission electron microscopy or CTEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a charge-coupled device. Transmission electron microscopes are capable of imaging at a significantly higher resolution than light microscopes, owing to the smaller de Broglie wavelength of electrons. This enables the instrument to capture fine detail, as small as a single column of atoms, which is thousands of times smaller than a resolvable object seen in a light microscope.

[0399] A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the detected signal to produce an image. SEM can achieve resolution better than 1 nanometer.

[0400] Circular dichroism (CD) is dichroism involving circularly polarized light, i.e., the differential absorption of left- and right-handed light. Left-hand circular (LHC) and right-hand circular (RHC) polarized light represent two possible spin angular momentum states for a photon, and so circular dichroism is also referred to as dichroism for spin angular momentum. It is exhibited in the absorption bands of optically active chiral molecules. CD spectroscopy has a wide range of applications in many different fields. Most notably, UV CD is used to investigate the secondary structure of proteins.

[0401] Density functional theory (DFT) is a computational quantum mechanical modelling method used in physics, chemistry and materials science to investigate the electronic structure (principally the ground state) of many-body systems, in particular atoms, molecules, and the condensed phases. Using this theory, the properties of a many-electron system can be determined by using functionals, i.e. functions of another function, which in this case is the spatially dependent electron density. DET is among the most popular and versatile methods available in condensed-matter physics, computational physics, and computational chemistry.

[0402] Dynamic light scattering (DLS) is a technique used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed by means of the intensity or photon auto-correlation function (also known as photon correlation spectroscopy or quasi-elastic light scattering). In the time domain analysis, the autocorrelation function (ACE) usually decays starting from zero delay time, and faster dynamics due to smaller particles lead to faster decorrelation of scattered intensity trace.

[0403] Ion-mobility spectrometry-mass spectrometry (IMS-MS), also known as ion-mobility separationmass spectrometry, is an analytical chemistry method that separates gas phase ions on a millisecond timescale using ion-mobility spectrometry and uses mass spectrometry on a microsecond timescale to identify components in a sample. It should be noted that this method may be used for evaluating and measuring the levels of the metabolite and thereby for determining metabolite accumulation.

[0404] Metabolic profiling or metabonomics or metabolomics is a study of chemical processes that are associated to and involve metabolites. It is a study of chemical fingerprints that are very unique and that any specific physiological processes in a cell always leave behind. Metabolic profiling can also be defined as the use of analytical methods in measurement and interpretation of various endogenous low molecular weight and intermediates from their samples. This study makes use of metabolome and it provides a critical view of the physiological characteristic of a cell, tissue or the whole organism as compared to proteomic analysis and mRNA analysis.

[0405] In some embodiments, the candidate active agent (also referred to as a candidate compound, test compound / agent, and the like), used for the screening method disclosed herein, is at least one of: an inorganic or organic molecule, a small molecule, a nucleic acid-based molecule, an aptamer, a polypeptide, or protein (L- as well as D-aa residues) or any combinations thereof.

[0406] A compound to be tested may be referred to as a test compound or a candidate compound.

[0407] A "Compound", or a "candidate compound", or “active agent” or “candidate active agent” is used herein to refer to any substance, agent (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Example of compounds applicable for the present disclosure, include, e.g., small molecules, nucleic acid molecules (e.g., RNAi agents, antisense oligonucleotide, gRNAs, aptamers), amino acid based molecules, for example, polypeptides, peptides, antibodies, lipids, polysaccharides, etc.

[0408] It should be understood that any compound described in connection with the present aspect is also applicable in all aspects of the disclosure. It should be further understood that the disclosure encompasses the use of any of the described compounds or any combinations or mixtures thereof. In general, candidate compounds may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the compound. A compound may be at least partly purified. In some embodiments a compound may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the compound, in various embodiments. In some embodiments a compound may be provided as a salt, ester, hydrate, or solvate. In some embodiments a compound is cell-permeable, e.g., within the range of typical compounds that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S -enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-) - and (-i-)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and / or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable.

[0409] Any compound may be used as a test or a candidate compound in various embodiments. In some embodiments a library of FDA approved compounds appropriate for humans may be used. Compound libraries are commercially available from a number of companies including but not limited to Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Microsource (New Milford, CT), Aldrich (Milwaukee, WI), AKos Consulting and Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia), Aurora (Graz, Austria), BioFocus DPI, Switzerland, Bionet (Camelford, UK), ChemBridge, (San Diego, CA), ChemDiv, (San Diego, CA), Chemical Block Ft, (Moscow, Russia), ChemStar (Moscow, Russia), Exclusive Chemistry, Etd (Obninsk, Russia), Enamine (Kiev, Ukraine), Evotec (Hamburg, Germany), Indofine (Hillsborough, NJ), Interbio screen (Moscow, Russia), Interchim (Montlucon, France), Fife Chemicals, Inc. (Orange, CT), Microchemistry Etd. (Moscow, Russia), Otava, (Toronto, ON), PharmEx Etd.(Moscow, Russia), Princeton Biomolecular (Monmouth Junction, NJ), Scientific Exchange (Center Ossipee, NH), Specs (Delft, Netherlands), TimTec (Newark, DE), Toronto Research Corp. (North York ON), UkrOrgSynthesis (Kiev, Ukraine), Vitas-M, (Moscow, Russia), Zelinsky Institute, (Moscow, Russia), and Bicoll (Shanghai, China). Combinatorial libraries are available and can be prepared. Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available or can be readily prepared by methods well known in the art. Compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, and marine samples may be tested for the presence of potentially useful pharmaceutical compounds, specifically, selective modulators of proteasome translocation. It will be understood that the agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. In some embodiments a library useful in the present disclosure may comprise at least 10,000 compounds, at least 50,000 compounds, at least 100,000 compounds, at least 250,000 compounds, or more.

[0410] In some specific embodiments, a candidate agent screened by the screening methods of the present disclosure or evaluated by any of the methods disclosed in the present disclosure, may be a small molecule. A "small molecule" as used herein, is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and / or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

[0411] In some specific embodiments, a candidate agent screened by the screening methods of the present disclosure or evaluated by any of the methods disclosed in the present disclosure, may be an aptamer. As used herein the term "aptamer" or “specific aptamers ” denotes single-stranded nucleic acid (DNA or RNA) molecules which specifically recognizes and binds to a target molecule. The aptamers according to the disclosure may fold into a defined tertiary structure and can bind a specific target molecule with high specificities and affinities. Aptamers may be usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and / or Molinex. In various embodiments, aptamers may include single-stranded, partially single-stranded, partially double-stranded or double-stranded nucleic acid sequences; sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges; synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and / or corresponding complementary sequence. In certain specific embodiments, aptamers used by the disclosure are composed of deoxyribonucleotides.

[0412] Still further, candidate compound that may be screened according to the methods of the disclosure include e.g., any proteins or polypeptides, for example, antibodies or an antigen-binding fragments thereof, receptors, enzymes, ligands, regulatory factors, aptamers, structural proteins, and any nucleic acid sequences encoding the same. Candidate substances also include nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoal antigens and parasitic antigens. Candidate compounds additionally comprise proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (for example, RNAs such as ribozymes or antisense nucleic acids). Proteins or polypeptides which can be screened using the methods of the present disclosure include chaperone proteins, hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasitic antigens and bacterial antigens.

[0413] In some specific embodiments, the amount of candidate active agent may vary between 0 to 1000 pM, specifically 0.1 pM, 0.5 pM, 1 pM, 5 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1000 pM.

[0414] In some other embodiments, the amount of candidate active agent may vary between 0 to 10 mM, specifically 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM , 0.7 mM, 0.8 mM , 0.9 mM , 1 mM, 5 mM, 10 mM.

[0415] In some alternative or additional embodiments, the cells form aggregates and / or clusters in said cell chamber, prior to exposure to said candidate agent.

[0416] As used herein an aggregate or cluster of cells refers to a group of cells in close proximity within a chamber and may comprise a number of cells that vary between 10 to 1000 cells, specifically 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 cells.

[0417] A cluster of cells may comprise cells having different phenotype for example living versus dying cells. In some embodiments, cluster of cells comprise more living cells than dying cells. In some further embodiments, a cluster of cells comprise only living cells.

[0418] As shown in the Examples, the cell phenotype may be modulated differently between cells forming a cluster or “cluster cells” or cells that do not form a cluster or “dispersed cells”.

[0419] In some other embodiment, the cell phenotype is different within the cluster specifically between the center and the periphery of the cluster. In some specific embodiment, more living cells may be found in the center of the cluster while more dying cells may be at the periphery of the cluster.

[0420] Still further, in some alternative or additional embodiments, the cells are cells of a subject suffering from a pathologic disorder. In yet some further alternative or additional embodiments, the pathologic disorder is any one of a malignant proliferative disorder, an inflammatory condition, a metabolic condition, an infectious disease, an autoimmune disease, protein misfolding disorder or deposition disorder.

[0421] In some further alternative or additional embodiments, the pathological disorder is a malignant proliferative disorder. According to such embodiments, the cells are primary cancer cells of the subject. In some alternative or additional embodiments, the malignant proliferative disorder is any one of carcinoma, melanoma, lymphoma, leukemia, myeloma and sarcoma. In yet some further alternative or additional embodiments, the cell viability is determined by using at least one cell-impermeant DNA- binding dyes and nuclear staining.

[0422] In some alternative or additional embodiments, the candidate active agent is at least one of a chemotherapeutic agent, a biological therapy agent, an immuno therapeutic agent, an hormonal therapy gent or any combination thereof.

[0423] Still further, in some alternative or additional embodiments, the candidate active agent is at least one of Alectinib, Crizotinib, doxorubicin, docetaxel, paclitaxel, methotrexate, and any combinations thereof.

[0424] In some embodiments, the disclosed methods may be used for screening for an anti-cancerous drug. Accordingly, the method comprises the following steps:

[0425] In step (a), exposing cancer cells in at least one cell chamber of at least one cell cultivation unit of the microfluidic platform, to at least one candidate active compound. In step (b), determining for the exposed cells of (a), cell viability, for at least one time interval; and (c), determining that the candidate drug is an anti-cancerous drug if cell viability is reduced as compared with the cell viability in the absence of the candidate active agent.

[0426] In some further embodiments, the candidate compound or therapeutic agent screened in the methods disclosed herein may be at least one of a chemotherapeutic agent, a biological therapy agent, an immuno therapeutic agent, an hormonal therapy gent or any combination thereof.

[0427] In some specific and non-limiting embodiments, the candidate active agent is at least one of Alectinib, Crizotinib, doxorubicin, docetaxel, paclitaxel, methotrexate, and any combinations thereof.

[0428] In some embodiments, the disclosed methods are suitable for assessing responsiveness of a subject suffering from a pathologic disorder to a treatment regimen comprising at least one therapeutic active agent, and optionally for monitoring disease progression. According to such embodiments, the method comprising the steps of: in step (a), exposing cells of the subject applied on at least one cell chamber of at least one cell cultivation unit of the microfluidic platform in accordance with the present disclosure (as specified herein above), to the therapeutic active agent. It should be noted that the therapeutic active agent is provided via the medium channel of each of the cell cultivation units. Next in step (b), determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one-time interval. The next step (c), involves classifying the subject:

[0429] Either as a (i), responsive subject to the treatment regimen, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with at least one of the cell viability and / or at least one cell phenotype in the absence of the therapeutic active agent. Alternatively, the subject is classified as (ii), a drug-resistant subject if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with at least one of the cell viability and / or at least one cell phenotype, in the absence of the active agent; thereby predicting, assessing and monitoring responsiveness of a mammalian subject to the treatment regimen. Modulation according to some embodiments, may comprise reduction of cell viability. Specifically, the subject is classified as a responder if the agents leads to reduction in the viability of the cells of the subject. Non -responsive subject may be in some embodiments a subject that display no change in cell viability (or increased cell viability) in response to the therapeutic agent. In some optional embodiments, where the disclosed methods further provide monitoring of the disease, the monitoring disease progression further comprises the steps of:

[0430] In step (d), repeating steps (a) and (b), to determine at least one of, cell viability and / or at least one cell phenotype for at least one cell of at least one more temporally-separated sample of the subject. In step (e), predicting and / or determining drug-resistance and / or reduction in drug effectiveness in the subject, if at least one cell of the at least one temporally separated sample, displays loss of the modulatory effect of the therapeutic active compound on at least one of, cell viability and / or at least one cell phenotype.

[0431] It should be understood that any cells disclosed in the present disclosure may be applicable for the present pethood. In some embodiments, cells suitable for the methods of the disclosure may originate from a biological sample. Biological sample is any sample obtained from the subject that comprises at least one cell or any fraction thereof. In some specific embodiments, sample applicable in the methods of the disclosure may include bone marrow, lymph fluid, blood cells, blood, serum, plasma, semen, spinal fluid or CSF, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, any sample obtained from any organ or tissue, any sample obtained by lavage, optionally of the - breast ductal system, or of the uterus, plural effusion, samples of in vitro or ex vivo cell culture and cell culture constituents.

[0432] In some specific embodiments, the biological sample may result from a biopsy. A biopsy is a medical test commonly performed by a surgeon. The process involves extraction of sample cells or tissues from the patient. The tissue obtained is generally examined under a microscope by a pathologist for initial assessment and may also be analyzed for cell phenotype as discussed by the present disclosure. When an entire lump or suspicious area is removed, the procedure is called an excisional biopsy. An incisional biopsy or core biopsy samples a portion of the abnormal tissue without attempting to remove the entire lesion or tumor. When a sample of tissue or fluid is removed with a needle in such a way that cells are removed without preserving the histological architecture of the tissue cells, the procedure is called a needle aspiration biopsy. Still further, the sample / s may be obtained from the described tissues ectomized from a patient (e.g., in case of therapeutic ectomy).

[0433] In certain embodiments, the cells used in the screening method of the present disclosure are cells of a subject suffering from a pathologic disorder.

[0434] In some further embodiments, the cells used in the screening method are of a subject suffering from pathologic disorder that may be any one of a malignant proliferative disorder, an inflammatory condition a metabolic condition, an infectious disease, an autoimmune disease, protein misfolding disorder or deposition disorder.

[0435] In more specific embodiments, the pathologic disorder is a malignant proliferative disorder. In yet some further particular embodiments, the cells are primary cancer cells of the diseased subject. In some embodiments, the cells may be a mixture of different primary cells, specifically, heterogeneous population of cells as originally present in the diseased tissue of the subject. The sample and the population of cells derived therefrom reflects the natural heterogeneity of the cells that are preserved in the cell chambers due to small volume of the sample and the low cell number required for operating the disclosed platforms and methods.

[0436] Still further, in some embodiments, the malignant proliferative disorder is any one of carcinoma, melanoma, lymphoma, leukemia, myeloma and sarcoma.

[0437] In other embodiments, the cells suitable for the methods of the disclosure originate from diseased subject with further malignancies that may comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The disclosure may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas / carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioblastoma; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS- related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, NonHodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone / Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic / Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia / Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer).

[0438] In some specific embodiments, the cells suitable for the methods of the disclosure originate from diseased subject with non-small cell lung cancer. In some other embodiments the cells suitable for the methods of the disclosure originate from diseased subject suffering from glioblastoma.

[0439] As indicated herein, the disclosed methods and microfluidic platforms are applicable for any type of cells or for any combination of any type of cells. In some embodiments, the cells are eukaryotic cells and may be derived either from a unicellular or a multicellular eukaryotic subject. In yet some further embodiments, the cells may be prokaryotic cells derived from any prokaryotic organism, for example, bacteria or archaea. In some embodiments, the cells may be bacterial cells. Bacterial cells in the context of the present aspect are any of the bacterial cells as defined herein after in connection with other aspects of the present disclosure.

[0440] In yet some further embodiments, the disclosed methods may be applicable for determining a personalized treatment regiment for a subject in need. According to such embodiments, the method comprising: in step (a), exposing cells of the subject applied on at least one cell chamber of at least one cell cultivation unit of the microfluidic platform of the present disclosure, to at least one therapeutic active agent provided via the medium channel of each of the cell cultivation units. In step (b), determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one time interval. Next in step (c), classifying the subject as: either as (i), a responsive subject to the treatment regimen, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with at least one of the cell viability and / or at least one cell phenotype in the absence of the candidate active agent; or alternatively as (ii), a drug-resistant subject if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with at least one of the cell viability and / or at least one cell phenotype in the absence of the therapeutic active agent. The next step (d), involves administering to a subject classified as a responder, an effective amount of the therapeutic active agent, or any compositions thereof.

[0441] In some embodiments, the subject is and / or was subjected to a treatment regimen comprising the therapeutic active agent and is monitored for disease progression. According to such embodiments, the method comprising the steps of: (a), exposing cells of the subject applied on at least one cell chamber of at least one cell cultivation unit of the microfluidic platform as described by the present disclosure, to the at least one therapeutic active agent provided via the medium channel of each of the cell cultivation units. It should be noted that the cell sample is obtained after the initiation of the treatment regimen. Next in step (b), determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one time interval. In step (c), determining at least one of: either (i), loss of responsiveness, and / or drug-resistance of the subject, if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with the cell viability and / or at least one cell phenotype in the absence of the candidate active agent. Alternatively (ii), determining responsiveness or maintained responsiveness of the subject, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with the cell viability and / or at least one cell phenotype in the absence of the candidate active agent. Next step (d), involves either ceasing a treatment regimen comprising the therapeutic active agent of a subject displaying disease relapse and / or loss of responsiveness, and / or drug-resistance. Alternatively, maintaining the treatment regimen of a subject displaying responsiveness or maintained responsiveness.

[0442] In some embodiments, the viability assay, or the cell phenotype assay, is performed in the active zone / s formed by the surface activation valves, as defined by the present disclosure. In such assay, cell assay material / s are collected from and / or produced by, the cells in each of the cell cultivation chambers, and transferred via the medium channel of each of the cell cultivation units to the active zone / s formed by the surface activation valves, as defined by the present disclosure, in each of the medium channel of each cell cultivation unit. In some embodiments, the cell assay material / s may be nucleic acids extracted from the cells in the cell cultivation chambers, and the medium with the nucleic acid preparation is transferred to the active zone that comprises probes or primers, or any detecting molecules or reagents that allow determination of cell viability or apoptosis of the cells.

[0443] The present disclosure provides personalized therapeutic methods and methods for assessing responsiveness of a subject for a specific treatment regimen, for monitoring a disease progression and for predicting relapse of the disease in a subject. As such, the present disclosure provides prognostic methods. It should be noted that "Prognosis", is defined as a forecast of the future course of a disease or disorder, based on medical knowledge. This highlights the major advantage of the disclosure, namely, the ability to assess responsiveness or drug-resistance and thereby predict progression of the disease, based on the cell phenotype / viability of the prognosed subject, in response to exposure to a certain therapeutic compound.

[0444] The term "response" or "responsiveness" to a certain treatment, specifically, treatment regimen that comprise at least one active agent, refers to an improvement in at least one relevant clinical parameter as compared to an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and / or classification of the pathology), or as compared to the clinical parameters of the same subject prior to treatment with said medicament.

[0445] The term "non responder'' or "drug resistance" to treatment with a specific medicament, specifically, treatment regimen that comprise at least one candidate active agent, refers to a patient not experiencing an improvement in at least one of the clinical parameter and is diagnosed with the same condition as an untreated subject diagnosed with the same pathology e.g., the same type, stage, degree and / or classification of the pathology), or experiencing the clinical parameters of the same subject prior to treatment with the specific medicament.

[0446] In some embodiments, the methods of the disclosure may be particularly useful for monitoring disease progression. In some embodiments, monitoring disease progression by the methods of the disclosure may comprise at least one of, predicting and determining disease relapse, and assessing a remission interval. The term "relapse", as used herein, relates to the re-occurrence of a condition, disease or disorder that affected a person in the past. Specifically, the term relates to the re-occurrence of a disease being treated with an active agent.

[0447] In some embodiments, the at least one more temporally-separated sample (e.g., sample taken at different time intervals) may be obtained after the initiation of at least one treatment regimen comprising at least one active agent.

[0448] It should be understood that in some particular embodiments, at least one sample may be obtained prior to initiation of the treatment. However, in some embodiments, the methods disclosed herein may be applied to subjects already treated by a treatment regimen comprising at least one therapeutic active agent. Such monitoring may therefore provide a powerful therapeutic tool used for improving and personalizing the treatment regimen offered to the treated subject.

[0449] As indicated above, in accordance with some embodiments of the disclosure, in order to assess the patient condition, or monitor the disease progression, as well as responsiveness to a certain treatment, at least two ''temporally-separated'' test samples must be collected from the examined patient and compared thereafter, in order to determine if there is any change or difference in the values between the samples. Such change may reflect a change in the responsiveness of the subject. In practice, to detect a change having more accurate predictive value, at least two "temporally-separated" test samples and preferably more, must be collected from the patient.

[0450] The cellular phenotype value is determined using the method disclosed herein, applied for each sample. As detailed above, the change in cellular phenotype is calculated by determining the change in cellular phenotype between at least two samples obtained from the same patient in different timepoints or time intervals. This period of time, also referred to as "time interval", or the difference between time points (wherein each time point is the time when a specific sample was collected) may be any period deemed appropriate by medical staff and modified as needed according to the specific requirements of the patient and the clinical state he or she may be in. For example, this interval may be at least one day, at least three days, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least one year, or even more.

[0451] The number of samples collected and used for evaluation and classification of the subject either as a responder or alternatively, as a drug resistant or as a subject that may experience relapse of the disease, may change according to the frequency with which they are collected. For example, the samples may be collected at least every day, every two days, every four days, every week, every two weeks, every three weeks, every month, every two months, every three months every four months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year or even more. Furthermore, to assess the disease progression according to the present disclosure, it is understood that the change in cellular phenotype value, may be calculated as an average change over at least three samples taken in different time points, or the change may be calculated for every two samples collected at adjacent time points. It should be appreciated that the sample may be obtained from the monitored patient in the indicated time intervals for a period of several months or several years. More specifically, for a period of 1 year, for a period of 2 years, for a period of 3 years, for a period of 4 years, for a period of 5 years, for a period of 6 years, for a period of 7 years, for a period of 8 years, for a period of 9 years, for a period of 10 years, for a period of 11 years, for a period of 12 years, for a period of 13 years, for a period of 14 years, for a period of 15 years or more.

[0452] As described hereinabove, the methods of the disclosure refer to determining the modulation of cell viability and / or at least one cell phenotype. In some further embodiments, the modulation of cell viability and / or at least one cell phenotype may be determined as a numeric value, thereby enabling assessment of a cut-off value.

[0453] Still further, an equivalent cellular phenotype in the presence or absence of a therapeutic active agent reflects non-responsiveness, or drug resistance. For example, in the case of assessing cell viability, an equal proportion of living cells versus dead cells (namely, 50% or more) indicates non-responsiveness. As such, a value of about 40% to 60%, specifically, 40%, 45%, 50%, 55%, 60%, may be used as a cutoff value. In yet some further embodiments, a value of about 50% of the proportion of living cells versus dead cells may be considered as a cutoff value.

[0454] As noted above, in some embodiments of the present disclosure, at least one control sample may be provided and / or used by the methods discussed herein. A "control sample" as used herein, may reflect a sample of at least one subject (a subject that is known to be a non-responder, or alternatively, known to be a responder, or sample displaying known cell phenotype at a certain predetermined degree), and preferably, a mixture at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more patients, specifically, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more patients. A control sample may alternatively or additionally comprise known cellular phenotype that can be used as a reference.

[0455] It should be appreciated that the effectiveness of the disclosed methods, in achieving both specificity and sensitivity in assessing drug responsiveness, derives from the quality of the sample, which preserves the heterogeneity of the various cell populations originally present in the subject. In particular, because only a small sample volume is required to perform the disclosed methods, the assay may be carried out immediately upon seeding the cells, without the need for prior in vitro expansion. This avoids artificial alterations in the relative abundance of different cellular subpopulations. As a result, the sample tested in each cultivation chamber more faithfully represents the cellular composition of the diseased subject, thereby providing a more accurate and rapid assessment of drug sensitivity and responsiveness.

[0456] In some embodiments, the therapeutic or candidate active agent assayed using the disclosed microfluidic platform may be a chemotherapeutic agent.

[0457] "Chemotherapeutic agent” or "chemotherapeutic drug” (also termed chemotherapy) as used herein refers to a drug treatment intended for eliminating or destructing (killing) cancer cells or cells of any other proliferative disorder. The mechanism underlying the activity of some chemotherapeutic drugs is based on destructing rapidly dividing cells, as many cancer cells grow and multiply more rapidly than normal cells. As a result of their mode of activity, chemotherapeutic agents also harm cells that rapidly divide under normal circumstances, for example bone marrow cells, digestive tract cells, and hair follicles. Insulting or damaging normal cells result in the common side-effects of chemotherapy: myelosuppression (decreased production of blood cells, hence also immuno-suppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss).

[0458] Various different types of chemotherapeutic drugs are available. A chemotherapeutic drug may be used alone or in combination with another chemotherapeutic drug or with other forms of cancer therapy, such as a biological drug, radiation therapy or surgery.

[0459] Certain chemotherapy agents have also been used in the treatment of conditions other than cancer, including ankylosing spondylitis, multiple sclerosis, hemangiomas, Crohn’s disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, lupus and scleroderma.

[0460] Chemotherapeutic drugs affect cell division or DNA synthesis and function and can be generally classified into groups, based on their structure or biological function. The present disclosure generally pertains to chemotherapeutic agents that are classified as alkylating agents, anti-metabolites, an thracy clines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents such as DNA- alkylating agents, anti-tumor antibiotic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple -helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial or exotoxic agents.

[0461] However, several chemotherapeutic drugs may be classified as relating to more than a single group. It is noteworthy that some agents, including monoclonal antibodies and tyrosine kinase inhibitors, which are sometimes referred to as “chemotherapy”, do not directly interfere with DNA synthesis or cell division but rather function by targeting specific components that differ between cancer cells and normal cells and are generally referred to as 'targeted therapies ', "biological therapy" or "immunotherapeutic agent'' as detailed below. Thus in some embodiments, the therapeutic or candidate active agent of the disclosure may refer to targeted therapy, biological therapy or “immunotherapeutic agent.

[0462] More specifically, as their name implies, alkylating agents function by alkylating many nucleophilic functional groups under conditions present in cells. Examples of chemotherapeutic agents that are considered as alkylating agents are cisplatin and carboplatin, as well as oxaliplatin. Alkylating agents impair cell function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in various biologically-significant molecules. Examples of agents which function by chemically modifying DNA are mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide. An additional agent acting as a cell cycle non-specific alkylating antineoplastic agent is the alkyl sulfonate agent busulfan (also known as Busulfex).

[0463] Still further, Anthracyclines (or anthracycline antibiotics) are a class of drugs used in cancer chemotherapy that are derived from the streptomyces bacterium. These compounds are used to treat many cancers, including leukemias, lymphomas, breast, uterine, ovarian, and lung cancers. These agents include, inter alia, the drugs daunorubicin (also known as Daunomycin), and doxorubicin and many other related agents (e.g., Valrubicin and Idarubicin). For example, the anthracycline agent Idarubicin acts by interfering with the enzyme topoisomerase II.

[0464] In some embodiments, the therapeutic or candidate active agent of the disclosure may be a biological therapy agent. It should be noted that the term “biological therapy agent” or “biological treatment” or “biological agent” or “biological drug”, as used herein refers to any biological material that affects different cellular pathways. Such agent may include antibodies, for example, antibodies directed to cell surface receptors participating in signaling, that may either activate or inhibit the target receptor. Such biological agents may also include any soluble receptor, cytokine, peptides or ligands.

[0465] Still further, the term "biological drug" refers to drugs consisting of or comprising biological molecules or material, i.e. both, proteins, polypeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, oligosaccharides and fragments thereof, as well as cells, tissues, biological fluids or extracts thereof, and which induce antibodies in a subject. In some embodiments, biological drugs may include proteins such as monoclonal antibodies, cytokines, soluble receptors, growth factors, hormones, enzymes, adhesion molecules and fusion proteins and peptides that are specific to certain targets known to modulate disease mechanisms. In yet some further embodiments, biological drugs may include or target any component participating in molecular and / or cellular processes such as, cell cycle, cell survival, apoptosis, immunity and the like. In more specific embodiments, biological drugs may be any checkpoint protein / s or any modulators or inhibitors thereof, or any combinations thereof. In yet some further embodiments, biological drugs (or their precursors or components) may be isolated from living sources human, animal, plant, fungal, or microbial.

[0466] Still further in some embodiments, "biological drug" or "biologies" refers to a class of therapeutics that are produced by means of biological processes involving recombinant DNA technology which are usually one of three types: (a) substances that are similar to the natural occurring proteins: (b) monoclonal antibodies; and (c) receptor constructs or fusion proteins, usually based on a naturally occurring receptor linked to the immunoglobulin frame. Major kinds of biologies include but are not limited to: Blood factors (such as Factor VIII and Factor IX), Thrombolytic agents (such as tissue plasminogen activator), Hormones (such as insulin, glucagon, growth hormone, gonadotrophins), Haematopoietic growth factors (such as Erythropoietin, colony stimulating factors), Interferons (such as Interferons-a, -P, -y), Interleukin-based products (such as Interleukin-2), Vaccines (such as Hepatitis B surface antigen) and monoclonal antibodies. Non-limiting examples of biological drugs made with recombinant DNA technology may include at least one of: abatacept (Orencia®), that is a fusion protein composed of the Fc region of the immunoglobulin IgGl fused to the extracellular domain of CTLA-4, used to treat autoimmune diseases like rheumatoid arthritis, by interfering with the immune activity of T cells; erythropoietin or Epoetin alfa (Epogen®), that is a human erythropoietin produced in cell culture using recombinant DNA technology, that stimulates erythropoiesis and is used to treat anemia, commonly associated with chronic renal failure and cancer chemotherapy; Muromonab-CD3 (Orthoclone OKT3®), that is a monoclonal antibody working as an immunosuppressant drug given to reduce acute rejection in patients with organ transplants. It binds to the T cell receptor-CD3-complex on the surface of circulating T cells thereby inducing blockage and apoptosis of the T cells; Abciximab (ReoPro®), that is a glycoprotein Ilb / IIIa receptor antagonist mainly used during and after coronary artery procedures; Basiliximab (Simulect®), that is a chimeric CD25 monoclonal antibody of the IgGl isotype, used as an immunosuppressant to prevent immediate transplant rejection; and Palivizumab (Synagis®), that is a humanized monoclonal antibody (IgG) directed against an epitope in the A antigenic site of the F protein of the respiratory syncytial virus (RSV).

[0467] Thus, in some embodiments, the therapeutic or candidate active agent applied in the disclosed methods and the microfluidic platforms of the present disclosure, may refer to antibody-mediated therapy. Antibody-mediated therapy as referred to herein refers to the use of antibodies that are specific to a cancer cell or to any protein derived there-from for the treatment of cancer. As a non-limiting example, such antibodies may be monoclonal or polyclonal which may be naked or conjugated to another molecule. Antibodies used for the treatment of cancer may be conjugated to a cytotoxic moiety or radioactive isotope, to selectively eliminate cancer cells. Non limiting examples for monoclonal antibodies that are used for the treatment of cancer include bevacizumab (also known as Avastin), rituximab (anti CD20 antibody), cetuximab (also known as Erbitux), anti-CTLA4 antibody and panitumumab (also known as Vectibix) and anti Grl antibodies as further detailed below. Thus, further non-limiting examples for such antibody that may be used in the methods of the disclosure include at least one of infliximab, etanercept, adalimumab, certolizumab pegol, golimumab, any biosimilar thereof and any combinations of the same.

[0468] In more specific embodiments, therapeutic or candidate active agent of the invention may also refer to biosimilar as further detailed below such as Remsima / INFLECTRA® (infliximab-dyyb), SB4 etanercept, SB2 infliximab and SB5 adalimumab.

[0469] In more embodiments, therapeutic or candidate active agent of the disclosure may refer to a TNF inhibitor. TNF inhibitors are pharmaceutical drugs that suppresses the physiologic response to tumor necrosis factor (TNF), which is part of the inflammatory response. Inhibition of TNF effects can be achieved using a monoclonal antibody such as infliximab REMICADE®, etanercept, ENBREE®, adalimumab HUMIRA®, certolizumab pegol CIMZIA®, golimumab, SIMPONI®, and any biosimilars thereof, to name but a few, Remsima / INFLECTRA® (infliximab-dyyb), SB4 etanercept, SB2 infliximab and SB5 adalimumab. Thalidomide (Immunoprin) and its derivatives lenalidomide (Revlimid) and pomalidomide (Pomalyst, Imnovid) are also active against TNF.

[0470] In some specific embodiments, the biological drug used by the methods of the disclosure may be infliximab. The term "infliximab" refers to the anti-TNF antibody marketed as REMICADE®, having FDA Unique Ingredient Identifier (UNII): B72HH48FLU and DRUG BANK Accession number DB00065. It is an Immunoglobulin G, (human-mouse monoclonal cA2 heavy chain), disulfide with human-mouse monoclonal cA2 light chain, dimer. More specifically, Infliximab is used to treat immune -mediated diseases such as Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and rheumatoid arthritis as well as Behget's disease and other conditions. Infliximab is administered by intravenous infusion, typically at six- to eight- week intervals, but cannot be given orally.

[0471] Infliximab is a purified, recombinant DNA-derived chimeric human-mouse IgG monoclonal antibody that consists of mouse heavy and light chain variable regions combined with human heavy and light chain constant regions. It has a serum half-life of 9.5 days and can be detected in serum 8 weeks after infusion treatment.

[0472] Infliximab neutralizes the biological activity of TNF-a by binding with high affinity to both the soluble and transmembranal forms of TNF-a thereby inhibiting the effective binding of TNF-a with its receptors.

[0473] Infliximab has high specificity for TNF-a, and does not neutralize TNF beta (TNFP, also called lymphotoxin a), an unrelated cytokine that uses different receptors from TNF-a. Blocked actions of TNF-a further leads to downregulation of local and systemic pro-inflammatory cytokines (i.e. IL-1, IL-6), reduction of lymphocyte and leukocyte migration to sites of inflammation, induction of apoptosis of TNF-producing cells (i.e. activated monocytes and T lymphocytes), increased levels of nuclear factor-KB inhibitor, and reduction of reduction of endothelial adhesion molecules and acute phase proteins. Infliximab also attenuates the production of tissue degrading enzymes synthesized by synoviocytes and / or chondrocytes.

[0474] In yet some further specific embodiments, the biological drug used by the methods of the disclosure may be etanercept. The term "etanercept" refers to the anti-TNF antibody marketed as ENBREL®, having FDA Unique Ingredient Identifier (UNII): OP401G7OJC and DRUG BANK Accession number DB00005. Etanercept is a fusion protein produced by recombinant DNA. It fuses the TNF receptor to the constant end of the IgGl antibody as follows: residues 1-235-are of Tumor necrosis factor receptor (human) fusion protein with residues 236-467 -immunoglobulin G1 (human yl -chain Fc fragment). It is a large molecule, with a molecular weight of 150 kDa.

[0475] In still further specific embodiments, the biological drug used by the methods of the disclosure may be adalimumab. The terms "adalimumab" refers to the anti-TNF antibody marketed as HUMIRA®, having FDA Unique Ingredient Identifier (UNII): FYS6T7F842 and DRUG BANK Accession number DB00051. It is an Immunoglobulin G 1 , (human monoclonal D2E7 heavy chain), disulfide with human monoclonal D2E7 light chain, dimer.

[0476] In yet some further specific embodiments, the biological drug used by the methods of the disclosure may be certolizumab pegol. The term "certolizumab pegol" refers to the anti-TNF antibody marketed as CIMZIA®, having FDA Unique Ingredient Identifier (UNII): UMD07X179E. It is a polyethylene- glycolated Fab' fragment of TNF antibody that binds specifically to TNFa and neutralizes it in a dosedependent manner.

[0477] In some further specific embodiments, the biological drug used by the methods of the disclosure may be golimumab. The term "golimumab" refers to the anti-TNF antibody marketed as SIMPONI®, having FDA Unique Ingredient Identifier (UNII): 91X1KLU43E. It is an Immunoglobulin Gl, (human monoclonal CNTO 148 gamma 1 -chain), disulfide with human monoclonal CNTO 148 kappa-chain, dimer. Its molecular weight is approximately 147 kDa.

[0478] In still further specific embodiments, the biological drug used by the methods of the disclosure may be Ustekinumab. The term "Ustekinumab" refers to a humanized monoclonal antibody that binds to IL- 12 and IL-23 marketed as STELARA®, having FDA Unique Ingredient Identifier (UNII): FU77B4U5Z0. It is an Immunoglobulin Gl, anti-(human interleukin 12 p40 subunit) (human monoclonal CNTO 1275 gammal -chain), disulfide with human monoclonal CNTO 1275 kappa-chain, dimer.

[0479] In still further specific embodiments, the biological drug used by the methods of the disclosure may be Etrolizumab. The term "Etrolizumab" or "rhuMAb Beta7" refers to a humanized monoclonal antibody against the P7 subunit of integrins a4p7 and aEp7, having FDA Unique Ingredient Identifier (UNII): I2A72G2V3J. It is an Immunoglobulin Gl, anti-(human integrin alpha47 / integrin alphaE7) (human-rat monoclonal rhuMAb Beta7 heavy chain), disulfide with human-rat monoclonal rhuMAb Beta7 light chain, dimer. It should be appreciated that in certain embodiment, any biosimilar of the above, specifically, any approved biosimilar, may be used by the methods of the disclosure as a target. In yet some further embodiments, the drug used by the methods of the disclosure may be Mirikizumab (LY3074828) that targets interleukin 23A and is in clinical use in treating inflammatory conditions such as Moderate-to-Severe Ulcerative Colitis. In yet some further embodiments the methods of the disclosure may use Risankizumab (ABBV-066) that is an anti-IL-23 antibody being clinically used for the treatment of multiple inflammatory diseases, including psoriasis, Crohn's disease and psoriatic arthritis.

[0480] Additional non-limiting examples of active agents suitable for the methods of the disclosure are further detailed below. More specifically, Axitinib (Inlyta®), a small molecule tyrosine kinase inhibitor, is used as a treatment option for kidney cancer. Bevacizumab (A vastin®), is a recombinant humanized monoclonal antibody that blocks angiogenesis by inhibiting VEGF-A. Avastin is used in the treatment of colorectal, kidney, and lung cancers. Cabozantinib (Cometriq®), is a small molecule inhibitor of the tyrosine kinases c-Met and VEGFR2, and also inhibits AXL and RET. Cabozantinib is used in the treatment of medullary thyroid cancer and kidney cancer. Lenalidomide (CC-5013; IMiD3; Revlimid®), having the formula C13H13N3O3, is an analogue of thalidomide, a glutamic acid derivative with anti-angiogenic properties and potent anti-inflammatory effects owing to its anti-tumor necrosis factor (TNF)a activity, and is therefore classified as an Imunomodulatory drug (IMiD). Lenalidomide is used as a treatment option for multiple myeloma and mantle cell lymphoma, which is a type of non-Hodgkin lymphoma. Lenvatinib mesylate (Lenvima®), having the formula C21H19CIN4O4, acts as a multiple kinase inhibitor against the VEGFR1 , VEGFR2 and VEGFR3 kinases, and is used for the treatment of certain kinds of thyroid cancer. Pazopanib (Votrient®), having the formula C21H23N7O2S, is a potent multi-targeted receptor tyrosine kinase inhibitor, that inhibits VEGFR, PDGFR, c-KIT and FGFR. Pazopanib is used as a treatment option for kidney cancer and advanced soft tissue sarcoma. Ramucirumab (Cyramza®), is a fully human monoclonal antibody (IgGl) that binds with high affinity to the extracellular domain of VEGFR2 and block the binding of natural VEGFR ligands (VEGF-A, VEGF-C and VEGF-D). Ramucirumab is used in the treatment of advanced stomach cancer; gastroesophageal junction adenocarcinoma, colorectal cancers; and non-small cell lung (NSCL) cancers. Regorafenib (Stivarga®), having the formula C21H15CIF4N4O3, is an oral multi-kinase inhibitor that display dual inhibitory activity on VEGFR2-TIE2. Regorafenib is used as a treatment option for colorectal cancer and gastrointestinal stromal tumors (GIST). Sorafenib (Nexavar®), having the formula C21H16CIF3N4O3, is a protein kinase inhibitor of various protein kinases, including VEGFR, PDGFR and RAF kinases. This drug is used in the treatment of kidney, liver, and thyroid cancers. Sunitinib (Sutent®), is an oral, small-molecule, multi-targeted receptor tyrosine kinase (RTK) inhibitor having the formula C22H27FN4O2, that blocks the tyrosine kinase activities of KIT, PDGFR, VEGFR2 and other tyrosine kinases. Sunitinib is used as a treatment option for kidney cancer, PNETs, and GIST. Thalidomide (Synovir, Thalomid®) (a-V-phthalimido-glutarimide), is a synthetic derivative of glutamic acid, which was know for causing birth defects when used as an antiemetic in pregnancy in the late 1950s and early 1960s. As indicated above, Thalidomide and its analogs are IMiDs. These drugs bind CRBN, a substrate receptor of CRL4 E3 ligase, to induce the ubiquitination and degradation of IKZF1 and IKZF3. Thalidomide is used in the treatment of multiple myeloma. Vandetanib (Caprelsa®), having the formula C22H24BrFN4O2, acts as a kinase inhibitor of a number of cell receptors, mainly the VEGFR, the EGFR, and the RET-tyrosine kinase. This drug is used as a treatment option for medullary thyroid cancer. Ziv-aflibercept (Zaltrap®), is a recombinant fusion protein consisting of VEGF-binding portions of the extracellular domains of human VEGF receptors 1 and 2, that are fused to the Fc portion of the human IgGl immunoglobulin. This drug is used in the treatment of wet macular degeneration and metastatic colorectal cancer. It should be appreciated that any of the anti- angiogenic agents disclosed herein are applicable as an additional therapeutic agent for any of the aspects of the present disclosure.

[0481] In some further embodiments, the therapeutic or candidate active agent of the disclosure may be an angiogenesis inhibitor. Non-limiting examples of angiogenesis inhibitors useful in the methods, of the present disclosure include at least one of: VEGF inhibitors, for example, anti- VEGF antibodies such as Bevacizumab (A vastin®), and Ramucirumab (Cyramza®), VEGF fusion proteins such as Ziv- aflibercept (Zaltrap®), kinase inhibitors such as Vandetanib (Caprelsa®), Sunitinib (Sutent®), Sorafenib (Nexavar®), Regorafenib (Stivarga®), Pazopanib (Votrient®), Cabozantinib (Cometriq®), Axitinib (Inlyta®), and agents involved with degradation of proteins (e.g., via interaction with E3 ligases) such as Thalidomide (Synovir, Thalomid®), and related drugs, for example, Lenalidomide (Revlimid®).

[0482] It should be appreciated that in certain embodiment, the biological drug used by the methods of the disclosure may be any biosimilar, specifically, any approved biosimilar of the aforementioned originator biologies.

[0483] The term "biosimilar" means a biological product that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.

[0484] A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and / or efficacy, or any combinations thereof. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in US is compared to a reference medicinal product which has been authorized by the FDA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the FDA in certain studies. Such studies include, for example, certain clinical and in vivo non-clinical studies.

[0485] In yet some further embodiments, such therapeutic active agent may be at least one of Alectinib, Crizotinib, doxorubicin, docetaxel, paclitaxel, methotrexate, and any combinations thereof.

[0486] In some embodiments, the therapeutic active agent may be Alectinib. Alectinib (marketed as Alecensa®) is an oral drug that blocks the activity of anaplastic lymphoma kinase (ALK) and is used to treat non-small-cell lung cancer (NSCLC). It can be given by mouth or by injection. It has FDA Unique Ingredient Identifier (UNII): LIJ4CT1Z3Y and DRUG BANK Accession number DB 11363. In some embodiments, the therapeutic active agent may be Crizotinib. Crizotinib, sold under the brand name Xalkori® among others, is an anti-cancer medication acting as an ALK (anaplastic lymphoma kinase) and ROS1 (c-ros oncogene 1) inhibitor, approved for treatment of some non-small cell lung carcinoma (NSCLC) in the US and some other countries, and undergoing clinical trials testing its safety and efficacy in anaplastic large cell lymphoma, neuroblastoma, and other advanced solid tumors in both adults and children. It can be given by mouth or by injection. It has FDA Unique Ingredient Identifier (UNII): 53AH36668S and DRUG BANK Accession number DB08700.

[0487] In some embodiments, the therapeutic active agent may be Doxorubicin. Doxorubicin, sold under the brand name Adriamycin® among others, is a chemotherapy medication used to treat cancer e.g. breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used together with other chemotherapy agents. Doxorubicin is given by injection into a vein. It has FDA Unique Ingredient Identifier (UNII): 80168379AG and DRUG BANK Accession number DB00997. In some embodiments, the therapeutic active agent may be Docetaxel. Docetaxel (DTX or DXL), sold under the brand name Taxotere® among others, is a chemotherapy medication used to treat a number of types of cancer e.g. breast cancer, head and neck cancer, stomach cancer, prostate cancer and non- small-cell lung cancer. It may be used by itself or along with other chemotherapy medication. It is administered by slow injection into a vein. It has an FDA Unique Ingredient Identifier (UNII): 699121PHCA and DRUG BANK Accession number DB01248.

[0488] In some embodiments, the therapeutic active agent may be Paclitaxel. Paclitaxel (PTX), sold under the brand name Taxol® among others, is a chemotherapy medication used to treat a number of types of cancer e.g. ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, and pancreatic cancer. It is given by injection into a vein. It has FDA Unique Ingredient Identifier (UNII): P88XT4IS4D and DRUG BANK Accession number DB01229.

[0489] In some embodiments, the therapeutic active agent may be Methotrexate. Methotrexate (MTX), formerly known as amethopterin, sold under the brand name Trexall®, Rheumatrex®, Otrexup® among others, is a chemotherapy agent and immune-system suppressant. It is used to treat cancer such as breast cancer, leukemia, lung cancer, lymphoma, gestational trophoblastic disease, and osteosarcoma, autoimmune diseases such as psoriasis, rheumatoid arthritis, and Crohn's disease, and ectopic pregnancy and for medical abortions. It can be given by mouth or by injection. It has FDA Unique Ingredient Identifier (UNII): YL5FZ2Y5U1 and DRUG BANK Accession number DB00563. In another embodiments, the therapeutic active agent may be Pemetrexed. Pemetrexed, sold under the brand name Alimta®) among others, is a chemotherapy medication for the treatment of pleural mesothelioma and non-small cell lung cancer (NSCLC). It has FDA Unique Ingredient Identifier (UNII): 04Q9AIZ7NO and DRUG BANK Accession number DB00642. In some specific and non-limiting embodiments, the present disclosure provides methods for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one malignant proliferative disorder in a subject in need thereof. These methods involve therapeutic and diagnostic steps. More specifically, the method comprising the steps of: (a), exposing cells of the subject applied on at least one cell chamber of at least one cell cultivation unit of the microfluidic platform as described by the present disclosure, to the at least one therapeutic active agent provided via the medium channel of each of the cell cultivation units. It should be noted that the cell sample is obtained after the initiation of the treatment regimen. Next in step (b), determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one time interval. In step (c), determining at least one of: either (i), loss of responsiveness, and / or drug-resistance of the subject, if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with the cell viability and / or at least one cell phenotype in the absence of the candidate active agent. Alternatively (ii), determining responsiveness or maintained responsiveness of the subject, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with the cell viability and / or at least one cell phenotype in the absence of the candidate active agent.

[0490] The next step (d) is the therapeutic step that involves selecting a treatment regimen based on the responsiveness, thereby treating the subject with the selected treatment regimen.

[0491] In some further embodiments, the selected treatment or therapeutic active agent of the disclosure may be administered and dosed by the methods of the disclosure, in accordance with good medical practice, systemically, for example by parenteral, e.g. intravenous. It should be noted, however, that the disclosure may further encompass additional administration modes. In other examples, the active agent can be introduced to a site by any suitable route including intraperitoneal, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular administration.

[0492] In yet some further embodiments, the therapeutic active agent applicable in the therapeutic methods disclosed herein is at least one of: an inorganic or organic molecule, a small molecule, a nucleic acidbased molecule, an aptamer, a polypeptide, or any combinations thereof.

[0493] In some embodiments, the cells used in the diagnostic step of the therapeutic methods disclosed herein form aggregates / clusters in the cell chamber, prior to exposure to the therapeutic active agent.

[0494] In some embodiments, the cells used in the diagnostic steps of the therapeutic methods disclosed herein are cells of a subject suffering from a pathologic disorder. In yet some further embodiments, the cells are of the subject that will be treated by the disclosed method, thereby providing tailored personalized therapeutic approach.

[0495] In some embodiments pathologic disorder treatable by the disclosed therapeutic method is any one of a malignant proliferative disorder, an inflammatory condition a metabolic condition, an infectious disease, an autoimmune disease, protein misfolding disorder or deposition disorder.

[0496] In yet some further embodiments, the pathologic disorder is a malignant proliferative disorder. In further embodiments, the cells used for the assessment prognostic stage of the disclosed method are primary cancer cells of the subject. In some embodiments, the cells may be a mixture of different primary cells.

[0497] In more specific embodiments, the malignant proliferative disorder is any one of carcinoma, melanoma, lymphoma, leukemia, myeloma and sarcoma.

[0498] Local administration to the area in need of treatment may be also achieved by, for example, by local infusion during surgery, topical application, direct injection into the specific organ, etc. More specifically, the active agent used in any of the methods of the disclosure, described herein before, may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes.

[0499] It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from at least one pathologic disorder. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease. The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, pathologic disorder involved with at least one short term cellular stress condition / process and any associated condition, illness, symptoms, undesired side effects or related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and / or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms. It should be appreciated that the terms "inhibition", "moderation", “reduction”, "decrease" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more. With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.

[0500] The term "amelioration" as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the methods according to the disclosure, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state.

[0501] The term "inhibit" and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with.

[0502] The term "eliminate" relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the disclosure described herein. The terms "delay", "delaying the onset", "retard" and all variations thereof are intended to encompass the slowing of the progress and / or exacerbation of a disorder associated with the at least one short term cellular stress condition / process and their symptoms, slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the disclosure. As indicated above, the methods provided by the present disclosure may be used for the treatment of a “pathological disorder”, i.e. pathologic disorder or condition involved with at least one short term cellular stress condition / process, which refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

[0503] It should be appreciated that any of the methods described by the disclosure may be applicable for treating and / or ameliorating any of the disorders disclosed herein or any condition associated therewith. It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, “disease”, “disorder”, “condition”, “pathology” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

[0504] It should be appreciated that the platforms, systems and methods of the present disclosure may be suitable for any subject that may be any multicellular organism, specifically, any vertebrate subject, and more specifically, a mammalian subject, avian subject, fish or insect. In some specific embodiments, the prognostic as well as the therapeutic, cosmetic and agricultural methods presented by the enclosed disclosure may be applicable to mammalian subjects, specifically, human subjects. By “patient” or “subject” it is meant any mammal that may be affected by the above-mentioned conditions, and to whom the treatment and prognosis methods herein described is desired, including human, bovine, equine, canine, murine and feline subjects. Specifically, the subject is a human.

[0505] In some embodiments, the disclosed methods provided herein are applicable for, and utilize a cellbased assay that is, in some specific embodiments, a target recognition assay to detect and / or quantify at least one target molecule produced by or produced from the cells in the cell cultivation chambers. According to such embodiments, the cell assay material / s, comprise material and / or components produced by the cells and / or materials and / or components produced from the cells, in each the cell cultivation chamber. Accordingly, the method comprises the step of: in step (a), transferring via the medium channel of each of the cell cultivation units to the active zone / s formed by the surface activation valves, as defined by the present disclosure. It should be noted that the cell assay material / s produced by the cells and / or materials and / or components produced from the cells. For example, any proteins produced or other products produced by the cells in each chamber and secreted to the medium, or alternatively, any proteaceous material or nucleic acid material prepared from the cells in each chamber. Next in step (b), contacting the cell assay material / s produced by the cells or produced from the cells, with at least one affinity moiety. The affinity moiety is immobilized into the contact zone in each of the medium channel of each cell cultivation unit, within each of the active zone in each of the medium channel. It should be noted that at least one affinity moiety is specific for the at least one target molecule, wherein upon recognition, the target binds the affinity moiety. Next step (c), involves determining the presence and / or the quantity of the target, if an end-point indication that reflects recognition and / or binding of the target molecule to the affinity moiety, is detected.

[0506] In the context of an affinity assay, the end-point indication refers to the measurable signal that reflects the extent of binding between a ligand and its target once the reaction has reached equilibrium or after a defined incubation time. The type of end-point readout depends on the detection system incorporated into the assay design.

[0507] End-point indication may be provided by optical signals such as absorbance, fluorescence, or luminescence, for example in ELISA-type assays where substrate conversion generates a colorimetric or chemiluminescent signal proportional to complex formation. Alternatively, label-free detection methods such as surface plasmon resonance, bio-layer interferometry, or quartz crystal microbalance yield end-point shifts in resonance angle, interference pattern, or frequency, respectively. In radiobinding assays, retained radioactivity on the solid phase serves as the end-point measure. In cell-based affinity systems, reporter gene activation, second-messenger levels, or staining intensity may constitute the indication.

[0508] In all cases, the end-point is defined as the measurable physical or chemical change that correlates directly with the amount of bound complex, enabling quantification of binding strength or comparative affinity. A non-limiting embodiment for the microfluidic platform or system that comprises the reaction zones as used in the disclosed method, is illustrated by Figure 11. A corresponding ELISA- like assay is exemplified in Example 6.

[0509] In some embodiments of the disclosed methods, the at least one affinity moiety comprises at least one antibody specific for the at least one target molecule, or any antigen-binding fragments thereof.

[0510] In some specific embodiments, an affinity moiety used by any of the methods disclosed in the present disclosure, may be an antibody, or any antigen-binding fragments thereof. The term "antibody" as used herein, means any antigen-binding molecule or molecular complex that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0511] Typically, an antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains. In humans, antibodies are encoded by three independent gene loci, namely kappa (K) chain (IgK) and lambda (X) chain (IgA) genes for the Eight chains and IgH genes for the Heavy chains, which are located on chromosome 2, chromosome 22, and chromosome 14, respectively.

[0512] The antibody used by the method of the disclosure may be any one of a polyclonal, a monoclonal or humanized antibody or any antigen-binding fragment thereof. The term "an antigen-binding fragment" refers to any portion of an antibody that retains binding to the antigen. Examples of antibody functional fragments include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2' and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen.

[0513] As appreciated by one of skill in the art, various antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin, or de novo synthesis. Antibody fragments are often synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries. The term antibody also includes bivalent molecules, diabodies, triabodies, and tetrabodies. References to “VH” or a “VH” refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, a disulfilde-stabilized Fv (dsFv) or Fab. References to “VL” or a “VE” refer to the variable region of an immunoglobulin light chain, including of an Fv, scFv, dsFv or Fab.

[0514] More specifically, the phrase “single chain Fv” or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for the stabilization of the variable domains without interfering with the proper folding and creation of an active binding site. A single chain antibody applicable for the disclosure, e.g., may bind as a monomer. Other exemplary single chain antibodies may form diabodies, triabodies, and tetrabodies. Antibodies may be used according to the methods of the disclosure to target an epitope of interest, within the target molecule produced by, or from the cells in the cultivating chambers. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0515] In yest some alternative or additional embodiments, the material / s produced by the cells, or produced from the cells comprises at least one protein-based molecule, that is or comprises in some embodiments , the target molecule.

[0516] In some further alternative or additional embodiments, the at least one target molecule is at least one protein product produced by the cells in each of the cell cultivation chamber / s. Still further, in some embodiments, the at least one target molecule is at least one protein product produced by, and secreted by the cells in each of the cell cultivation chamber / s.

[0517] In yet some further embodiments of the disclosed methods, the at least one affinity moiety comprises at least one nucleic acid-based affinity molecule.

[0518] In some alternative or additional embodiments, the at least one nucleic acid-based affinity molecule comprises at least one of: at least one primer, at least one probe, and / or at least one nucleic acid aptamer.

[0519] Still further, in some alternative or additional embodiments, the material / s produced by the cells, or produced from the cells comprises at least one protein-based molecule and / or a nucleic acid-based molecule.

[0520] In some alternative or additional embodiments, the material / s produced from the cells comprises at least one nucleic acid-based molecule. Accordingly, at least one target molecule is or comprises at least one nucleic acid -based target molecule, for example, DNA or RNA molecules serving as a target molecule.

[0521] In yet some alternative or additional embodiments, the nucleic acid-based target molecule may be identified by at least one nucleic acid-based affinity molecule. Still further, the at least one nucleic acid-based affinity molecule is at least one primer and / or prob. Accordingly, the at least one primer and / or probe comprise at least one nucleic acid sequence complementary to at least one nucleic acid sequence within the at least one nucleic acid target molecule. Still further, according to alternative or additional embodiments, the preparation of the nucleic acidbased or protein-based material from the cells in each the cell cultivation chamber is performed using at least one extraction reagent. For example, any of the reagents disclosed by the present disclosure.

[0522] Still further, in some alternative or additional embodiments, the at least one extraction reagent is provided to each the cell cultivation chamber, via the at least one cell channel.

[0523] In some embodiments, prior to performing the at least one target-recognition assay, the cells in each of the cell cultivation chambers, are exposed to at least one compound or therapeutic agent (e.g., a compound affecting cell viability). In some embodiments, such a compound or agent is provided via the medium channel of each of the cell cultivation units.

[0524] A further aspect of the present disclosure provides a method for performing a cell-cell interaction assay. The method comprises the steps of:

[0525] In step (a), applying at least one sample comprising at least two types of cells into a first microfluidic platform of a microfluidic system as defined in the present disclosure. The cells are applied into at least one of the cell cultivation units, via the cell channel. Each of the cell cultivation units is applied with one type of cells, such that the cell cultivation chambers in each the cell cultivation unit are seeded with the same one type of cells. Next in step (b), counting the number of the cells in each of the cell cultivation chambers in each the cell cultivation unit. Step (c), involves transferring cells from at least two cell cultivation units, each in a desired cell number, to the cell mixing module via dedicated input / output lines of at least one of the cell cultivation units of the first microfluidic platform. It should be noted that at least two cell cultivation units comprise at least two different types of cells. Still further, a desired number of cells from each type of cell is transferred to the mixing module, each from one or more the cell cultivation unit comprising the cell type. Next in step (d), mixing the at least two types of cells, each in a desired number to obtain a cell mixture comprising at least two types of cells in a predetermined ratio. Next in step (e), transferring the cell mixture obtained in step (d), from the cell mixing module via the output port that is coupled to the at least one inlet of the second the microfluidic platform of the system, such that each of the cell cultivation chambers in each of the cell cultivation units is applied with a mixture of at least two types of cells at a predetermined ratio. In step (f), performing at least one cell-cell interaction assay in at least one of the cell cultivation units of the second microfluidic platform of the microfluidic system. The present disclosure this provides platforms, systems and methods for performing various cell-cell interaction assays that involve one or more, and specifically, at least two type of cells. A cell-cell interaction assay, in the context of the present disclosure, may be defined as an in vitro or ex vivo experimental system designed to evaluate the functional, structural, or biochemical consequences of contact or communication between two or more cell populations. Such assays are intended to reproduce, in a controlled setting, the physiological or pathological interactions that occur within a subject, thereby allowing the study of mechanisms of disease progression, immune surveillance, therapeutic response, or tissue regeneration. The present method is based on co-culture systems, that allow direct contact between different cell types, such as immune effector cells and tumor cells, stromal and epithelial cells, or endothelial and leukocyte populations, in order to monitor activation, adhesion, cytotoxicity, or differentiation events. It should be understood that the microfluidic platforms, systems and methods disclosed herein may allow multicellular arrangements (aggregates, e.g., organoid-like aggregates) that more closely mimic tissue architecture. The cells involved may be primary cells directly isolated from a subject, immortalized or engineered cell lines, or stem cell- derived populations, selected according to the biological question under investigation. The defining characteristic is that the assay provides a measurable outcome, such as viability, proliferation, cytokine release, gene expression, or morphological change, that reflects the nature of the interaction between distinct cellular populations.

[0526] In some embodiments, the cell-cell interaction assays are conducted for defined periods of time sufficient to permit measurable interaction between the participating cell populations. The time intervals may range from several minutes to several weeks, depending on the nature of the assay and the biological response being evaluated. For example, in certain embodiments, the interaction is monitored for between about 1 minute and about 60 minutes, such as 1, 2, 5, 10, 15, 30, 45, or 60 minutes. In further embodiments, the interaction period extends from about 1 hour to about 24 hours, including 1, 2, 4, 6, 8, 12, or 24 hours. In yet other embodiments, the assay is conducted over multiple days, for example between about 1 day and about 14 days, such as 1, 2, 3, 5, 7, 10, or 14 days. Still further embodiments contemplate longer-term monitoring over a period of about 2 to about 6 weeks, such as 2, 3, 4, 5, or 6 weeks.

[0527] The selection of the specific time interval may be dictated by the cellular behavior of interest. For instance, rapid adhesion events or receptor signaling may be detectable within minutes or hours, while proliferation, differentiation, or cytotoxic effects may require multi-day cultures. Prolonged interactions, extending into weeks, may be employed in organoid or three-dimensional culture systems to assess chronic or sustained effects of cellular communication.

[0528] In some embodiments, a microfluidic system applicable for performing the cell-cell interaction assays are illustrated by Figures 14-18. Specifically, systems comprising mixing modules are according to some nonO-limiting embodiments illustrated by Figures 14, 15, 16 and 18. Still further, a non-limiting example for a cell-cell interaction assay is exemplified in examples 4 and 5.

[0529] Thus, in some embodiments, the cell-cell interaction assay performed in step (f) in the second microfluidic platform, is any of the cell-cell interaction assays disclosed herein before by the present disclosure, for example, at least one killing assay. Accordingly, at least one of the at least two types of cells is effector cells and at least one of the at least two types of cells is target cells.

[0530] In some embodiments, the disclosed methods provide a cell killing assay. Accordingly, the disclosed method comprises the step of: In step (i), incubating a cell mixture comprising at least one effector cell and at least one target cell in a predetermined ratio, in each cell cultivation chamber of each cell cultivation unit. It should be understood that the cells are incubated for any suitable time period, specifically, minutes, hours, or days, as discussed above. Next, after a suitable incubation period, (ii), performing a cell viability assay to determine the number of the target cells in each of the cell cultivation chambers.

[0531] In some embodiments, the ratio of effector cells to target cells in the assay ranges from about 1 : 1 to about 1: 1000. At the lower end of the spectrum, a ratio of 1: 1 reflects a condition in which each effector cell is paired with a single target cell, thereby allowing highly precise measurement of individual effector activity. In some embodiments, the ratio of effector cells to target cells is defined with precision, extending from 1:1 through 1: 1000. At the lower end of the spectrum, the ratio may be 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10. Additional contemplated ratios include 1: 15, 1:20, 1:25, 1:30, 1:40, and 1:50. Higher ranges may include 1:60, 1:75, 1:80, 1:90, and 1:100. Progressively larger target-dominant conditions may be represented by ratios of 1: 150, 1:200, 1:250, 1:300, 1:400, 1:500, 1:600, 1:750, 1:800, 1:900, and 1: 1000.

[0532] The selection of these ratios permits systematic evaluation of effector function across conditions where effector cells are equal in number to targets, moderately outnumbered, or vastly outnumbered. The continuum from 1:1 to 1: 1000 ensures that the assay encompasses both physiologically relevant and stress-testing scenarios, enabling accurate determination of effector cell potency, persistence, and responsiveness under controlled conditions. In killing assays, the effector cells are typically immune-derived populations capable of mediating cytotoxic activity, including but not limited to cytotoxic T lymphocytes (CD8+T cells), natural killer (NK) cells, chimeric antigen receptor (CAR)-engineered T cells or NK cells, and other lymphocyte subsets with cytolytic potential. Depending on the context, effector cells may be derived directly from patient peripheral blood mononuclear cells, from in vitro-expanded or engineered cell lines, or from stem cell-derived immune progenitors. The target cells in such assays are generally those intended to represent diseased or aberrant populations, including tumor cell lines, primary cancer cells isolated from patients, virus-infected cells, or engineered cell lines expressing defined antigens. In certain embodiments, non-malignant cells such as fibroblasts, epithelial cells, or other primary tissue-derived cells may be used as negative controls to assess specificity of effector-mediated killing. In some embodiments, the effector cells used in the killing assay comprise chimeric antigen receptor (CAR) T cells or CAR-NK cells, which are genetically engineered T lymphocytes or NK cels, designed to express synthetic receptors that specifically recognize a chosen antigen on the surface of target cells. The target cells in such assays may include malignant cells, such as leukemic or solid tumor cell lines, or primary tumor cells obtained from a subject, wherein the tumor cells express the antigen of interest recognized by the CAR construct. In certain cases, the target cells may be engineered cell lines that stably express the relevant antigen, thereby providing a defined and reproducible system for assessing CAR-T cytotoxicity. Thus, in some embodiments, effector cells are CAR-T or CAR-NK cells and the target cells are cells expressing the target antigen of said CAR. In yet some further embodiments the target cells are of a subject suffering from at least one pathologic disorder.

[0533] In yet some further embodiments, the cell-cell interaction assay performed in step (f) in the second microfluidic platform, is at least one cell activation assay, wherein at least one of the at least two types of cells comprise effector cells and wherein at least one of the at least two types of cells comprise target cells.

[0534] According to such embodiments, the activation assay comprises detection and / or quantification of at least one target molecule produced by the effector cells in response to interaction with the target cell. It should be noted that the at least one target molecule is detected and / or quantified by a target recognition assay as defined by the disclosed methods, using the affinity moiety discussed above. As indicated above, the cell-cell interaction assay provided by the present disclosure may be conducted between effector and target cells as a killing assay, wherein the degree of target cell death serves as a direct measure of effector function. In other embodiments, the assay is configured as an activation assay, wherein the functional state of the effector cells is determined based on the production and release of specific effector molecules. Such effector molecules may include cytokines and chemokines that mediate immune signaling, cytotoxic proteins such as perforin, granzymes, Fas ligand (FasL), or TRAIL that induce target cell apoptosis, antibodies generated by B cell-derived effectors, as well as antimicrobial mediators including reactive oxygen species (ROS), nitric oxide (NO), and enzymes with microbicidal properties. The detection and quantification of these molecules, specifically, as discussed herein above using the disclosed microfluidic platforms, systems and methods, provides a reliable readout of effector cell engagement and activity upon contact with target cells, thereby enabling the assay to serve as a versatile platform for evaluating cytotoxicity, specificity, and immune activation in a physiologically relevant context.

[0535] It should be understood that the disclosed cell-cell interaction assays are carried out using the microfluidic system in accordance with the present disclosure that comprise a first microfluidic platform in communication with a mixing module, the mixing module being in communication with a second microfluidic platform. The mixing module is configured to enable counting of cells and to apply at least two different cell types, at predetermined numbers and ratios, onto the second microfluidic platform that contains the cultivation chambers. However, it should further be appreciated that the same cell-cell interaction assays may alternatively be performed using a single microfluidic platform as disclosed by the present disclosure, provided that the cells are introduced directly into the cultivation chambers at the desired ratios.

[0536] A further aspect of the present disclosure provides a method for performing nucleic acid-based assay and / or a protein-based assay in a population of cells comprising at least one type of cells. The disclosed method comprises the steps of: First in step (a), applying at least one sample comprising at least one type of cells into a first microfluidic platform of a microfluidic system as defined by the present disclosure. The cells are applied into at least one of the cell cultivation units, via the cell channel. Still further, each of the cell cultivation units are applied with at least one type of cells, such that the cell cultivation chambers in each the cell cultivation unit, is seeded with the same at least one type of cells. An optional step involves counting the number of the cells in each of the cell cultivation chambers in each said cell cultivation unit. Next in step (b), transferring cells from one or more cell cultivation units, to the cell mixing module via dedicated input / output lines of at least one the cell cultivation unit of the first microfluidic platform as defined above. In step (c), mixing the at least one type of cells, and subjecting the cell mixture to at least one protein preparation procedure and / or at least one nucleic acid preparation procedure. In step (d), applying the nucleic acid preparation and / or protein preparation obtained in step (c) on at least one protein-cased and / or nucleic acid-based assay.

[0537] A further aspect of the present disclosure relates to a method for detecting and / or quantify at least one target molecule in at least one sample comprising at least one type of cells. The method comprising: (a), applying at least one sample comprising at least one type of cells into a first microfluidic platform of a microfluidic system as defined by the present disclosure. The system comprises at least one microfluidic platform and at least one sensor, and the cells are applied into at least one of the cell cultivation unit, via the cell channel. Each of the cell cultivation units is applied with at least one type of cells, such that the cell cultivation chambers in each of the cell cultivation unit are seeded with the same at least one type of cells. Example 7 provides a non-limiting example for the discussed system. Still further, in some embodiments, Example 7 provides an example for the disclosed assay.

[0538] In some optional embodiments, the method further involves the step of counting the number of cells in each of the cell cultivation chambers in each said cell cultivation unit.

[0539] In yet some further embodiments, the disclosed method may optionally comprise the step of exposing the cells to any agent (candidate compound, therapeutic compound or any of the agents and compounds disclosed by the present disclosure) or performing any cell-cell interaction in the cells. Next in step (b), transferring cells from one or more cell cultivation units, to the sensor module via dedicated input / output lines of at least one the cell cultivation unit of the first microfluidic platform. The sensor is configured to detect and / or quantify at least one target molecule in the fluid present in the dedicated input / output line. It should be further noted that the sensor comprises at least one affinity moiety specific for said target molecule.

[0540] In step (c), determining the presence and / or the quantity of the target, if an end-point indication that reflects recognition and / or binding of the target molecule to the affinity moiety, is detected.

[0541] The versatility of the disclosed platforms and methods allows the combination of one or more microfluidic platforms with one or more accessory modules, such as mixing module / s, and sensor module / s, as discussed above. For example, in some embodiments, the system optionally comprises at least a first microfluidic platform, a mixing module in communication with the first microfluidic platform, and a second microfluidic platform in communication with the mixing module, wherein the second microfluidic platform is operatively connected to a sensor configured to detect, monitor, or quantify one or more parameters of the cell-cell interaction assay, in accordance with the methods disclosed herein.

[0542] In some embodiments, the cells used herein in the disclosed methods, platforms and systems, are cells of a subject suffering from a pathologic disorder.

[0543] In yet some further embodiments, the method for determining a personalized treatment regimen disclosed herein is applicable for a subject suffering from any one of a malignant proliferative disorder, an inflammatory condition, a metabolic condition, an...

Claims

CLAIMS:

1. A microfluidic platform, comprising: a primary block of substrate material, the primary block defining a first plurality of cell cultivation units, and a control system for enabling control of fluid flows with respect to the first plurality of cell cultivation units; wherein the cell cultivation units are fluidly isolated from one another; wherein each said cell cultivation unit comprises a medium channel, a cell channel and a second plurality of filter pathways, wherein: the medium channel is laterally spaced with respect to the cell channel; the second plurality of filter pathways laterally interconnects the medium channel with the cell channel; the cell channel defines a second plurality of serially adjacent cell cultivation zones, wherein each cell cultivation zone is in selective fluid communication with the medium channel via a respective said filter pathway; wherein for each said cell cultivation unit, the control system is configured for selectively blocking fluid communication via the cell channel between adjacent cell cultivation zones to thereby provide respective cell cultivation chambers, wherein each said cell cultivation chamber is in selective fluid communication with the medium channel exclusively via the respective said filter pathway; wherein each said cell cultivation unit comprises a dedicated input / output line providing selective exclusively fluid communication between an outside of the device and each one of the cell channel and the medium channel of the respective cell cultivation unit; wherein for each cell cultivation unit: the medium channel is configured for enabling fluids to be received therein from the dedicated input / output line or to be delivered therefrom to the dedicated input / output line; the cell channel is configured for enabling cells to be received therein from the dedicated input / output line or to be delivered therefrom to the dedicated input / output line; the filter pathways are configured for selectively providing fluid communication between the medium channel and the cell channel, while concurrently preventing transit of cells therethrough from the cell channel to the medium channel.

2. The microfluidic platform according to claim 1, wherein the control system comprises a plurality of microfluidic valves, each said microfluidic valve being configured for selectively allowing or preventing flow therethrough under the control of the control system.

3. The microfluidic platform according to any one of claims 1 to 2, wherein each said cell cultivation unit extends between a first unit end and a second unit end.

4. The microfluidic platform according to claim 3, wherein each said dedicated input / output line is operatively coupled to the first unit end of the respective cell cultivation unit.

5. The microfluidic platform according to claim 4, wherein each said dedicated input / output line is operatively and separately coupled to the respective cell channel and the respective medium channel at a first cell channel port and a first medium channel port, respectively, at the first unit end of the respective cell cultivation unit.

6. The microfluidic platform according to claim 5, wherein the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective first cell channel address valve overlying the respective first cell channel port, wherein the control system is operable to selectively open or close the first cell channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said cell channel and the respective said dedicated input / output line.

7. The microfluidic platform according to claim 6, wherein the control system is configured for concurrently operating all the first cell channel address valves to thereby concurrently selectively open or close all the corresponding first cell channel ports.

8. The microfluidic platform according to any one of claims 5 to 7, wherein the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective first medium channel address valve overlying the respective first medium channel port, wherein the control system is operable to selectively open or close the first medium channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said medium channel and the respective said dedicated input / output line.

9. The microfluidic platform according to claim 8, wherein the control system is configured for concurrently operating all the first medium channel address valves to thereby concurrently selectively open or close all the corresponding first medium channel ports.

10. The microfluidic platform according to any one of claims 2 to 9, wherein for each said cell cultivation unit the respective said medium channel extends longitudinally between the first unit end and the second unit end, and the respective cell channel extends longitudinally between the first unit end and the second unit end.

11. The microfluidic platform according to any one of claims 1 to 10, wherein each said filter pathway comprises a third plurality of lateral channels in selective fluid communication between the respective said medium channel and the respective said cell channel, each said lateral channel having a flow cross-section so dimensioned as to prevent transit of the cells therethrough from the cell channel to the medium channel.

12. The microfluidic platform according to any one of claims 1 to 11 , wherein the control system comprises a said microfluidic valve in the form of a respective horizontal segmentation valve overlying each said filter pathway of each said cell cultivation unit, wherein the control system is operable to selectively open or close each said horizontal segmentation valve to thereby allow or prevent fluid communication, respectively, between the respective said cell cultivation chamber and the respective medium channel.

13. The microfluidic platform according to claim 12, wherein the control system is configured for concurrently operating all the horizontal segmentation valves to thereby concurrently selectively open or close all the horizontal segmentation valve.

14. The microfluidic platform according to any one of claims 1 to 13, wherein the control system comprises a pair of said microfluidic valve in the form of a respective pair of longitudinally spaced vertical segmentation valves longitudinally enclosing each said cell cultivation zone of each said cell cultivation unit, wherein the control system is operable to selectively close each said pair of vertical segmentation valves to thereby provide the respective said cell cultivation chambers.

15. The microfluidic platform according to claim 14, wherein the control system is configured for concurrently operating all the vertical segmentation valves of each said cell cultivation unit to thereby concurrently selectively close all the corresponding vertical segmentation valves of the respective cell cultivation unit, thereby concurrently providing all the cell cultivation chambers of the respective cell cultivation unit.

16. The microfluidic platform according to claim 15, wherein the control system is configured for concurrently operating all the vertical segmentation valves of said first plurality of cell cultivation unit to thereby concurrently selectively close all the corresponding vertical segmentation valves of said first plurality of cell cultivation units, thereby concurrently providing all the cell cultivation chambers of said first plurality of cell cultivation units.

17. The microfluidic platform according to any one of claims 2 to 16, further comprising a fluid inlet system operatively coupled to the respective second unit ends of the cell cultivation units.

18. The microfluidic platform according to claim 17, wherein the fluid inlet system comprises a manifold arrangement, the manifold arrangement having at least one inlet coupled to a first plurality of outlets via a succession of bifurcating flow paths defining flow path branches, wherein each said outlet is operatively coupled to a respective said second unit end of a different said cell cultivation unit.

19. The microfluidic platform according to claim 18, wherein the control system comprises a plurality of said microfluidic valves in the form of a respective plurality of manifold address valves, each overlying a respective said branch, wherein the control system is operable to selectively open or close each said manifold valve to thereby allow or prevent fluid communication, respectively, through the respective branch.

20. The microfluidic platform according to claim 19, wherein the control system is operative to selectively concurrently open or close all the manifold address valves to thereby enable or prevent, respectively, fluid communication between the at least one inlet and the first plurality of outlets.

21. The microfluidic platform according to any one of claims 19 to 20, wherein the control system is operative to selectively concurrently open or close selected said manifold address valves to thereby enable fluid communication between the at least one inlet and only a selected one said outlet of said first plurality of outlets.

22. The microfluidic platform according to any one of claims 18 to 21 , wherein each said outlet is operatively and separately coupled to the respective cell channel and the respective medium channel at a second cell channel port and a second medium channel port, respectively, at the second unit end of the respective cell cultivation unit.

23. The microfluidic platform according to claim 22, wherein the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective second cell channel address valve overlying the respective second cell channel port, wherein the control system is operable to selectively open or close the second cell channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said cell channel and the respective said outlet.

24. The microfluidic platform according to claim 23, wherein the control system is configured for concurrently operating all the second cell channel address valves to thereby concurrently selectively open or close all the corresponding second cell channel ports.

25. The microfluidic platform according to any one of claims 22 to 24, wherein the control system comprises, for each said cell cultivation unit, a said microfluidic valve in the form of a respective second medium channel address valve overlying the respective second medium channel port, wherein the control system is operable to selectively open or close the second medium channel address valve to thereby allow or prevent fluid communication, respectively, between the respective said medium channel and the respective said outlet.

26. The microfluidic platform according to claim 25, wherein the control system is configured for concurrently operating all the second medium channel address valves to thereby concurrently selectively open or close all the corresponding second medium channel ports.

27. The microfluidic platform according to any one of claims 22 to 26, wherein the control system comprises, for each said cell cultivation unit, a plurality of said microfluidic valves in the form of a respective plurality of surface activation valves overlying a portion of the respective medium channel, wherein the control system is operable to selectively open or close all surface activation valves together, and wherein when closed each respective said surface activation valve abutting seals against a respective contact zone on a surface of the medium channel, while concurrently permitting fluid communication through the medium channel.

28. The microfluidic platform according to claim 27, wherein each contact zone is capable of having applied thereto at least one affinity moiety, when the respective surface activation value is open, and wherein responsive to subsequently closing the respective activation value, the respective at least one affinity moiety is fluidly isolated from the respective medium channel.

29. The microfluidic platform according to claim 28, wherein application of the at least one affinity moiety on the respective contact zone is performed by the steps of:(a) with the surface activation valve open, applying surface activating material to said surface of the medium channel;(b) subsequently closing the surface activation valve;(c) with the surface activation valve closed, applying a surface blocking material to said surface of the medium channel, excluding the now isolated contact zone;(d) subsequently opening the surface activation valve;(e) with the surface activation valve open, applying said at least one affinity moiety to the now exposed contact zone; thereby immobilizing the affinity moiety thereat to create a reaction zone at the contact zone.

30. The microfluidic platform according to any one of claims 27 to 29, wherein the control system is configured for concurrently operating the surface activation valves of all the cell cultivation units to thereby concurrently selectively open or close all the corresponding surface activation valves.

31. The microfluidic platform according to any one of claims 1 to 30, further comprising a gas channel configured for delivering a gas optionally including at least one of carbon dioxide (CO2),oxygen (O2) and nitrogen (N) in close proximity to the medium channels, and wherein the gas channel is laterally spaced from all the cell channels .

32. The microfluidic platform according to claim 31 , wherein the gas channel comprises a channel inlet, a channel outlet, a plurality of longitudinal channel segments and a plurality of lateral channel segments, and wherein the longitudinal channel segments are in overlying relationship with respect to the medium channels.

33. The microfluidic platform according to any one of claims 1 to 32, wherein the cell cultivation units are in adjacent laterally spaced relationship with respect to one another within the primary block.

34. The microfluidic platform according to claim 33, wherein each pair of laterally adjacent said cell cultivation units are laterally separated by a solid wall of said substrate material that provides fluid isolation between the cell cultivation units of the respective pair.

35. The microfluidic platform according to any one of claims 2 to 34, wherein said control system comprises a plurality of microfluidic control lines, each said microfluidic control line configured for controlling operation of one or more said microfluidic valves associated with the respective said microfluidic control line.

36. The microfluidic platform according to any one of claims 1 to 35, including one of the following:- wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension;- wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a material transparent to electromagnetic radiation at least in the visible spectrum;- wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a material transparent to electromagnetic radiation at least in the visible spectrum, and, wherein said material is or comprises at least one of polydimethylsiloxane, polycarbonate, polystyrene;- wherein said primary block comprises a block member in overlying fixed relationship with a base member, and wherein said block member comprises an outer-facing first block surface and an outer-facing second block surface, wherein the second block surface is spaced from the first block surface by a block member thickness dimension, and, wherein said block member comprises a first block layer in overlying abutting relationship with a second block layer, wherein the second block layer comprises said control system, and said first block layer comprises said first plurality of cell cultivation units, and said dedicated input / output lines.

37. The microfluidic platform according to any one of claims 1 to 36, wherein said first plurality of said cell cultivation units are arranged in an array with respect to the primary block of said substrate material.

38. The microfluidic platform according to any one of claims 1 to 37, wherein said first plurality is within a first integer range, the first integer range being between 2 and 20.

39. The microfluidic platform according to any one of claims 1 to 38, wherein said first plurality is 16.

40. The microfluidic platform according to any one of claims 1 to 39, wherein said second plurality is within a second integer range, the second integer range being between 2 and 20.

41. The microfluidic platform according to any one of claims 1 to 40, wherein said second plurality is 11.

42. A microfluidic system comprising at least one microfluidic platform as defined in any one of claims 1 to 41, and an accessory module configured for being fluidly coupled with respect to said at least one microfluidic platform.

43. The microfluidic system according to claim 42, wherein the accessory module is in the form of a cell mixing module, wherein the mixing module comprises a secondary block, defining a cell mixing chamber, an inlet port array, a medium input port, an output port, a pump system, and an air purging system, wherein: the inlet port array comprising a plurality of inlet ports selectively connectable to the dedicated input / output lines of said cell cultivation units, such that when thus connected each such connected inlet port and dedicated input / output line are in fluid communication with one another, and wherein the inlet ports are in open fluid communication with the cell mixing chamber; the cell mixing chamber defining a mixing volume in open fluid communication with the medium input port, the output port and the air purging system; the pump system comprising at least one microfluidic pump operative to selectively mix contents of the cell mixing chamber when present; the output port configured for enabling outputting therefrom the contents of the mixing volume; the input port configured for enabling inputting desired media into the mixing volume; the air purging system configured for enabling selectively purging air that may be present in the mixing volume.

44. The microfluidic system according to claim 43, wherein the pump system comprises at least two spaced microfluidic valves operative to periodically open and close one said microfluidic valve while concurrently closing and opening the other microfluidic valve to thereby induce a pumping action within the mixing volume.

45. The microfluidic system according to any one of claims 43 to 44, comprising one said microfluidic platform operatively coupled with the cell mixing module, wherein a respective said dedicated input / output lines of at least one said cell cultivation unit is fluidly coupled to a corresponding said inlet port of the cell mixing module.

46. The microfluidic system according to claim 45, for use in preparation and / or extraction of protein extract / material and / or nucleic acid extract / material, for use in at least one protein-based assay and / or nuclei acid-based assay.

47. The microfluidic system according to any one of claims 43 to 44, comprising a first said microfluidic platform and a second microfluidic platform, wherein the first microfluidic platform is operatively coupled with the cell mixing module such that a respective said dedicated input / output lines of at least one said cell cultivation unit of the first said microfluidic platform is fluidly coupled to a corresponding said inlet port of the cell mixing module, and wherein the cell mixing module is coupled to the second said microfluidic platform such that the output port is coupled to the at least one inlet of the second said microfluidic platform.

48. The microfluidic system according to claim 47, wherein cells mixed in the cell mixing module are for use in carrying out at least one cell-cell interaction assay.

49. The microfluidic system according to claim 42, wherein the accessory module is in the form of a sensor module, and comprises at least one sensor configured for being selectively operatively coupled to at least one said dedicated input / output line; wherein the at least one sensor is configured for detecting and / or quantifying at least one parameter and / or at least one target molecule of a fluid present in said dedicated input / output line.

50. The microfluidic system according to claim 49, wherein said platform comprises a the gas channel as defined in claims 32 to 32, and further comprising a controller and a gas source including at least one of CO2, O2 and N, wherein the controller is operatively coupled with the said at least one sensor and is configured to operate the gas source to control the flow of said at least one of CO2, O2 and N, to the platform, to provide a desired level of said at least one parameter.

51. The microfluidic system according to claim 50, wherein said at least one parameter comprises pH of the fluid present in said dedicated input / output line.

52. The microfluidic system according to claim 49, wherein said sensor is configured to detect and / or quantify at least one target molecule in the fluid present in said dedicated input / output line, wherein said sensor comprises at least one affinity moiety specific for said target molecule.

53. A cell mixing module, comprising a block defining a cell mixing chamber, an inlet port array, a medium input port, an output port, a pump system, and an air purging system, wherein: the inlet port array comprising a plurality of inlet ports in open fluid communication with the cell mixing chamber; the cell mixing chamber defining a mixing volume in open fluid communication with the medium input port, the output port and the air purging system; the pump system comprising at least one microfluidic pump operative to selectively mix contents of the cell mixing chamber when present; the output port configured for enabling outputting therefrom the contents of the mixing volume; the input port configured for enabling inputting desired media into the mixing volume; the air purging system configured for enabling selectively purging air that may be present in the mixing volume.

54. A method for performing a cell-based assay, the method comprising the steps of: a. applying at least one sample comprising at least one type of cells into a microfluidic platform as defined in any one of claims 1 to 41 ; wherein the cells are applied into at least one of said cell cultivation unit, via the cell channel, such that each of the cell cultivation chambers in each said cell cultivation unit is seeded with the same at least one type of cells; b. providing at least one cell assay reagent / s and / or cell assay material / s via said medium channel into, or from each of said cell cultivation chambers in each of said cell cultivation units, wherein: i. said cell assay reagent / s are applied on cells grown in each of the cell cultivation chambers, via said medium channel; or ii. said cell assay material / s are collected from and / or produced by, the cells in each of the cell cultivation chambers, and transferred via said medium channel of each of said cell cultivation units to the active zone / s formed by the surfaceactivation valves, as defined by any one of claims 27 to 29, in each of said medium channel of said each cell cultivation unit; and c. determining at least one test parameter in each said cell cultivation unit:• on cells of each cell cultivation chambers of (i); or• on cell assay material / s that were collected from and / or produced by, the cells on each cell cultivation chambers of (ii), and reacted with said reaction zone / s.

55. The method according to claim 54, wherein said cell-based assay is a cell viability assay, and wherein said cell assay reagent / s comprise at least one candidate compound or therapeutic agent; said method comprising:(a) exposing cells applied in step (a), in each of the cell cultivation chambers, to at least one candidate compound or therapeutic agent, wherein said compound or agent is provided via said medium channel of each of said cell cultivation units; and(b) determining cell viability and / or cell number in each of the cell cultivation chamber;Wherein the cells in all cell cultivation chambers of each said cell cultivation unit / s are exposed to the same at least one candidate compound or therapeutic agent.

56. The method according to any one of claims 54 to 55, wherein at least one of:(a) said cells are of a subject suffering from at least one pathologic disorder;(b) said pathologic disorder is any one of a malignant proliferative disorder, an inflammatory condition a metabolic condition, an infectious disease, an autoimmune disease, protein misfolding disorder or deposition disorder;(c) said pathologic disorder is a malignant proliferative disorder, and wherein said cells are primary cancer cells of said subject;(d) said candidate active agent is at least one of a chemotherapeutic agent, a biological therapy agent, an immuno therapeutic agent, an hormonal therapy gent or any combination thereof; and(e) said candidate active agent is at least one of Alectinib, Crizotinib, doxorubicin, docetaxel, paclitaxel, methotrexate, and any combinations thereof.

57. The method according to any one of claims 54 to 56, for use in screening for an active agent that affects cell viability and / or at least one cell phenotype, the method comprising the steps of:(a) exposing cells in at least one cell chamber of at least one cell cultivation unit of a microfluidic test platform as defined in any one of claims 1 to 41, to at least one candidate active agent, that is provided via said medium channel of each of said cell cultivation units;(b) determining that said candidate is an agent that affects cell viability and / or phenotype if at least one of: cell viability and / or at least one cell phenotype is modulated as compared with the cell viability and / or at least one cell phenotype in the absence of said candidate active agent.

58. The method according to claim 57, wherein at least one of:(a) said candidate active agent is at least one of: an inorganic or organic molecule, a small molecule, a nucleic acid-based molecule, an aptamer, a polypeptide, or any combinations thereof;(b) said cells form aggregates and / or clusters in said cell chamber, prior to exposure to said candidate agent;(c) said cells are cells of a subject suffering from a pathologic disorder;(d) said pathologic disorder is any one of a malignant proliferative disorder, an inflammatory condition a metabolic condition, an infectious disease, an autoimmune disease, protein misfolding disorder or deposition disorder;(e) said pathologic disorder is a malignant proliferative disorder, and wherein said cells are primary cancer cells of said subject;(f) said malignant proliferative disorder is any one of carcinoma, melanoma, lymphoma, leukemia, myeloma and sarcoma;(g) wherein cell viability is determined by using at least one cell-impermeant DNA-binding dyes and nuclear staining;(h) said candidate active agent is at least one of a chemotherapeutic agent, a biological therapy agent, an immuno therapeutic agent, an hormonal therapy gent or any combination thereof; and(i) said candidate active agent is at least one of Alectinib, Crizotinib, doxorubicin, docetaxel, paclitaxel, methotrexate, and any combinations thereof.

59. The method according to any one of claims 57 to 58, for screening for an anti-cancerous drug, the method comprising the steps of:(a) exposing cancer cells in at least one cell chamber of at least one cell cultivation unit of said a microfluidic platform, to at least one candidate active compound;(b) determining for the exposed cells of (a), cell viability, for at least one time interval; and(c) determining that said candidate drug is an anti-cancerous drug if cell viability is reduced as compared with the cell viability in the absence of said candidate active agent.

60. The method according to any one of claims 54 to 56, for assessing responsiveness of a subject suffering from a pathologic disorder to a treatment regimen comprising at least one therapeutic active agent, and optionally for monitoring disease progression, the method comprising the steps of:(a) exposing cells of said subject applied on at least one cell chamber of at least one cell cultivation unit of said microfluidic platform according to any one of claims 1 to 41, to said therapeutic active agent, wherein the therapeutic active agent is provided via said medium channel of each of said cell cultivation units;(b) determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one-time interval; and(c) classifying said subject as:(i) a responsive subject to said treatment regimen, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with at least one of the cell viability and / or at least one cell phenotype in the absence of said therapeutic active agent; or(ii) a drug-resistant subject if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with at least one of the cell viability and / or at least one cell phenotype, in the absence of said active agent; thereby predicting, assessing and monitoring responsiveness of a mammalian subject to said treatment regimen, optionally, wherein said monitoring disease progression further comprises the steps of:(d) repeating steps (a) and (b), to determine at least one of, cell viability and / or at least one cell phenotype for at least one cell of at least one more temporally-separated sample of said subject; and(e) predicting and / or determining drug-resistance and / or reduction in drug effectiveness in said subject, if at least one cell of said at least one temporally separated sample, displays loss of the modulatory effect of said therapeutic active compound on at least one of, cell viability and / or at least one cell phenotype.

61. The method according to any one of claims 54 to 56, for determining a personalized treatment regiment for a subject in need, the method comprising:(a) exposing cells of said subject applied on at least one cell chamber of at least one cell cultivation unit of said microfluidic platform according to any one of claims 1 to 41, toat least one therapeutic active agent provided via said medium channel of each of said cell cultivation units;(b) determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one time interval;(c) classifying said subject as:(i) a responsive subject to said treatment regimen, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with at least one of the cell viability and / or at least one cell phenotype in the absence of said candidate active agent; or(ii) a drug-resistant subject if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with at least one of the cell viability and / or at least one cell phenotype in the absence of said therapeutic active agent; and(d) administering to a subject classified as a responder, an effective amount of said therapeutic active agent, or any compositions thereof.

62. The method according to claim 61, wherein said subject is and / or was subjected to a treatment regimen comprising said therapeutic active agent, and is monitored for disease progression, the method comprising the steps of:(a) exposing cells of said subject applied on at least one cell chamber of at least one cell cultivation unit of said microfluidic platform according to any one of claims 1 to 41, to said at least one therapeutic active agent provided via said medium channel of each of said cell cultivation units, wherein said cell sample is obtained after the initiation of said treatment regimen;(b) determining for the exposed cells of (a), cell viability and / or at least one cell phenotype, for at least one time interval;(c) determining at least one of:(i) loss of responsiveness, and / or drug-resistance of said subject, if at least one of, cell viability and / or at least one cell phenotype is not modulated as compared with the cell viability and / or at least one cell phenotype in the absence of said candidate active agent; or(ii) responsiveness or maintained responsiveness of said subject, if at least one of, cell viability and / or at least one cell phenotype is modulated as compared with the cell viability and / or at least one cell phenotype in the absence of said candidate active agent; and(d) ceasing a treatment regimen comprising said therapeutic active agent of a subject displaying disease relapse and / or loss of responsiveness, and / or drug-resistance; or maintaining said treatment regimen of a subject displaying responsiveness or maintained responsiveness.

63. The method according to claim 54, wherein said cell-based assay is a target recognition assay to detect and / or quantify at least one target molecule, and wherein said cell assay material / s, comprise material and / or components produced by the cells and / or materials and / or components produced from the cells, in each said cell cultivation chamber; and wherein the method comprises the step of:(a) transferring via said medium channel of each of said cell cultivation units to the active zone / s formed by the surface activation valves, as defined by any one of claims 27 to 29, said cell assay material / s produced by the cells and / or materials and / or components produced from the cells;(b) contacting said cell assay material / s with at least one affinity moiety within each said active zone in each said medium channel; wherein said at least one affinity moiety is specific for said at least one target molecule, and wherein upon recognition, the target binds said affinity moiety;(c) determining the presence and / or the quantity of said target, if an end-point indication that reflects recognition and / or binding of the target molecule to the affinity moiety, is detected.

64. The method according to claim 63, wherein at least one of:(a) said at least one affinity moiety comprises at least one antibody specific for said at least one target molecule, or any antigen-binding fragments thereof;(b) wherein said material / s produced by the cells, or produced from the cells comprises at least one protein-based molecule;(c) wherein said at least one target molecule is at least one protein product produced by the cells in each said cell cultivation chamber / s; and(d) wherein said at least one target molecule is at least one protein product produced by, and secreted by the cells in each said cell cultivation chamber / s.

65. The method according to claim 63, wherein at least one of:(a) said at least one affinity moiety comprises at least one nucleic acid-based affinity molecule;(b) wherein said at least one nucleic acid-based affinity molecule comprises at least one of: at least one primer, at least one probe, and / or at least one nucleic acid aptamer;(c) wherein said material / s produced by the cells, or produced from the cells comprises at least one protein-based molecule and / or a nucleic acid-based molecule;(d) wherein said material / s produced from the cells comprises at least one nucleic acid-based molecule, and wherein said at least one target molecule is or comprises at least one nucleic acid target molecule;(e) wherein said at least one nucleic acid-based affinity molecule is at least one primer and / or prob; wherein said at least one primer and / or probe comprise at least one nucleic acid sequence complementary to at least one nucleic acid sequence within said at least one nucleic acid target molecule;(f) wherein preparation of said nucleic acid-based or protein-based material from the cells in each said cell cultivation chamber is performed using at least one extraction reagent; and(g) wherein said at least one extraction reagent is provided to each said cell cultivation chamber, via said at least one cell channel.

66. The method according to any one of claims 63 to 65, wherein prior to performing said at least one target-recognition assay, the cells in each of the cell cultivation chambers, are exposed to at least one compound or therapeutic agent, said compound or agent is provided via said medium channel of each of said cell cultivation units.

67. A method for performing a cell-cell interaction assay, the method comprising the steps of:(a) applying at least one sample comprising at least two types of cells into a first microfluidic platform of a microfluidic system as defined in any one of claims 42 to 44, 47 to 52, wherein the cells are applied into at least one of said cell cultivation unit, via the cell channel, wherein each of said cell cultivation unit is applied with one type of cells, such that the cell cultivation chambers in each said cell cultivation unit is seeded with the same one type of cells;(b) counting the number of the cells in each of the cell cultivation chambers in each said cell cultivation unit;(c) transferring cells from at least two cell cultivation units, each in a desired cell number, to said cell mixing module via dedicated input / output lines of at least one said cell cultivation unit of the first said microfluidic platform, wherein said at least two cell cultivation units comprise at least two different type of cells, and wherein a desired number of cells from each type of cells is transferred to the mixing module, each from one or more said cell cultivation unit comprising said cell type;(d) mixing said at least two types of cells, each in a desired number to obtain a cell mixture comprising at least two types of cells in a predetermined ratio; and(e) transferring the cell mixture obtained in step (d), from said cell mixing module via the output port that is coupled to the at least one inlet of the second said microfluidic platform of said system, such that each of the cell cultivation chambers in each of said cell cultivation units is applied with a mixture of at least two types of cells at a predetermined ratio; and(f) performing at least one cell-cell interaction assay in at least one of said cell cultivation units of said second microfluidic platform of said microfluidic system.

68. The method according to claim 67, wherein the cell-cell interaction assay performed in step (f) in said second microfluidic platform, is at least one killing assay, wherein at least one of said at least two types of cells is effector cells and wherein at least one of said at least two types of cells is target cells.

69. The method according to claim 68, wherein said killing assay comprise the step of:(i) incubating a cell mixture comprising at least one effector cell and at least one target cell in a predetermined ratio, in each cell cultivation chamber of each cell cultivation unit; and(ii) performing a cell viability assay to determine the number of the target cells in each of said cell cultivation chambers.

70. The method according to claim 67, wherein the cell-cell interaction assay performed in step (f) in said second microfluidic platform, is at least one cell activation assay, wherein at least one of said at least two types of cells is effector cells and wherein at least one of said at least two types of cells is target cells.

71. The method according to claim 70, wherein said activation assay comprises detection and / or quantification of at least one target molecule produced by said effector cells in response to interaction with said target cell, wherein said at least one target molecule is detected and / or quantified by a target recognition assay as define in any one of claims 63 to 65.

72. A method for performing nucleic acid-based assay and / or a protein-based assay in a population of cells comprising at least one type of cells, the method comprising the steps of:(a) applying at least one sample comprising at least one type of cells into a first microfluidic platform of a microfluidic system as defined in any one of claims 42 to 46, and 50 to 52, wherein the cells are applied into at least one of said cell cultivation unit, via the cell channel, wherein each of said cell cultivation unit is applied with at least one type of cells, such that the cell cultivation chambers in each said cell cultivation unit is seeded with the same at least one type of cells;(b) transferring cells from one or more cell cultivation units, to said cell mixing module via dedicated input / output lines of at least one said cell cultivation unit of the first said microfluidic platform;(c) mixing said at least one type of cells, and subjecting the cell mixture to at least one protein preparation procedure and / or at least one nucleic acid preparation procedure; and(d) applying the nucleic acid preparation and / or protein preparation obtained in step (c) on at least one protein-cased and / or nucleic acid-based assay.

73. A method for detecting and / or quantify at least one target molecule in at least one sample comprising at least one type of cells, the method comprising:(a) applying at least one sample comprising at least one type of cells into a first microfluidic platform of a microfluidic system as defined in any one of claims 42, 49 and 52, said system comprises at least one microfluidic platform and at least one sensor, wherein the cells are applied into at least one of said cell cultivation unit, via the cell channel, wherein each of said cell cultivation unit is applied with at least one type of cells, such that the cell cultivation chambers in each said cell cultivation unit is seeded with the same at least one type of cells;(b) transferring cells from one or more cell cultivation units, to said sensor module via dedicated input / output lines of at least one said cell cultivation unit of the first said microfluidic platform, wherein said sensor is configured to detect and / or quantify at least one target molecule in the fluidpresent in said dedicated input / output line, said sensor comprises at least one affinity moiety specific for said target molecule; and(c) determining the presence and / or the quantity of said target, if an end-point indication that reflects recognition and / or binding of the target molecule to the affinity moiety, is detected.

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