A method to independently analyze multiple biological processes in encapsulated 3D cell co-cultures

Abstract

The present invention relates to a multiplexed and encapsulated 3D cell co-culture drug testing or screening method. The present invention also discloses an in vitro drug testing kit suitable for testing the effect of one or more drugs of interest on multiple biological processes in one or more target cell types.

Description

[Technical field]

[0001] The present invention relates to a drug testing or screening method in multiplexed and encapsulated 3D cell co-cultures. The present invention also discloses an in vitro drug testing kit suitable for testing the effect of one or more drugs of interest on multiple biological processes in one or more target cell types. [Background technology]

[0002] In vitro screening or testing of molecular substances in living cells constitutes an important procedure used in the development of novel pharmaceuticals and, more recently, during "precision medicine" testing to define the best drug to treat patients. In vitro molecular screening can be used in the early stages of drug discovery to identify "hit" molecules or, at a later stage, to define a set of "lead" substances from hit molecules defined as hit molecules with multiple advantageous properties. Due to the large number of chemicals to be screened, in vitro drug testing is very costly in time and resources. Despite these large investments, in vitro drug testing is not always successful in defining good candidates with the desired biological effect for many reasons, mainly due to the low clinical relevance of the cell cultures used. Typical monolayer cell cultures used in drug screening do not reflect 3D tissue physiology, the diversity of cell subsets or the multi-organ interactions that occur in vivo. Moreover, drug screening is usually performed using a single experimental readout or a maximum of two readouts per test. Thus, multiple rounds of screening focusing on different properties of the molecules are required to identify drugs with the best overall drug development profile. For example, drug screening typically begins with an evaluation of biological activity on target cells, followed by additional rounds of screening to identify hepatotoxicity or neurotoxicity, including metabolites that are only produced in the presence of hepatocytes. Important physiological interactions also exist in vivo, such as the metabolism of prodrugs, such as tamoxifen, by the liver for activation in other tissues, in the case of tamoxifen, in the breast. In particular, hepatocytes are often not present in the original drug screen, or if present, may be in direct contact with other cell types, which does not reflect in vivo cell biology. Not including these important physiological interactions between cell types may lead to missing efficient therapies in vivo.Thus, a problem in the current drug screening landscape, where multiple tests are required to evaluate various properties and effects of potential drug candidates, is the lack of methods that can simultaneously address all of the above phenomena in cell culture drug testing. Specifically, improved drug screening or testing methods would have the ability to multiplex the evaluation process and utilize more physiologically relevant cell culture systems.

[0003] A distinction can be made between target-based and phenotypic drug screening. Target-based drug screening approaches rely on the prior identification of specific, often unique, molecular pathways, receptors or other biomolecules that can be targeted to achieve the desired biological effect on the cells of interest. For example, estrogen receptors in (tumorous) breast-derived cells have been demonstrated to be viable targets for endocrine therapy.

[0004] Experimental cell lines can be engineered to express an estrogen-dependent reporter, making the effect of drugs on this receptor easier to detect or study in vitro. In contrast, phenotypic screening does not depend on prior knowledge of the exact molecular pathway or cellular target that a drug should act on to achieve the desired biological effect in the cells of interest. In phenotypic screening, common biological processes such as cell death, cell proliferation, cytotoxicity and immune cell activation can be detected using genetic or protein markers established for these common phenotypic changes. The exact molecular partners targeted by drugs identified using phenotypic screening remain unknown until additional testing is performed, which is beyond the scope of this patent.

[0005] Previous attempts to improve in vitro drug screening and testing have only partially addressed the aforementioned problems, and to the applicant's knowledge have never simultaneously addressed all of the aforementioned problems. For example, European Patent No. 1235935B1 (Rosetta Inpharmatics LLC) describes the use of a "multiplexed" (multiple per cell) gene reporter cell culture system to identify drugs that inhibit the function of target genes related to cell fitness, i.e., target-based drug screening. Although multiple genes can be detected after co-culture of different cell types, the teachings of that literature are not extended to 3D spheroid cell cultures, and the ability to independently analyze various types of co-cultured cells is not possible.

[0006] A multiplexed phenotypic drug test has also been published previously (see PMID 20116850), showing the possibility to study drug-induced modulation of a large panel of proteins in cell supernatants. Again, this drug testing methodology does not include the use of 3D cell cultures such as spheroids to improve the physiological relevance of the system. Furthermore, it is not possible to detect in which cells biological processes are modulated, as the assay readout consists of values ​​of soluble proteins.

[0007] There is an increasing number of prior art and academic publications related to cell culture systems with improved physiological relevance, particularly 3D spheroid cultures. For example, European Patent No. 2491386B1 (Plasticell Ltd) describes cell culture in multiple microcapsules, each labeled with two or more labels, either in the encapsulant or inside the capsule. This document also includes "split pool cell" or "combined cell" cultures, which are primarily aimed at defining how cells of the same type but with different differentiation states have different phenotypic or genotypic characteristics from each other, such as different proliferation capabilities or expression of specific genes or proteins. Similarly, Korean Patent No. 101726063B1 (UNIV SUNGKYUNKWAN RES&BUS [KR]) describes the labeling of liposomes (lipid bilayer like capsules) with various fluorescent dyes, which are used to encapsulate 3D tumor cell cultures. These documents are similar to the present technology in that different fluorescent dyes (quantum dots) can be used to label the capsules, but neither discloses a means to detect multiple biological processes per test, nor does it disclose that the different capsules can be easily distinguished by microscope, as liposomes are mainly used for genetic diagnosis of tumors. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] European Patent No. 1235935B1 [Patent Document 2] European Patent No. 2491386B1 [Patent Document 3] Korean Patent No. 101726063B1 [Non-patent literature]

[0009] [Non-Patent Document 1] PMID20116850 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention aims to improve in vitro drug testing, i.e., the screening of new or previously approved chemical or biological agents, to identify treatments that have the desired activity in at least two target cells and the fewest adverse side effects. These improvements include (1) improving translational relevance by using co-cultures of multiple cell types that better represent a multi-tissue organism, (2) improving the amount and quality of information that can be obtained from the test by increasing the number of biological processes or readouts and the number of different cell types that can be independently analyzed in the test, and (3) improving the effectiveness of drug testing by reducing the time, cost, and amount of testing that needs to be performed in vivo, all of which are relevant attributes in the shortlisting of better drug candidates. The present invention, as a result of the features described in items (1) and (2), allows for improved selection of drug candidates to proceed more reliably to the subsequent in vivo testing stage.

[0011] It is an object of the present invention to provide an in vitro method suitable for drug testing that is capable of analyzing multiple biological processes in one or more cell types, the method comprising: a) expressing in each of said one or more cell types analyzed in the same in vitro drug testing assay at least one fluorescent or bioluminescent reporter gene for each biological process analyzed; b) encapsulating each of said one or more cell types to be analyzed in the same in vitro drug testing assay with an alginate biopolymer, and in the case of multiple cell types, labeling all or all but one of the capsules with a different type of fluorophore, respectively; step c) of optionally co-culturing all encapsulated cell types of step b) that are to be analyzed in the same in vitro drug testing assay with one or more additional unlabeled cell types, optionally encapsulated in labeled or unlabeled alginate biopolymer; Optionally, step d) exposing said co-culture to one or more drugs; e) measuring the activity of said multiple biological processes in each cell type analyzed in the same in vitro drug testing assay of step c) by analyzing the fluorescence or bioluminescence intensity of each fluorescent or bioluminescent reporter gene individually by optical imaging before and / or after exposure to said optional one or more drugs to be tested. When multiple cell types are co-cultured, each cell type being analyzed is identified by optical imaging based on the type of fluorophore label associated with the alginate capsule containing the cell type being analyzed.

[0012] Another object of the present invention is to provide an in vitro drug testing kit suitable for testing the effect of one or more drugs of interest on multiple biological processes in one or more target cell types, the kit comprising: At least one ready-to-use microwell plate containing one or more alginate-encapsulated target cell types expressing a fluorescent or bioluminescent reporter gene for each biological process to be analyzed in said one or more target cell types, where in the case of multiple cell types, all or all but one of the capsules are each labeled with a different type of fluorophore.

[0013] Other objects and advantages of the present invention will become apparent to those skilled in the art from a review of the following detailed description, taken in conjunction with the following illustrative drawings and the appended claims. [Brief description of the drawings]

[0014] [Figure 1]Pharmacological inhibition of estrogen receptor alpha (ERα) by tamoxifen (Tam) versus a tamoxifen metabolite (4-hydroxytamoxifen, 4OHT) in breast cancer (BC) cells co-cultured with liver cancer (LC) cells (hepatocytes). BC cells stably expressing luciferase under the control of estrogen receptor alpha (ERα) were cultured in the presence or absence of LC cells and treated with increasing concentrations of Tam or one of its metabolites, 4-OHT, for 48 hours. BC cells were lysed and bioluminescence was measured to determine ERα activity, which can be considered as a readout for the pre-cancerous activity of BC cells. The graph shows the percentage of ERα activity as a function of Log[inhibitor concentration], taking the activity of ERα without inhibitor as 100%. Data represent three independent repeats. [Diagram 2] Quantification of two cell proliferation states (active proliferation and latency) of two different cell types (BC and LC) in co-culture is shown. Proliferation of co-cultures of 3D encapsulated BC and LC cells genetically engineered to express the dual fluorescent reporter gene FUCCI is shown. BC and LC cells stably expressing the FUCCI reporter (proliferating cells are green and non-proliferating cells are red) were encapsulated in far-red labeled alginate capsules (BC) or unlabeled alginate capsules (LC). BC and LC capsules were mixed to prepare co-cultures and incubated with different concentrations of fetal bovine serum (FBS) for 96 h. The fluorescence intensity of cells and capsules was then measured with an ImageXpress Micro Confocal microscope (Molecular Devices) and processed and analyzed with the software MetaXpress (Molecular Devices). The graph shows the fluorescence intensity (arbitrary units) of the reporter gene corresponding to either BC or LC cells. Data represent three independent replicates. [Diagram 3]Multiple cultures of encapsulated BC cells expressing the dual reporter gene FUCCI, encapsulated BC cells expressing the reporter gene zipGFP-Casp3, and encapsulated LC cells are shown. LC cells were encapsulated in green-labeled alginate capsules, BC cells stably expressing the dual reporter gene FUCCI (actively proliferating cells are green, non-proliferating cells (latent) are red) were encapsulated in unlabeled alginate capsules, and BC cells stably expressing the reporter gene zipGFP-Casp3 (all cells are red (reporter expression control) and turn green upon induction of apoptosis) were encapsulated in far-red labeled alginate capsules. The encapsulated cells were mixed together in a 96-well plate with a cover glass bottom and incubated at 37 °C for 96 h. Image acquisition was performed on an automated confocal microscope and images were annotated directly. [Figure 4]Quantification of BC cell apoptosis treated with increasing concentrations of tamoxifen either in monoculture or in multiplex culture with LC cells. LC cells were encapsulated in green-labeled alginate capsules, BC cells stably expressing the dual reporter gene FUCCI (actively proliferating cells are green and non-proliferating cells (latent) are red) were encapsulated in unlabeled alginate capsules, and BC cells stably expressing the reporter gene zipGFP-Casp3 (all cells are red (reporter expression control) and turn green upon induction of apoptosis) were encapsulated in far-red labeled alginate capsules. Encapsulated cells were mixed together with (multiplex culture) or without (monoculture) LC cells in 96-well plates with a cover glass bottom and treated or not with increasing concentrations of tamoxifen in the micromolar range and incubated at 37 °C for 96 h. Image acquisition was performed with an automated confocal microscope. Cell segmentation and quantification of cell fluorescence intensity were performed with the software MetaXpress (Molecular Devices). For quantification of apoptosis, cell fluorescence intensity from far-red labeled capsules was measured and green fluorescence was normalized to red fluorescence. The graph shows the normalized fluorescence intensity of the zipGFP-Casp3 reporter gene upon treatment with increasing concentrations of tamoxifen. Data represent three independent replicates. [Diagram 5]Quantification of BC cell proliferation treated with increasing concentrations of tamoxifen either in monoculture or in multiplex culture with LC cells. LC cells were encapsulated in green-labeled alginate capsules, BC cells stably expressing the dual reporter gene FUCCI (actively proliferating cells are green and non-proliferating cells (latent) are red) were encapsulated in unlabeled alginate capsules, and BC cells stably expressing the reporter gene zipGFP-Casp3 (all cells are red (reporter expression control) and turn green upon apoptosis induction) were encapsulated in far-red labeled alginate capsules. Encapsulated cells were mixed together with (multiplex culture) or without (monoculture) LC cells in 96-well plates with a coverslip bottom and treated or not with increasing concentrations of tamoxifen in the micromolar range and incubated at 37 °C for 96 h. Image acquisition was performed with an automated confocal microscope. Cell segmentation and quantification of cell fluorescence intensity was performed with the software MetaXpress (Molecular Devices). For proliferation quantification, cell fluorescence intensity from unlabeled capsules was measured. The graph shows the fluorescence intensity (arbitrary units) of the FUCCI dual reporter gene upon treatment with increasing concentrations of tamoxifen. Data represent three independent replicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] To the applicant's knowledge, multiplexed drug testing methods have not been combined with 3D spheroid cultures that simulate solid in vivo tissue structures, nor with co-culture models that aim to recapitulate the multi-organ nature of biological systems such as the human body. Unexpectedly, by combining various cell culture drug testing methods, we obtain unique advantages that are not present in any of the methods that only address one or two of the major limitations of current cell culture drug testing detailed below.

[0016] The advantages of a drug testing method combining multiplexed detection of biological processes with physical separation of different cell types include obvious and simple economic measures of testing costs, labor, and time. In addition, it also includes the advancement of non-obvious scientific concepts inherent to current technology. This includes the ability to identify drugs with potentially different effects on cells of distinct human organs. For example, hepatocyte metabolizing drugs have very strong anti-tumor growth effects while being highly toxic to lung cells at very low concentrations and inducing unexpected inflammatory responses from immune cells. Thus, the ability to simultaneously detect the modulation of multiple biological processes in different cell types allows for unparalleled high content screening of drug activity. Thus, the present invention provides a unique advantage over prior techniques, as it allows for a narrower selection of drug candidates that would have progressed to the next round of the development process without the simultaneous evaluation of biological processes. An additional advantageous feature of the present technology is that it allows for more effective identification of drugs with multiple desired characteristics, such as strong biological activity in the target cells of interest and low toxicity or inflammation of cells from vital organs.

[0017] Encapsulation of cells to prevent direct contact of bacteria or hepatocytes with most other cell types is also advantageous since these cells do not normally come into direct contact with most other cell types in vivo. For example, hepatocytes often metabolize circulating drugs but do not come into direct contact with neurons, lung cells, etc. As mentioned above, most other cell culture systems do not include the means to individually analyze multiple biological processes in separate cell types, making the present technology uniquely positioned to detect the effects of chemicals tested in drug screens on cells from various tissues with the aim of better recapitulating an entire biological system.

[0018] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0019] In case of conflict, the present specification, including definitions, shall prevail.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the subject matter of this specification belongs.As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0020] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0021] The term "comprise" is generally used in an inclusive sense, that is to say permitting the presence of one or more features or components.

[0022] As used herein, the terms "subject" or "patient" are well known in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some embodiments, the subject is a subject in need of treatment or a subject having a disease or disorder. However, in other embodiments, the subject may be a normal subject. The term does not denote a particular age or sex. Thus, adult and neonatal subjects, both male and female, are intended to be covered.

[0023] The term "multiplexed" refers to drug tests that can simultaneously detect multiple biological processes via associated chemical or biological tags.

[0024] The term "spheroid" refers to a roughly spherical 3D aggregate of cells.

[0025] The term "hepatocyte" refers to any primary hepatocyte or any cell line derived from healthy or transformed hepatocytes, including hepatoma (LC) cell lines, etc.

[0026] "Capsule" refers to a polymeric sheet or layer produced by a microfluidics device, typically including the alginate of the present invention used to encapsulate or encase cells. Capsules are known in the art to be a protective barrier surrounding a cell unit. The term is commonly used in the art to refer to semi-permeable or impermeable structures, and in the context of the present invention, microcapsules are semi-permeable, allowing the passage of components of the medium and other reagents, but retain labels and tags to allow identification of the cell unit.

[0027] Microencapsulation or encapsulation is the surrounding of cell units within microcapsules.

[0028] As used herein, the term "culture conditions" refers to the environment in which cells are placed or exposed to promote their growth or differentiation. Thus, the term refers to the medium, temperature, atmospheric conditions, substrate, agitation conditions, etc., that may affect cell growth and / or differentiation. More specifically, the term refers to specific agents incorporated into the culture medium that may affect cell growth and / or differentiation. This includes supernatants or lysates after culture of primary cells, cell lines, or microorganisms, or biological fluids or extracts derived from living organisms.

[0029] As referred to herein, a "cell" is defined as the smallest structural unit of an organism capable of independent function, or a single-celled organism consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semi-permeable cell membrane or cell wall. A cell may be a prokaryote, eukaryote, animal or plant, or archaea. For example, a cell may be a eukaryotic cell. A cell may be natural or modified, such as by genetic engineering or subculture, to achieve desired characteristics. Stem cells are defined in more detail below and are totipotent, pluripotent, or multipotent cells that can give rise to multiple differentiated cell types. Stem cells can be differentiated in vitro to give rise to differentiated cells that are themselves multipotent or can be terminally differentiated. In vitro differentiated cells are cells that have been artificially created by exposing stem cells to one or more agents (agents) that promote cell differentiation.

[0030] The term "cell" is used in its general and broad context to define a cell as either a cell line, a primary cell from an organism or human, or a bacterial, fungal, or plant cell. Properties of encapsulated cell cultures, such as specific pressures applied to encapsulated cells or the formation of 3D tissue-like structures, may favor differentiation or growth of cells that are otherwise difficult to achieve in vitro. For example, encapsulation may promote proliferation of multipotent neural stem cells or chondrocytes and facilitate long-term maintenance of hepatocytes, thus providing additional translational relevance to the proposed drug testing methods.

[0031] A "group" of cell units (or cells) is a plurality of such cell units that are not interconnected. For example, a cell unit is not a cell group, but one or more cells clustered together into one single unit. Single cells and individual cell units may be pooled to form a group of cells or cell units. A group can be divided by splitting the group into two or more groups of cells or cell units.

[0032] The term "drug testing" is a general term that includes drug screening. In accordance with the present invention, drug testing can include screening molecular libraries to identify new chemical entities with desired biological effects, and can include in vitro testing of known or previously approved drugs to identify which drug or drug combination is most appropriate for a patient, or to characterize the mechanism or non-specific effects of new or previously identified drugs.

[0033] One object of the present invention is a method for detecting different biological processes, such as cell proliferation, cytotoxicity, neoplastic growth or arrest, immune cell activation, inflammatory response, antibiotic resistance, drug synergy, in one or more cells grown in 3D cell co-cultures with or without the addition of a test compound for drug testing. The method uses encapsulated cell lines genetically engineered to contain fluorescent reporter genes corresponding to the different biological processes mentioned above, and different fluorescent colors are used for each reporter gene, so that the magnitude readouts of these biological processes can be analyzed separately. Biological activity can also be distinguished between different cell types by encapsulating them in alginate bound to different fluorescent compounds. The method aims to improve drug screening assays by (1) incorporating different cell lines, for example, hepatic cells capable of metabolizing drugs together with breast cancer cells used as cellular drug targets, (2) providing synchronized information on multiple biological readouts in a single test, in particular toxicity to liver or other cells and / or antitumor activity or other drug efficacy metrics, and (3) using 3D cell cultures of target cells to provide additional physiological relevance when testing the efficacy of drugs.

[0034] As used herein, a "label" or "tag" is a means for identifying a cell unit and / or determining the culture condition or set of culture conditions to which the cell unit was exposed. Thus, a label may be a set of labels, each added at a specific culture step, or a label added at the beginning or in an experiment that is modified according to the culture step to which the cell unit is exposed or tracked during the culture step, or simply a location reference from which the culture step used can be inferred. A label or tag may also be an element that reports or records the location or identity of a cell unit at any one time point, or assigns a unique identifier to the cell unit. Examples of labels or tags include molecules of a unique sequence, structure or mass, or fluorescent molecules or objects such as beads, or radio frequency and other transponders, or objects with a unique marking or shape. Since different fluorescent dyes may have overlapping emission spectra, the selection of different tags should be performed so that there is minimal or no overlap in fluorescence between the different fluorescent dyes, or the measurements are corrected using stained control samples, so that the interpretation of the imaging results is not compromised.

[0035] An "identifying label" is a label that allows one to determine the nature of the cell unit to which it is attached. It allows one to record the exposure of a cell unit to various culture conditions by adding an identifying label at each exposure and then deconvoluting by analysis of the label.

[0036] Cells are "exposed to culture conditions" when the cells are placed in contact with a medium or grown under conditions that affect one or more cellular processes, such as the growth, differentiation, or metabolic state of the cells.

[0037] Thus, when the culture conditions include culturing cells in a medium, the cells are placed in the medium for a sufficient period of time to exert an effect.Similarly, when the conditions are temperature conditions, the cells are cultured at a desired temperature.The "pooling" of one or more groups of cell units includes mixing groups to create a single group or pool that includes cell units exposed to multiple backgrounds, i.e., multiple different sets of culture conditions.Pools may be further divided into groups, either randomly or non-randomly, and such groups are not themselves "pools" for the purposes of the present invention, but may themselves be pooled by combination, for example, after exposure to different sets of culture conditions.

[0038] "Cell growth" and "cell proliferation" are used interchangeably herein to refer to an increase in cell number without differentiation into a distinct cell type or lineage. In other words, these terms refer to an increase in the number of viable cells. Preferably, proliferation is not accompanied by an appreciable change in phenotype or genotype.

[0039] "Cell differentiation" is the development of one cell type into a different cell type. For example, bipotent, pluripotent or totipotent cells can differentiate into neural cells. Differentiation may be accompanied by proliferation or may be independent of proliferation. The term "differentiation" generally refers to the acquisition of a mature cell type phenotype from a developmentally undefined cell type, such as a neuron or lymphocyte, but does not exclude transdifferentiation, so that one mature cell type can be transformed into another mature cell type, such as a neuron into a lymphocyte or a monocyte into a macrophage.

[0040] The term "plurality" means more than one. In the context of labels, plurality describes the fact that each encapsulation adds at least one more label, such that multiple encapsulated cell units can be labeled with at least one label per encapsulation. In the context of cell units within a microcapsule, two, three, four, five, six or more cell units can be contained within a single microcapsule.

[0041] The term "phenotypic screen" refers to a type of screen used in biological research and drug discovery to identify substances such as small molecules, peptides, antibodies or RNAi that alter the phenotype of a cell or organism in a desired way.

[0042] Fluorescent reagents are commonly used in life science laboratories to measure the activity of biological processes. For example, the combination of the fluorescent reagents DiOC6 and propidium iodide allows for the detection and quantification of live (green fluorescence) and dead (red fluorescence) cells in a cell population. Dihydroethidium is a fluorescent probe for measuring the production of intracellular reactive oxygen species, an indicator of cellular stress, by turning red after oxidation. Coumarin boronate or fluorescein boronate can be used to measure the cellular production of nitric oxide, involved in many physiological processes including inflammation, by emitting blue or green fluorescence, respectively. Fura-2 is a fluorescent probe for measuring the intracellular concentration of calcium, which fluctuates upon stimulation of signaling pathways.

[0043] The incorporation of such reagents in the present invention helps to broaden the spectrum of biological activities that can be analyzed. However, instead of a reporter gene, the fluorescent reagent indiscriminately reports biological activity in all cells of a cell population. Indeed, the fluorescent reagent stains all cells, since it is a soluble molecule that diffuses freely after being added to the cell culture medium. The fluorescent reagent can be used in combination with a fluorescent or bioluminescent reporter gene to measure additional biological activities that may not be present in the absence of a reporter gene. To do so, the activity of the fluorescent reporter genes must first be measured, since they specifically report biological activity in the cell type in which they are expressed. Then, the addition of the fluorescent reagent can be performed to stain all cells. The relevant biological activity of a specific encapsulated cell type in a cell co-culture can be measured, since the color of the capsule can still be used to measure the fluorescence of the reagent individually. Also, several fluorescent reagents can be used simultaneously to the extent that their fluorescence spectra do not overlap.

[0044] In particular, the present invention aims to provide an in vitro drug testing method capable of analyzing multiple biological processes in one or more cell types, the method comprising: a) expressing in each of said one or more cell types analyzed in the same in vitro drug testing assay at least one fluorescent or bioluminescent reporter gene for each biological process analyzed; b) encapsulating each of said one or more cell types to be analyzed in the same in vitro drug testing assay with an alginate biopolymer, and in the case of multiple cells, labeling all or all but one of the capsules with a different type of fluorophore, respectively; step c) of optionally co-culturing all encapsulated cell types of step b) that are to be analyzed in the same in vitro drug testing assay with one or more additional unlabeled cell types, optionally encapsulated in labeled or unlabeled alginate biopolymer; Optionally, step d) of exposing said co-culture to one or more drugs; e) measuring the activity of said multiple biological processes in each cell type analyzed in the same in vitro drug testing assay of step c) by analyzing the fluorescence or bioluminescence intensity of each fluorescent or bioluminescent reporter gene separately by optical imaging before and / or after exposure to said optional one or more drugs to be tested. When multiple cell types are co-cultured, each cell type to be analyzed is identified by optical imaging based on the type of fluorophore label associated with the alginate capsule containing the cell type to be analyzed.

[0045] In addition to testing drugs, the in vitro methods of the present invention are also suitable for testing biological fluids, including supernatants from cells or tissues, or lysates from cell or microbial cultures.

[0046] Advantageously, although the invention is intended for drug testing, other molecular families with expected or confirmed effects on human health can be tested. For example, the invention can be used to predict the effects of unknown compounds on human health and / or to expand the knowledge of known compounds by selecting the relevant biological activity and cell type to be tested. Other chemicals such as nutrients, cosmetics, pollutants, and exogenous actives can be tested to the extent that they can be added in soluble form to the cell culture medium. The invention can also be used on fluids of undetermined and / or complex composition, such as contaminated liquids (e.g., downstream water from chemical plants or hospitals, etc.) or physiological fluids (e.g., urine, serum, etc.), to determine specific biological activities and their effects on cell types, without the need to identify the molecule or molecules they contain and that are responsible for the measured effect. With such applications, the invention can be used comprehensively as a predictive tool to anticipate the potential harmful and / or beneficial effects of compounds and / or fluids on human health.

[0047] A number of biological activities, also defined as biological processes, can be reported by both fluorescent or bioluminescent reporter genes. Some bioluminescent proteins, called luciferases, can be used as reporter genes. For example, firefly luciferase, Renilla luciferase, and Gaussia luciferase are commonly used as bioluminescent reporter genes. Bioluminescence is measured in a bioluminometer and is often more sensitive than fluorescence. Some reporter genes are only available with bioluminescent reporter genes because they report weak biological activities that are poorly detected by fluorescent reporter genes. Thus, bioluminescent reporter genes are a good alternative to fluorescent reporter genes for measuring biological activities that are poorly detected. Multiplexing with some bioluminescent reporter genes is difficult due to the limited set of bioluminescent colors available, and fluorescent reporter genes are often preferred.

[0048] However, dual reporter systems can be designed using firefly luciferase and Renilla luciferase, allowing the bioluminescence of both bioluminescent reporter proteins to be measured consecutively in the same sample. There is a possibility to combine a fluorescent reporter gene with a bioluminescent reporter gene, but the fluorescence must be measured first before measuring the bioluminescence. Indeed, the bioluminescent signal is very strong and impairs the detection of the fluorescence. The bioluminescence can be easily controlled, since a substrate is required and can be added to the cell co-culture immediately after the fluorescence acquisition. Another possibility is to use a single bioluminescent reporter gene in one cell type in co-culture with the other cell type to measure a specific biological activity, while taking into account possible biological communication between the cell types that may affect the specific biological activity reported (see Example 1).

[0049] The present invention includes the possibility of encapsulating several cell types per capsule. For example, liver capsules can be prepared using hepatocytes along with other cells normally found in the liver to better recapitulate liver function in vitro, and thus metabolize drugs or study hepatotoxicity in a manner that more closely reflects in vivo biology.

[0050] Alternatively, spheroids can be generated that contain cancer cells (tumor cell lines or patient-derived tumor cells) together with immune system or stromal cells (e.g., monocytes, T lymphocytes or endothelial cell lines, or primary cells from the patient) in a single capsule to test whether drugs that can (re)program non-cancerous tumor-associated cells promote tumor regression, e.g., tumor-associated immune cells become more inflammatory or mount a cytotoxic immune response against the tumor, or tumor endothelial cells reduce the secretion of angiogenic factors.

[0051] Therefore, the possibility of using several cell types per capsule is particularly interesting if different cells can be recognized (i.e. one cell type is green and another cell type is red in the same capsule, or one biological activity is read in green and a second biological activity is read in red).

[0052] Preferably, the fluorophores are conjugated to the amine-alginate biopolymer, however, a wide variety of fluorescent dyes with simple covalent conjugation chemistry are known that can replace the amine-based dyes if they have more desirable physicochemical or assay-dependent properties or better cross-compatibility with other fluorescent labels when combining more fluorophores in the same test (e.g., four different fluorescent capsule labels, one unlabeled capsule, and four different reporter gene fluorescent labels), all of which can be precisely distinguished from each other via the specific filter and laser configuration of the imaging microscope.

[0053] In the present invention, said multiple biological processes are selected from the list including cell proliferation, inflammation, tumor growth or inhibition, drug toxicity, angiogenesis, immune stimulation, detoxification response, hormonal response, xenobiotic response, genotoxic stress response and apoptosis.

[0054] In one embodiment of the invention, the one or more cell types are selected for co-culture based on predicted or confirmed physiological interactions between the cell types and / or based on predicted or confirmed effects of the one or more cell types on the one or more drugs being tested.

[0055] For example, predicted or previously described physiological interactions between one or more cell types may include co-culture of hepatocytes with a cancer cell type based on the prediction that the drug may be metabolized by hepatocytes and alter the responsiveness of the drug to the targeted cancer cell type, or co-culture of ovarian cancer cells with breast cancer cells based on the knowledge that ovarian cancer cells produce estrogens that induce proliferation of breast cancer cells, or co-culture of hepatocytes with endometrial cancer cells and breast cancer cells to test an anti-estrogenic drug based on the knowledge that a drug that is an anti-estrogenic drug in breast cancer cells exhibits pro-estrogenic effects in the endometrium, which is a predicted adverse side effect of an anti-estrogenic drug.

[0056] In a preferred embodiment of the invention, said in vitro drug testing method comprises a step d) of exposing said co-culture to one or more drugs.

[0057] In particular, in vitro drug testing methods capable of analyzing multiple biological processes in one or more cell types include a) expressing in each of said one or more cell types analyzed in the same in vitro drug testing assay at least one fluorescent or bioluminescent reporter gene for each biological process analyzed; b) encapsulating each of said one or more cell types to be analyzed in the same in vitro drug testing assay with an alginate biopolymer, and in the case of multiple cell types, labeling all or all but one of the capsules with a different type of fluorophore, respectively; step c) of optionally co-culturing all encapsulated cell types of step b) that are to be analyzed in the same in vitro drug testing assay with one or more additional unlabeled cell types, optionally encapsulated in labeled or unlabeled alginate biopolymer; d) exposing the co-culture to one or more drugs; e) measuring the activity of said multiple biological processes in each cell type analyzed in the same in vitro drug testing assay of step c) by analyzing the fluorescence or bioluminescence intensity of each fluorescent or bioluminescent reporter gene separately by optical imaging before and / or after exposure to said one or more drugs to be tested. When multiple cell types are co-cultured, each cell type to be analyzed is identified by optical imaging based on the type of fluorophore label associated with the alginate capsule containing the cell type to be analyzed.

[0058] In a preferred embodiment, said in vitro drug testing method further comprises the additional step f) of exposing said co-culture to one or more fluorescent reagents to measure one or more additional biological processes.

[0059] Preferably, optical imaging is performed by a microscope and / or a luminometer. If appropriate instrument nozzles are used and the size of the cell capsules is small enough, optical imaging may also be performed by flow cytometry. Using flow cytometry, more fluorescence parameters can be easily analyzed compared to standard imaging microscopes, allowing for better multiplexing of the assay, and can even be analyzed without the use of image analysis software, but a smaller number of capsules per plate well is more suitable for image-based microscopy.

[0060] In another embodiment of the invention, the at least one or more cell types are selected from the group comprising liver cells, tumor cells, brain cells, epithelial cells, endothelial cells, immune system cells, pluripotent stem cells, embryonic stem cells, lung cells, kidney cells, arterial cells, bone cells, chondrocytes, muscle cells, pancreatic cells, intestinal cells, skin cells, fibroblasts, fungal cells or bacterial cell lines or primary tissue cell subsets.

[0061] Preferably, at least one of said one or more cell types analysed in the in vitro drug testing assay, or at least one of the unlabelled cells optionally added in step c), is a hepatocyte.

[0062] In one embodiment of the invention, at least one of the one or more encapsulated cell types is derived from a patient cancer sample.

[0063] In one embodiment of the invention, at least one of the one or more cell types is a bacterial or fungal cell. In the latter case, the drug being tested is an antibiotic or bactericide. In this embodiment, the number of biological processes being analyzed is selected from the list including antimicrobial resistance or susceptibility, bacterial growth, bactericidal or bacteriostatic activity.

[0064] In another embodiment, the in vitro method of the invention is designed for drug screening or testing, and the multiple fluorescent reporter genes are selected for the biological process that is studied during the phenotypic drug discovery process.

[0065] For example, a genetic reporter may be selected because it is a particular target that has been previously identified through mechanistic biology studies, namely a receptor that plays an important role in the pathogenesis of a particular cancer or other particular disease.

[0066] Preferably, the gene reporter and the regulatory element are flanked by CTCF insulator sequences.

[0067] Fluorescent dyes are selected such that spectral overlap between the alginate fluorophore and the fluorescent reporter gene construct is minimized or eliminated using appropriate controls when configuring the detection device (microscope or cytometer).

[0068] In other words, one subject of the present invention consists of eukaryotic (e.g. mammalian) or prokaryotic cells (e.g. bacteria) expressing a fluorescent reporter gene encapsulated in a fluorescently labeled alginate capsule. The combination of these two different types of fluorescent label (intracellular reporter gene and alginate capsule label) allows the simultaneous monitoring of multiple biological processes in one or more cell types.

[0069] Specifically, cells are encapsulated in an alginate biopolymer. The capsule exerts a physical constraint on the cells that mimics normal physiological pressure. This pressure causes the multicellular aggregates within the capsule to remain at a size less than 300 μm that is compatible with the diffusion of oxygen and nutrients through the cells, improving the physiological relevance of this cell culture model (examples of improved biological relevance are described above and include neural stem cells and chondrocytes).

[0070] Each cell type is encapsulated in an alginate shell that is chemically modified with one specific fluorophore, thus allowing identification of capsules containing one specific cell type during analysis for in vitro drug testing.

[0071] The encapsulated cells typically consist of cell lines that have been genetically engineered to express different fluorescent or other reporter molecules, allowing the monitoring of key activities involved in the "biological process" being studied.

[0072] Panels of genes that can be simultaneously monitored include commonly studied features that can be detected in drug tests, i.e. key regulators or biomarkers of biological processes. These features include cytotoxicity, cell proliferation, inflammation, hormonal response, xenobiotic response, genotoxic stress response and antibiotic resistance.

[0073] In particular, the present invention includes a method for characterizing the effect of a drug on multiple cellular processes in multiple cell types simultaneously within the same study. To accomplish this, the method uses encapsulated cells that have been genetically engineered to be able to monitor one or more cellular processes via regulatory elements fused to fluorescent reporter genes whose fluorescent activity indicates biological process activity. The cell lines used in the co-culture can be engineered to express one or more reporter genes involved in biological processes including, but not limited to, cell proliferation, xenobiotic stress response, inflammation, tumor growth or inhibition, and drug toxicity or detoxification responses.

[0074] The same cell type can be genetically engineered to express reporter genes for different biological processes. To multiplex the analysis of multiple reporter genes, cells are split into the required number of separate cell batches, with each batch engineered to contain a single reporter gene corresponding to a single biological process. The separate cell batches are either pooled together for encapsulation in the same capsule, or each encapsulated separately to measure the activity of multiple biological processes in either a mixed co-culture or a physically separated co-culture. Multiplexed analysis of genes corresponding to different cellular processes aims to provide many advantages, including improved ability to predict the best drug candidates and savings in reagents, consumables and time required to perform detailed characterization of drug candidates.

[0075] Each type of capsule typically contains one or more cell types found in a single human organ, or in a distinct physiological site, such as a tumor. Cell encapsulation involves the preparation of separate single-cell suspensions of the different cell types. The suspensions are then encapsulated in a thin layer of alginate that has been pre-labeled with one of many fluorescent markers that allow identification of the capsule contents by microscopy.

[0076] The encapsulated cells are then incubated to promote the formation of 3D tissue-like structures called spheroids. Different encapsulated cell spheroids are mixed to create co-cultures of different cell subtypes, which may include, but are not limited to, tumor cell lines, primary tumor cells from patients, and cell lines, particularly hepatocytes that can metabolize drugs.

[0077] The use of encapsulated 3D cell culture (i) prevents cell-cell contact of different cell types that is not normally found in the same human organ, and (2) allows for the simultaneous analysis of different cell types in the same well, e.g., the toxicity of a drug can be evaluated separately in hepatocytes and cancer cells in the same co-culture.

[0078] The purpose of co-culture of multiple cell types in the same well is to reproduce the multicellular microenvironment, tissue or inter-organ system that occurs in the human body to improve the clinical relevance of this drug testing approach. For example, the purpose of co-culture of tumor cells and liver cells in separate capsules is to simulate the general process following drug administration in vivo, in which the liver first metabolizes the drug, and then the drug metabolites become the main effectors of anti-tumor responses at distant anatomical sites.

[0079] The present invention also saves time and resources because multiple effects, such as antitumor effects and hepatotoxicity, can be studied simultaneously, which is important because drug-induced liver injury (DILI) is one of the main reasons why many drug candidates do not progress through the development process.

[0080] One unique advantage of the present invention is the ability to measure multiple biological processes in co-cultures of mixed cell types, which improves the efficiency and accuracy of drug testing (and more translational relevance to in vivo models).

[0081] The common advantage of the present invention with other 3D co-culture or encapsulated cell-based drug testing methodologies is that it improves the clinical relevance of drug testing through the following features common to existing techniques: The alginate capsule acts as a physical separation of different cell types that normally do not contact each other in vivo. 3D spheroid cell cultures behave more like in vivo tissues compared to cell culture monolayers.

[0082] Furthermore, the present invention makes it possible to increase the number of biological processes that can be studied simultaneously and to increase the repertoire of biological processes, i.e. to create libraries of reporter genes for the quantification of a large proportion of the biological processes commonly studied by pharmaceutical companies.

[0083] The present invention combines many existing individual elements or processes currently known in the fields of cell biology and applied drug discovery, as well as elements that are completely novel. These technical elements or processes are described in the following subsections a to d. a, Encapsulation of cells to form 3D cell spheroids with tissue-like properties. Preferably, the method for alginate-based encapsulation of cells is carried out using a microfluidics device, as described in Alessandri K, Feyeux M, Gurchenkov B, Delgado C, Trushko A, Krause KH, Vignjevic D, Nassoy P, Roux A. Lab Chip, 26 Apr 2016, 16(9):1593-1604, doi:10.1039 / c61c00133e. b, Co-culture of different cell types to generate in vivo-like cell interactions that help achieve more clinically relevant results in drug screening. Preferably, the present invention uses hepatocytes to metabolize drugs in cell co-culture. c, Labeling of cell capsule with fluorescent probes.Techniques for labeling alginate with common fluorescent molecules are widely known in the art. d. Detection of multiple regulatory activities related to genetic manipulation and key cellular processes. The invention relates to the simultaneous (multiplexed) analysis of the fluorescence intensity of different fluorescent reporter genes in the same cell. The method of the invention aims to perform "phenotypic drug screens" that do not depend on knowing the identity of a specific molecular drug target, or "target-based" drug screens, where the regulatory activity of a specific protein is measured in a cell. This element represents a unique feature of the invention.

[0084] Another object of the present invention is to provide an in vitro drug testing kit suitable for testing the effect of one or more drugs of interest on multiple biological processes in one or more target cell types, the kit comprising: At least one ready-to-use microwell plate containing one or more alginate-encapsulated target cell types expressing a fluorescent or bioluminescent reporter gene for each biological process to be analyzed in said one or more target cell types, where in the case of multiple cell types, all or all but one of the capsules are each labeled with a different type of fluorophore.

[0085] Preferably, at least one of said one or more target cell types is a hepatocyte.

[0086] In one embodiment of the invention, the fluorophore-labeled, alginate-encapsulated target cell type or types are co-cultured with one or more patient-derived cell types, preferably engineered to contain at least one fluorescent reporter gene for each of the multiple biological processes to be analyzed.

[0087] More preferably, each of said one or more patient-derived cell types is encapsulated in a fluorophore-labeled alginate biopolymer.

[0088] The in vitro drug testing kit has one advantage that it can be used for phenotypic (affecting a common biological process with a reporter common to almost all cells) or target-based (modulation of a specific pathway previously identified as an ideal therapeutic target that may only be present in some cells, e.g., estrogen receptor) drug discovery.

[0089] In another embodiment of the invention, the multiple biological processes analyzed are selected from the list comprising cell proliferation, inflammation, tumor growth or inhibition, drug toxicity, angiogenesis, immune stimulation, detoxification response, hormonal response, xenobiotic response, genotoxic stress response and apoptosis.

[0090] In a further embodiment of the invention, the one or more patient-derived cell types are selected from the list comprising liver cells, tumour cells, brain cells, epithelial cells, endothelial cells, immune system cells, pluripotent stem cells, embryonic stem cells, lung cells, kidney cells, arterial cells, bone cells, chondrocytes, muscle cells, pancreatic cells, intestinal cells, fibroblasts or skin cells.

[0091] In one embodiment of the invention, one or more drugs are tested per well of the at least one ready-to-use microwell plate to identify the effect of the one or more drugs on a biological process in the encapsulated target cell type.

[0092] In another embodiment of the invention, multiple drugs are tested per well of said at least one ready-to-use microwell plate to identify interactions between said drugs.

[0093] In particular, one or more drugs are tested per well to identify the effect of a drug on a biological process within the encapsulated cells of interest, or interactions between multiple drugs, such as synergistic or non-synergistic effects in regulating a biological process, including but not limited to cytotoxicity or cell proliferation.

[0094] Those skilled in the art will understand that the invention described herein may be modified and altered other than those specifically described. The invention should be understood to include all such modifications and alterations without departing from its spirit or essential characteristics. The invention also includes all steps, features, compositions and compounds referred to or shown herein, individually or collectively, and any and all combinations of any two or more of said steps or features. Thus, the present disclosure is to be considered in all respects as illustrative and not limiting, and the scope of the invention should be considered as indicated by the appended claims, and all changes that come within the meaning and range of equivalents are intended to be included within the scope of the invention.

[0095] Various references are cited throughout the specification, each of which is incorporated herein by reference in its entirety.

[0096] The foregoing will be more fully understood with reference to the following examples, which are illustrative of methods of practicing the invention, but are not intended to limit the scope of the invention. EXAMPLES

[0097] Example 1 explanation Monolayered breast cancer (BC) cells (MELN cells, Tables 1 and 2), stably expressing a luciferase reporter gene under the control of an estrogen response element, are treated with or without co-culture with encapsulated hepatoma (LC) cells with increasing concentrations of tamoxifen or 4-hydroxytamoxifen for 48 h. The activity of estrogen receptor alpha in BC cells is determined by measuring the activity of luciferase (bioluminescence intensity).

[0098] Materials and Methods Cell seeding of BC cells Aspirate the media onto the MELN cells (see Table 1). Wash once with PBS and aspirate the PBS. Trypsin is added to the cells and excess trypsin is aspirated. Incubate at 37 °C for 5 min.

[0099] The cells are detached by repeated pipetting with white cell culture medium (phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum), collected in a tube, and the cells are seeded in a 96-well plate at 10,000 cells / well in white cell culture medium. The cells are incubated at 37°C.

[0100] [Table 1]

[0101] LC cell encapsulation protocol -Preparing cells for encapsulation LC cells from confluent dishes (see Table 1) are washed with PBS, detached by trypsinization, and collected using cell culture medium Count the cells and prepare a cell suspension containing 5 million cells per ml. The cell suspension is poured through a 40 μm Corning filter into a 50 ml Falcon tube. Place 1 ml of cell suspension (i.e. 5 million cells) into a 1.5 mL centrifuge tube. Pellet the cells by centrifugation at 1000 rpm for 5 minutes and resuspend in 300 μM sorbitol (90 μl). Place the cells on ice.

[0102] -Encapsulation The encapsulation is carried out with a patented device (WO 2013 / 113855) described in Alessandri K, Feyeux M, Gurchenkov B, Delgado C, Trushko A, Krause KH, Vignjevic D, Nassoy P, Roux A.Lab Chip, 26 Apr 2016, 16(9):1593-1604, doi:10.1039 / c61c00133e. Collect the capsules in a 10 cm Petri dish containing calcium solution and incubate for 30 min. Replace the calcium solution with cell culture medium. Allow the capsules to settle to the bottom of the dish. Aspirate 90% of the calcium solution using an 18 gauge needle being careful not to aspirate the capsules at the bottom of the dish. Fill a 10 cm capsule dish completely (40 mL) with cell culture medium. Allow the capsules to settle and aspirate and replenish the culture medium. Repeat two times, leaving 15 ml of cell culture medium in the last repetition. The incubation capsules are incubated at 37°C.

[0103] Loading and processing LC capsules onto plates Aspirate the medium from the capsule, leaving approximately 5 ml of medium. Using a 1 ml pipette with a tip that allows the capsules to pass through, wash the surface of the dish with the remaining medium. Collect as many capsules as possible into a 15 ml tube. Add 5 ml of medium and collect the remaining capsules with a 1 ml pipette.

[0104] Centrifuge the capsules at 300 rpm for 1 minute, aspirate off the medium and resuspend the capsules in 10 ml of cell culture medium without phenol red (phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum). Repeat once more. · Centrifuge the capsules at 300 rpm for 1 minute and aspirate the medium, leaving approximately 200 μl of medium on top of the capsule. Dispense the capsules on top of the BC cell monolayer in a 96-well plate by pipetting up and down continuously to keep the capsules in suspension before transferring the appropriate amount to the wells. Half of the wells are not filled with capsules (BC cells only). All wells are treated with 10 pM 17β-estradiol (E2) followed by the addition of different concentrations of tamoxifen or 4-hydroxytamoxifen ranging from 30 pM to 1 μM. Plates are incubated at 37° C. for 48 hours.

[0105] Measurement of luciferase activity Aspirate the capsules, wash once with PBS, and aspirate to completely remove the capsules. Add 20 μl of Passive Lysis Buffer (Promega) to the BC cells and incubate at 100 rpm at room temperature on a plate shaker for 15 minutes. 10 μl of lysate is transferred to a luminometer compatible white 96 well plate and 10 μl of luciferase substrate (Promega) is added. Bioluminescence intensity is read in a luminometer.

[0106] result As can be seen in Figure 1, the inclusion of encapsulated LC cells in in vitro co-culture with BC cells growing in monolayer increased the anticancer activity of tamoxifen (ERα activity was inhibited and the inclusion of LC cells resulted in a lower IC50). This suggests that co-culture of BC cells with encapsulated LC cells can recapitulate the physiological production of a secondary active drug metabolite (endoxifen) by LC cells, which was missed in a monoculture system containing only the prodrug (tamoxifen) and the BC target cell line. See Figure 1 for more details.

[0107] Example 2 explanation Co-cultures of encapsulated BC cells stably expressing the dual reporter FUCCI (see Table 2) in far-red labeled capsules and encapsulated LC cells stably expressing the dual reporter FUCCI in unlabeled capsules were incubated at different serum concentrations. The amount of actively proliferating and non-proliferating cells (latent) was determined by quantifying the percentage of red (G1 phase) and green (G2 / S phase) nuclei simultaneously in both LC and BC cells in the same co-culture well.

[0108] Materials and Methods Protocol for establishing stable reporter cell lines Seed HEK293 cells in 10 cm dishes at 4 million cells / dish. Incubate at 37 °C for 24 h. The cells are transfected with a mixture of plasmids pMD2G, psPAX2 and the lentiviral reporter plasmid of interest (pBOB-EFl-fastFUCCI-puro) using the polyethylenimine transfection method. The transfection solution is added to the cells and incubated overnight at 37°C. Perform a complete medium change. The cells are incubated at 37° C. for 8 hours. Collect the medium containing the lentivirus into a tube. Complete medium is added to the cells and the cells are incubated at 37° C. for 16 hours. Repeat collection two more times using the medium at 8-16 h intervals. Cells used to generate stable reporter cell lines (MDA-MB134 and HepG2, Table 1) are seeded directly into lentivirus-containing medium at the same density as in regular cell culture for 96 h at 37°C. Aspirate the lentivirus-containing medium and replace it with complete cell culture medium. Select antibiotic-resistant (reporter+) cells by adding puromycin at a final concentration of 2 µg / mL directly to the medium for 24 h. Remove the puromycin medium containing dead cells and replace with complete cell culture medium containing 1 µg / mL puromycin. Stable reporter cell lines are maintained in medium containing 1 μg / mL puromycin.

[0109] [Table 2]

[0110] Staining of alginate with amine-containing fluorophores (see Table 3) Prepare 25 mL of 1% (w / v) alginate solution in 0.1 M MES at pH 6.0 and mix overnight on a rotator at room temperature (RT). Add 1 mg of dye dissolved in 200 µl of DMSO and mix with the alginate for 10-30 min at RT on a rotator. Add sulfo-NHS dissolved in 200 µl 0.1 M MES, pH 6.0 to a final concentration of 2 mM in the alginate solution and mix for 30 min at RT on a rotator. Add EDC dissolved in 200 μl of 0.1 M MES (pH 6.0) to the alginate solution so that the final concentration is 5 mM, and mix and react on a rotator at room temperature for 1 to 2 hours. Dialyze in a Slide-A-Lyser cassette (10K) against 1 L of distilled water for 30 min. Then, change the water and place the cassette in 5 L of distilled water and leave overnight at 4 °C with gentle agitation. Collect the stained alginate from the cassette and transfer it to a plastic tube and store at 4 °C. Prior to encapsulation, mix the stained alginate with unstained alginate (prepared by performing step 1 only) in a ratio of 1:10 stained alginate:unstained alginate.

[0111] [Table 3]

[0112] Stable reporter cell encapsulation protocol -Preparing cells for encapsulation Same protocol as in Example 1, "Preparation of cells for encapsulation." -Encapsulation Same protocol as "Encapsulation" in Example 1, except Applicant used far-red labeled alginate to encapsulate BC cells and unlabeled alginate to encapsulate LC cells.

[0113] Loading of BC and LC capsules onto plates for co-culture Aspirate the medium from the capsule, leaving approximately 5 ml of medium. Using a 1 ml pipette with a tip that allows the capsules to pass through, wash the surface of the dish with the remaining medium. Collect as many capsules as possible into a 15 ml tube. Add 5 ml of medium and collect the remaining capsules with a 1 ml pipette. The capsules are centrifuged at 300 rpm for 1 minute, the medium is aspirated and the capsules are resuspended in 10 ml of cell culture medium. · Centrifuge the capsules at 300 rpm for 1 minute and aspirate the medium, leaving approximately 200 μl of medium on top of the capsule. Dispense the capsules into the wells of a 96-well imaging plate by pipetting up and down continuously to keep the capsules suspended before transferring the appropriate amount into the wells. Co-cultures are incubated at 37° C. for 96 hours with or without the addition of fetal bovine serum to the wells.

[0114] Measurement of fluorescence intensity of BC and LC cells in co-culture Image acquisition of the 96-well plates containing encapsulated 3D cell co-cultures is performed with an automated confocal microscope (IXM-C, Molecular Devices™) using a 4x objective and performing 10 z-steps in 12 different areas per well for each fluorescence channel.

[0115] Moreover, the method used herein is a typical high content screening (HCS) procedure, which is currently the method of choice for drug discovery in the pharmaceutical industry. After image acquisition, image analysis is performed in two steps: first, to identify the different cell reporters and capsules in the image by applying a series of segmentation processes that generate relevant object masks, and second, to extract several parameters (fluorescence intensity, size, shape, etc.) from the original image to which the masks were applied. This image analysis process allows for the accurate quantification of each proliferation state (as reported by the FUCCI reporter) of both co-cultured cell types simultaneously.

[0116] result Figure 2 shows that two different cell states reported by two different fluorescent reporters (the FUCCI reporter contains both a green fluorescent reporter gene for proliferating cells and a red fluorescent reporter gene for non-proliferating cells) can be quantified simultaneously in a 3D co-culture of two different cell types encapsulated in two different alginate capsules (far-red labeled alginate capsules for BC cells and unlabeled alginate capsules for LC cells). The results in Figure 2 also show that the HCS procedure of automated microscope acquisition followed by image processing and analysis with the software MetaXpress (Molecular Devices) allows the fluorescence of the reporters to be distinguished from each other and from the fluorescence of the labeled alginate capsules.

[0117] Example 3 explanation Cocultures of encapsulated BC cells stably expressing the dual reporter FUCCI (Table 2) in unlabeled capsules and encapsulated BC cells stably expressing the reporter zipGFP-Casp3 (Table 2) in far-red labeled capsules, with or without coculture with encapsulated LC cells in green labeled capsules, are treated with increasing concentrations of tamoxifen for 96 h. Induction of apoptosis is measured by automated confocal microscopy (HCS) in BC cells stably expressing the reporter zipGFP-Casp3 and proliferation is measured simultaneously in BC cells stably expressing the dual reporter FUCCI.

[0118] Materials and Methods Protocol to establish stable reporter cell lines of the two BCs The protocol is the same as "Protocol for establishing stable reporter cell lines" in Example 2, except that in addition to generating BC cell lines stably expressing the dual reporter FUCCI, applicant also generated BC cell lines stably expressing the reporter zipGFP-Casp3 (Table 2) using a homemade lentiviral construct containing the reporter incorporated into the pHAGE lentiviral vector (pHAGE-fEF1-zipGFP-Casp3).

[0119] Staining of alginate with amine-containing fluorophores (see Table 3) This is the same protocol as in Example 2, "Staining of alginic acid with fluorophores having amine groups (see Table 3)."

[0120] Stable reporter cell encapsulation protocol -Preparing cells for encapsulation Same protocol as in Example 1, "Preparation of cells for encapsulation." -Encapsulation Same protocol as "Encapsulation" in Example 1, except that unlabeled alginate is used to encapsulate BC cells stably expressing the dual reporter FUCCI, far-red labeled alginate is used to encapsulate BC cells stably expressing the reporter zipGFP-Casp3, and green labeled alginate is used to encapsulate LC cells.

[0121] Loading and processing of capsules onto plates for co-culture Same protocol as in Example 1 "Loading and processing LC capsules onto plates" with the following exceptions. The capsules are dispensed into the wells of a 96-well imaging plate by pipetting up and down continuously to keep them in suspension before transferring the appropriate amount of capsules into the wells. Half of the wells are not filled with LC capsules (BC cells only). All wells are treated with 10 pM 17β-estradiol (E2) followed by the addition of different concentrations of tamoxifen or 4-hydroxytamoxifen ranging from 1 μM to 10 μM. Plates are incubated at 37° C. for 96 hours.

[0122] Measurement of fluorescence intensity of BC cells in co-culture This is the same protocol as in Example 2, "Measurement of fluorescence intensity of BC cells and LC cells in co-culture," except that the image analysis process allows for accurate simultaneous quantification of each proliferation state (reported by the FUCCI reporter) and induction of apoptosis (reported by the zipGFP-Casp3 reporter) in BC cells expressing the FUCCI reporter and BC cells expressing the zipGFP-Casp3 reporter, respectively.

[0123] result Figure 3 shows a multiplex culture of three types of encapsulated cells: LC cells, BC cells stably expressing the reporter gene zipGFP-Casp3, and BC cells stably expressing the dual reporter gene FUCCI. The capsule color allows identification of each type of cell, and the fluorescence intensity of the cells can be measured to quantify a specific biological activity in each type of capsule (here, proliferation of BC cells in unlabeled capsules or apoptosis of BC cells in far-red capsules). In this particular experiment, applicants used capsules of different colors to distinguish between two different reporters with the same fluorescent color, and an additional capsule color was used to identify the second cell type (LC cells).

[0124] Figures 4 and 5 show that the applicant can use reporters of two different biological activities (cell proliferation and apoptosis) to measure two different biological activities simultaneously in multiple culture (co-culture) wells. In this experiment, the applicant used concentrations of tamoxifen in the micromolar range. At these concentrations of tamoxifen, the inhibition of cell proliferation is saturated, while apoptosis is induced in a dose-dependent manner. In Figure 4, the applicant can observe that the induction of apoptosis in BC cells is increased by tamoxifen in a dose-dependent manner only in co-culture with LC cells. In Figure 5, the cell proliferation inhibition is already maximal at 1 μM and cannot be further increased by higher concentrations of tamoxifen. This experiment shows that using the method of the invention, the applicant can independently and simultaneously measure two different biological activities during multiple culture. The applicant again showed that the presence of LC cells improves the efficiency of tamoxifen on BC cells. Importantly, this experiment allows to reliably distinguish two different pharmacological effects of the same molecule in the same culture well (inhibition of cell proliferation and induction of apoptosis).

Claims

1. An in vitro drug testing method capable of analyzing two or more biological processes in a co-culture of three or more cell types, comprising: a) expressing at least one fluorescent reporter gene for each biological process analyzed in two or more of said three or more cell types analyzed in the same in vitro drug testing assay; b) encapsulating each of the three or more cell types to be analyzed in the same in vitro drug testing assay using an alginate biopolymer and labeling all or all but one of the capsules with a different type of fluorophore; step c) of co-culturing all encapsulated cell types of step b) that are to be analyzed in the same in vitro drug testing assay with one or more additional unlabeled cell types encapsulated in labeled or unlabeled alginate biopolymer; d) exposing the co-culture to one or more drugs; e) measuring the activity of said two or more biological processes in each cell type analyzed in the same in vitro drug testing assay of step c) by analyzing the fluorescence intensity of each fluorescent reporter gene separately by high content screening before and / or after exposure to one or more drugs to be tested; wherein each cell type to be analyzed is identified by high content screening based on the type of fluorophore label associated with the alginate capsule containing said cell type to be analyzed.

2. 2. The in vitro drug testing method of claim 1, wherein the two or more biological processes are selected from the list comprising cell proliferation, inflammation, tumor growth or inhibition, drug toxicity, angiogenesis, immune stimulation, detoxification response, hormonal response, xenobiotic response, genotoxic stress response and apoptosis.

3. 3. The in vitro drug testing method of claim 1 or 2, wherein the three or more cell types are selected for co-culture based on predicted or confirmed physiological interactions between the cell types and / or based on predicted or confirmed effects of the three or more cell types on the one or more drugs being tested.

4. 4. The in vitro drug testing method according to any one of claims 1 to 3, further comprising a step f) of exposing said co-culture to one or more fluorescent reagents to measure one or more additional biological processes.

5. 5. The in vitro drug testing method according to any one of claims 1 to 4, characterized in that the three or more cell types are selected from the group comprising liver cells, tumour cells, brain cells, epithelial cells, endothelial cells, immune system cells, pluripotent stem cells, embryonic stem cells, lung cells, kidney cells, arterial cells, bone cells, chondrocytes, muscle cells, pancreatic cells, intestinal cells, skin cells, fibroblasts, fungal cells or bacterial cell lines or primary tissue cell subsets.

6. 6. In vitro drug testing method according to any one of claims 1 to 5, characterized in that at least one of the three or more cell types analysed in the in vitro drug testing assay or at least one of the additional unlabelled cell types of step c) is a hepatocyte.

7. An in vitro drug testing method according to any one of claims 1 to 6, characterized in that at least one of the three or more encapsulated cell types is derived from a cancer sample of a patient.

8. The in vitro drug testing method according to any one of claims 1 to 5, characterized in that at least one of the three or more cell types is a bacterial or fungal cell.

9. 9. The in vitro drug testing method of claim 8, wherein the drug being tested is an antibiotic or a bactericide.

10. 9. The in vitro drug testing method of claim 8, wherein at least one of the two or more biological processes analyzed is selected from the list comprising antimicrobial resistance or susceptibility, bacterial growth, bactericidal activity or bacteriostatic activity.

11. An in vitro drug testing kit suitable for testing the effects of one or more drugs of interest on two or more biological processes in three or more target cell types, comprising: An in vitro drug testing kit comprising at least one ready-to-use microwell plate containing co-cultures of three or more alginate-encapsulated target cell types expressing fluorescent reporter genes for each biological process to be analyzed in said three or more target cell types, wherein all or all but one of the capsules are each labeled with a different type of fluorophore.

12. 12. The in vitro drug testing kit of claim 11, wherein at least one of the three or more target cell types is a hepatocyte.

13. 13. An in vitro drug testing kit according to claim 11 or 12, wherein the three or more fluorophore-labelled, alginate-encapsulated target cell types are co-cultured with one or more patient-derived cell types.

14. 14. The in vitro drug testing kit of claim 13, wherein the one or more patient-derived cell types are genetically engineered to contain at least one fluorescent reporter gene for each of two or more biological processes to be analyzed.

15. 15. The in vitro drug testing kit of claim 13 or 14, wherein said one or more patient-derived cell types are each encapsulated in a fluorophore-labeled alginate biopolymer.

16. 16. An in vitro drug testing kit according to any one of claims 11 to 15, wherein the two or more biological processes analysed are selected from the list comprising cell proliferation, inflammation, tumour growth or inhibition, drug toxicity, angiogenesis, immune stimulation, detoxification response, hormonal response, xenobiotic response, genotoxic stress response and apoptosis.

17. 17. An in vitro drug testing kit according to any one of claims 11 to 16, wherein the three or more target cell types are selected from the list comprising liver cells, tumour cells, brain cells, epithelial cells, endothelial cells, immune system cells, pluripotent stem cells, embryonic stem cells, lung cells, kidney cells, arterial cells, bone cells, chondrocytes, muscle cells, pancreatic cells, intestinal cells, fibroblasts or skin cells.

18. 18. The in vitro drug testing kit of any one of claims 11 to 17, wherein the one or more drugs are tested per well of the at least one ready-to-use microwell plate to identify an effect of the one or more drugs on a biological process in the encapsulated target cell type.

19. 18. The in vitro drug testing kit of any one of claims 11 to 17, wherein a plurality of drugs are tested per well of said at least one ready-to-use microwell plate to identify interactions between said drugs.

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