An open-top microphysiological system for use in 2d and 3D cell and tissue cultures and the operating method of this system

Abstract

The invention relates to an open-top microphysiological system (MPS) for use in 2D and 3D cell and tissue cultures, in which the local responses of cells to gradients and other cells can be determined, and to an operating method of this system.

Description

[0001] AN OPEN-TOP MICROPHYSIOLOGICAL SYSTEM FOR USE IN 2D AND 3D CELL AND TISSUE CULTURES AND THE OPERATING METHOD OF THIS

[0002] SYSTEM

[0003] Technical Field of the Invention

[0004] The invention relates to an open-top microphysiological system for use in 2D and 3D cell and tissue cultures, where cellular responses to localized gradients and other cells can be determined, and to the operating method of this system. In the open-top system subject to the invention, different contents can be loaded into each channel thanks to a divider with a non-flat cross-section. This divider also prevents delamination that may occur during the loading process. Furthermore, the open-top design of the system allows for direct bioprinting into the channels.

[0005] State of the Art

[0006] Cell cultures are models that enable the growth of cells in the laboratory under various test conditions to investigate a specific biological mechanism or process. The quality of these models and how well they represent the behavior of cells in actual tissue play a significant role in the value of the data produced and how it is used

[0001] . In vitro cell cultures are frequently used to understand the underlying mechanisms of cell behavior, including differentiation, migration, and growth, which are influenced in vivo by biochemical and biomechanical microenvironments. Deciphering the mechanisms behind these behaviors is of great importance for understanding in vivo processes that lead to the formation and function of tissues and organs [2],

[0007] Cell culture studies are applications widely used across various fields, from scientific research to industry. With the increasing number of cell lines, advancements in cell culture techniques have also brought improvements in growth, imaging, data collection, and analysis methods. Although flat, two-dimensional (2D) cell culture is commonly used, recent studies are shifting toward culturing with three-dimensional (3D) structures and more realistic biochemical and biomechanical microenvironments [3]. Traditional 2D cell culture relies on cells adhering to a flat surface — typically a glass or polystyrene Petri dish — to provide mechanical support [4], Cell growth in 2D layers allows for equal access to nutrients and growth factors present in the culture medium, resulting in homogeneous growth and proliferation

[0008] [5]. However, 2D cell cultures used as an in vitro testing method fall short in mimicking human physiology.ln 2D culture media, components such as oxygen and nutrients are intended to diffuse into the cells. In reality, however, cells receive components like oxygen and nutrients under flow, and under chemical and mechanical stress. Additionally, when cultured in Petri dishes, cells lose some of their properties. Moreover, in 2D cultures, in v / vo-like cell-cell interactions are absent, and administered drugs interact directly with the cells. Therefore, to overcome these limitations, 3D cell culture platforms that better mimic in vivo conditions are being employed.3D cell culture is a model system in which cell aggregates form as tissue spheroids or embedded cells within a scaffold that mimics the extracellular matrix (ECM) of living tissues, containing structural proteins and other biological molecules — or using liquid-based methods. 3D cell culture approaches, which aim to model in vivo interactions of tissues and organs, also enable the investigation of biochemical and biomechanical signals. Additionally, drug discovery, cell growth, cytotoxicity, genotoxicity, and studies on protein and gene expression are among the key areas where 3D cell culture systems are frequently used.

[0009] Current fundamental research in human biology and the development of therapeutic drugs are generally based on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately reflect the structure, function, and physiology of living tissues, nor the highly complex and dynamic three- dimensional (3D) environments found in vivo. In contrast, microfluidic technology offers microscale complex structures and well-controlled parameters to mimic the in vivo environment of cells. Moreover, the combination of microfluidic technology with 3D cell culture presents significant potential for in v / vo-like, tissue-based applications such as organ-on-a-chip systems used in current techniques [6].

[0010] Additionally, microfluidic systems require only small sample volumes and consume minimal amounts of reagents, which significantly reduces costs in bioanalysis, drug discovery, and development. Organ-on-a-chip devices are microfluidic cell culture systems that model aspects of organ-level functionality by mimicking the microenvironmental features of tissues, including three-dimensional geometries and biophysical stimuli, and the main goal of this technology is to develop effective microphysiological models for investigating physiological events that characterize the interactions between organs, the immune system, and exogenous stimuli such as pharmaceuticals and nutraceuticals in both healthy and diseased states. These biomimetic models can be achieved by reconstructing the key structure and functions of a specific human tissue or an in vitro functional organ network [7], and these systems are typically fabricated using lithographic or molding processes with materials like polydimethylsiloxane (PDMS) and glass, which represent common manufacturing substrates due to their optical properties that support live-cell imaging.

[0011] In the prior art, patent application number US10961496B2 relates to a microfluidic device that includes an open-top cavity with structural anchors designed to prevent delamination caused by gel contraction on vertical wall surfaces, and a porous membrane (optionally stretchable) placed over a central microfluidic channel(s). The device is particularly suitable for the growth of cells mimicking dermal layers and enables testing the efficacy of cosmetics and drug candidates (including aerosols), as well as anti-cancer therapeutics. It is also disclosed that the device is suitable for co-cultures and multi-cell-type cultures. Furthermore, the OOC (organ-on-chip) device described in document D1 consists of an upper body segment and a lower body segment. It is stated that the upper and lower body segments can be made from PDMS material. Additionally, the device may optionally include a base such as a glass slide, and for different tissue types, the structural anchors may have different geometries. However, the structural anchors in the referenced device, which are designed to prevent delamination caused by gel contraction on vertical wall surfaces, rely on the device's ability to stretch when vacuum is applied. With the stretching motion, the gel located in the anchors can detach from the device walls. Also, in this system, the mentioned multi-culture is performed with cells grown on both surfaces of the membrane. Furthermore, the vacuum chambers seen adjacent to the anchors do not open into the channel containing biological material. These channels are not culture channels; they are used to enable stretching motion within the system if needed. Therefore, in this invention, cells are cultured in a vertically opposing arrangement, on both sides of the membrane.

[0012] Another patent application in the prior art, W02019153004A1 , describes an in vitro, open-top microfluidic "organ-on-a-chip" device. Here, there is an air-liquid interface medium (3) to support the use of multiple cell types in the cultures. Said device is made from PDMS material. However, since PDMS forms a tight seal with glass and can bond to plastic polymers either reversibly or irreversibly, it is also stated that hybrid devices containing rigid components could be produced. In the mentioned document, cells can be cultured in different channels. By using different designs, cells can be placed in the system with a membrane, hydrogel, or liquid between them. However, in this study, there is no defined separator region between cells. As a result, the applied drug can affect the entire system, not locally, creating a gradient effect.

[0013] The limitations and inadequacies of existing solutions in the current technology, such as the effect of drugs influencing the entire system in microphysiological systems used in cell cultures, the inability to load different contents into each channel within the system, the inability to perform multi-cell cultures in different channels along the horizontal axis, the lack of clear boundaries between different channels, the presence of structures like membranes between channels leading to a non-physiological interface, air entrapment due to separators with flat crosssections between adjacent channels, and the use of multiple layers, necessitate the need for improvements in microphysiological systems.

[0014] Brief Description and Objectives of the Invention

[0015] The invention describes an open-top microphysiological system and its working method for use in 2- and 3-dimensional cell and tissue cultures, where the local responses of cells to gradients and other cells can be determined.

[0016] The objective of the invention is to provide a microphysiological system for use in 2- and 3-dimensional cell and tissue cultures, where different contents can be loaded into each channel. In the system subject to the invention, different contents are loaded into each channel through separators with non-flat cross-sections between adjacent channels.

[0017] Another objective of the invention is to observe the local effect of the tested drug within the microphysiological system. In the system subject to the invention, adjacent channels are clearly defined, and there is an interface between these channels. Due to the height of the interface being smaller than that of the adjacent channels, the applied drug remains effective in the local region, depending on the culture duration.

[0018] Another objective of the invention is to enable direct loading into the channels of microphysiological systems and to prevent delamination during the loading process. In the invention, delamination caused by air entrapment between adjacent channels with separators having non-flat cross-sections is prevented. Additionally, the opentop design of the system allows for direct loading into the channel.

[0019] Description of the Figures

[0020] Figure 1. A top view representation of the 3-channel version of the microphysiological system when it is empty.

[0021] Figure 2. A side view representation of the 3-channel version of the microphysiological system when it is empty.

[0022] Figure 3. Examples of interface profiles in the microphysiological system.

[0023] Figure 4. A top view representation of the 5-channel version of the microphysiological system when it is empty.

[0024] Figure 5. A side view representation of the 5-channel version of the microphysiological system.

[0025] Figure 6. Images and graphs showing the results of the simulation experiments.

[0026] Explanation of References in the Figures

[0027] 1. Side wall

[0028] 2. Bottom surface

[0029] 3. Culture medium

[0030] 4. Geometric interface

[0031] 5. Matrix 6. Cells

[0032] 7. Channel

[0033] K1 : Channel 1

[0034] K2: Channel 2

[0035] K3: Channel 3

[0036] K4: Channel 4

[0037] K5: Channel 5

[0038] Detailed Description of the Invention

[0039] The invention relates to an open-top microphysiological system and its working method for use in 2- and 3-dimensional cell and tissue cultures, where the local responses of cells to gradients and other cells can be determined. In the open-top system subject to the invention, different contents can be loaded into each channel through a separator with a non-flat cross-section. This separator also prevents delamination that could occur during the loading process. Additionally, the open-top design of the system allows for direct bio-printing into the channel.

[0040] The invention relates to an open-top microphysiological system (MPS) for use in 2- and 3-dimensional cell and tissue cultures, comprising at least two geometric interfaces (4) with a non-flat cross-section, at least four side walls (1 ) compatible with the geometric interfaces (4), at least 3 channels (7) whose boundaries are defined by the interface (4) and walls (1 ) where cells (6) and / or culture medium (3) are loaded, and a bottom surface (2) on which the walls (1 ) are positioned. This type of MPS includes at least three channels (7). The geometric interface (4) mentioned can have the structure of a triangle, trapezoid, or any other geometric shape with a non-flat cross-section. The geometric interface (4), side walls (1 ), and bottom surface (2) can be made of glass, polydimethylsiloxane (PDMS), polystyrene (PS), or cyclic olefin copolymer (COC), polyethylene (PE), cyclic olefin polymer (COP), polymethylpentene (PMP), poly(1 -trimethylsilane), poly(1 -trimethylsilane-1 - propyne) (PTMSP), or (di-phenylacetylene) methylated polymers, metals, ceramics, or combinations thereof. The mold containing at least two geometric interfaces (4) with a non-flat cross-section can be joined to the bottom surface (2) using pressure, temperature, chemical bonding, etc. techniques. The entire MPS can be produced from the same material, or the bottom surface (2) and other parts may be made from different materials. The mold used for the MPS or its casting can be produced using 3D printers, micro-injection molding, vacuum molding, or a combination of these methods. During use, the MPS can be temporarily or permanently closed with a thin film or another piece, or used with a cap.

[0041] In an embodiment of the invention, the subject microphysiological system is a three- channel MPS, where cells (6) and / or culture medium (3) are loaded. In the case where the system consists of three channels, named Channel 1 , Channel 2, and Channel 3 (Figure 1 ), cells (6), patient samples, spheroids, or organoids are added to the middle channel (Channel 2) within the matrix or culture medium (3). Then, cell-based or acellular culture medium (3) is added to the side channels (Channel 1 and Channel 3), and the MPS operates under static conditions or flow conditions on a rotator. For example, to vascularize brain organoids, brain organoids are loaded into the middle channel (Channel 2) in hydrogel and mixed with endothelial cells, while endothelial cells are loaded into the side channels (Channel 1 and Channel 3). After endothelial cells are added to the side channels, the OOC is tilted at an angle to allow the endothelial cells to accumulate at the interface between the middle channel (Channel 2) and the side channels. For flow conditions, the MPS is placed on a rotator. The rotator is moved up and down at desired angles along the long axis of the MPS to facilitate the movement of liquids between K1 and K3. The MPS is incubated in a cell culture incubator that provides conditions suitable for the cells (6) it contains, without the rotator for static conditions and on the rotator for flow conditions. In this application, drugs that increase or decrease angiogenesis can be added to all channels, and their effects can be studied. In another application, a scaffold mimicking bone and the relevant cells (6) are placed in the middle channel (Channel 2), and culture medium (3) is added to the side channels. Bone regeneration is studied under static or flow conditions. In this embodiment, drugs that increase or decrease ossification can be added to all channels, and their effects can be studied.

[0042] In another embodiment of the three-channel MPS, a cell-based or acellular matrix is added to the middle channel (K2), while a culture medium (3) providing chemical gradients is added to one side channel (K1 ), and the cells (6) to be studied for chemical orientation are added to the other side channel (K3) in plain culture medium (3). The migration of the cells (6) to the middle channel can be microscopically examined at desired time points. In this embodiment, drugs that increase or decrease cell migration can be added to all channels, and their effects can be studied.

[0043] In another embodiment of the three-channel MPS, a cell-based matrix is added to the middle channel (K2), a culture medium (3) providing chemical or biological gradients is added to one side channel (K1 ), and plain culture medium (3) is added to the other side channel (K3). The migration of cells (6) to the right or left channels (K1 , K3) can be compared microscopically at desired time points. In this embodiment, drugs that increase or decrease cell migration can be added to all channels, and their effects can be studied.

[0044] The operating method of the microphysiological system subject to the invention includes the following steps: i. Loading cells (6) into the K2 channel within a matrix and / or culture medium (3), ii. Loading cell-based or acellular culture medium (3) into the K1 and K3 channels, iii. Incubating the MPS in a cell culture incubator for at least one day, iv. Determining the behavior of the cells (6) using microscopy and / or spectroscopy.

[0045] In the method related to the invention described above, the cells (6) mentioned in step (i) may be cell lines, cells from healthy or diseased individuals, spheroids, organoids, animal cells, microorganisms, or plant cells. Additionally, the culture medium mentioned may contain drugs and / or chemokines and / or cytokines that direct the orientation of the cells (6). Furthermore, the cellular behaviors referred to in step (iv) may include viability, movement, clustering, or similar characteristics. For example, when endothelial cells are used in step (i), the behavior to be examined in step (iv) may be vascularization. In an embodiment of the invention, for the five-channel microphysiological system (MPS), the two outermost channels (K1 and K5) of the mentioned channels contain culture medium (3), the central channel (K3) contains a cell-free matrix, and the two adjacent channels to the central one (K2 and K4) contain cells (6) to be studied within a matrix. In this setup, the cell-free matrix is first added to the central channel (K3) and polymerized at 37 °C. Alternatively, collagen, laminin, agarose, polyacrylamide, biocompatible matrices, biological or synthetic gels, or combinations of these can be used instead of a cell-free matrix. In another embodiment of the invention, the central channel (K3) contains only the culture medium (3). The cells (6) are prepared at the desired density and mixed with an equal volume of matrix, then loaded into the two adjacent channels (K2 and K4) on either side of the central channel (K3). Finally, all channels are filled with culture medium (3). If separation of the cell-laden or cell-free matrix from the MPS walls is observed during the culture period, the MPS is first coated with aminopropyltriethoxysilane (APTES). To prevent culture medium loss due to evaporation in the MPS, cell culture is performed in a humid environment within glass petri dishes. If excessive evaporation is observed in the culture medium (3), additional covers made from thin PDMS sheets or 6 cm petri dish lids are used for the MPS. With this method, the effects of one drug on cells (6) in K2 and another drug on cells (6) in K4 can be studied simultaneously. Additionally, in this method, drug applications can be performed from the start, or one drug may be applied to all channels while another drug is applied only to specific channels. This allows the investigation of both general and localized drug applications. In another application of the invention, for the operation of the 5-channel microphysiological system, cellladen or cell-free matrix (5) is first loaded into channels K2 and K4 without filling the entire volume. Then, the remaining volumes of K2 and K4 along with channels K1 and K5 are filled with culture medium (3). Cells (6) intended for directional study are loaded into the K3 channel, and the migration of these cells (6) toward channels K2 and K4 is microscopically compared at desired time points.

[0046] Industrial Applicability of the Invention

[0047] The invention relates to an open-top microphysiological system for use in 2D and 3D cell and tissue cultureallowing the determination of cellular responses to localized gradients and other cells and the operating method of this system, and is industrially applicable.

[0048] The invention is not limited to the above description, and a person skilled in the art can easily come up with different embodiments of the invention. These should be considered within the scope of the protection claimed by the claims.

[0049] REFERENCES

[0050] [1] Knight E, Przyborski S. Advances in 3D cell culture technologies enabling tissuelike structures to be created in vitro. Journal of anatomy. 2015;227(6):746-756

[0051] [2] Huh D, Hamilton GA, Ingber DE. From 3D cell culture to organs-on-chips. Trends in cell biology. 2011 ;21 (12):745-754.

[0052] [3] Duval K, Grover H, Han L-H, Mou Y, Pegoraro AF, Fredberg J, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology. 2017;32(4):266-277.

[0053] [4] Freshney R. Culture of Animal Cells: A Manual of Basic Technique: Wiley-Liss; 2005.

[0054] [5] Edmondson R, Broglie JJ, Adcock AF, Yang L. Threedimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay and drug development technologies. 2014; 12(4):207-218.

[0055] [6] Li XJ, Valadez AV, Zuo P, Nie Z. Microfluidic 3D cell culture: potential application for tissue-based bioassays. Bioanalysis. 2012 Jun;4(12):1509-25. doi: 10.4155 / bio.12.133. PMID: 22793034; PMCID: PMC3909686.

[0056] [7] Yildiz-Ozturk, E. ve Yesil-Celiktas, O. 2015. “Diffusion phenomena of cells and biomolecules in microfluidic devices”, Biomicrofluidics, vol. 9, no. 052606, p.1932-

Claims

CLAIMS1. An open-top microphysiological system (MPS) for use in 2D and 3D cell and tissue culture, characterized by comprising:• at least two geometric interfaces (4) with non-flat cross-sections,• at least four sidewalls (1 ) conforming to the geometric interfaces (4),• at least 3 channels (7) defined by the interfaces (4) and walls (1 ), into which cells (6) and / or culture medium (3) are loaded,• a bottom surface (2) on which the walls (1 ) are positioned.

2. The system according to Claim 1 , characterized in that the mentioned geometric interface (4) has a structure of any geometric shape with a nonflat cross-section.

3. The system according to Claims 1 and 2, characterized in that the said geometric interface (4), the mentioned sidewall (1 ), and the mentioned bottom surface (2) are made of glass, polydimethylsiloxane (PDMS), polystyrene (PS), or cyclic olefin copolymer (COC), polyethylene (PE), cyclic olefin polymer (COP), polymethylpentene (PMP), poly(1 -trimethylsilyl), poly(1 -trimethylsilyl-1 -propyne) (PTMSP), or (di-phenylacetylene) methylated polymer, metals, ceramics, or any combination thereof.

4. An operating method of an open-top microphysiological system for use in 2D and 3D cell and tissue culture, characterized by comprising the steps of: i. Loading cells (6) into the K2 channel within a matrix and / or culture medium (3), ii. Loading cell-containing or cell-free culture medium (3) into the K1 and K3 channels, iii. Incubating the MPS in a cell culture incubator for at least one day, iv. Determining the behavior of the cells (6) using microscopy and / or spectroscopy.

5. The method according to claim 4, characterized in that the cells (6) mentioned in step (i) are cell lines, cells from healthy or diseased individuals, spheroids, organoids, animal cells, microorganisms, or plant cells.

6. The method according to claim 4, characterized in that the culture medium (3) mentioned contains drugs and / or chemokines and / or cytokines that guide the orientation of the cells (6).

7. The method according to claim 4, characterized in that the cell behavior mentioned in step (iv) includes viability, motility, or clustering.

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