WO2025040692A1

WO2025040692A1 - Color- and position-based identification of multicellular structures for the multiplexed and individualized analysis of multi-tissue co-cultures

Info

Publication number: WO2025040692A1
Application number: PCT/EP2024/073385
Authority: WO
Inventors: Grégory SEGALA; Clélia BOURGOINT; Théo DEREGNAUCOURT
Original assignee: Fluosphera Sa
Current assignee: Fluosphera Sa
Priority date: 2023-08-22
Filing date: 2024-08-20
Publication date: 2025-02-27
Anticipated expiration: 2026-02-22

Abstract

The invention relates to a method to independently identify by fluorescence microscopy different cell types forming multicellular structures in co-culture thanks to a fluorescently-labelled extracellular matrix, and the individual identification of each multicellular structure of the co-culture with a spatial positioning of those structures in microcavities, so as to individually and independently measure multiple biological activities in each multicellular structure of each type of tissue of the co-culture.

Description

Color- and Position-based Identification of multicellular structures for the multiplexed and individualized analysis of multi-tissue cocultures

FIELD OF THE INVENTION

The invention relates to a method to independently identify by fluorescence microscopy different cell types forming multicellular structures in co-culture thanks to a fluorescently- labelled extracellular matrix, and the individual identification of each multicellular structure of the co-culture with a spatial positioning of those structures in microcavities, so as to individually and independently measure multiple biological activities in each multicellular structure of each type of tissue of the co-culture.

BACKGROUND OF THE INVENTION

Drug testing and chemical testing involve the use of in vitro methods such as cell-based assays intended to mimic human body responses. The goal of in vitro testing is to predict the effects of drugs and/or compounds to develop products that are efficient for their intended use while being safe for human beings. Today, drug discovery and chemical safety testing are limited by the lack of predictivity of in vitro models. Indeed, the reliability of in vitro methods fully depends on their faithfulness /capacity to recapitulate the organization of cells and tissues in the human body.

The vast majority of human cell biology studies still relies on cell culture monolayer which involves the growth of human cells, e.g. primary cells or cancer cell lines, on a flat and rigid plastic surface in a nutritive medium. In such a configuration, the cells are far from a physiological environment: they are spatially organized in 2D unlike a 3D organization in the human body, and the stifness of the plastic surface is incredibly stronger than the one of the extracellular matrix in which the cells are naturally evolving. Moreover, in cell culture monolayer, cells from a specific tissue are isolated and cultured without the presence of other tissue types. However, the communications between different tissues is absolutely essential for human body functions like metabolic regulations and hormonal systems and it is important to recapitulate such systemic organization in vitro to develop accurate clinical applications. Multiple methods and technologies were invented to tackle down the lack of relevance of in vitro models. The most significant advances were in the field of 3D cell culture biology where different methods were designed to force the cells to structure and thus to self-organize in 3D (JPH 06327462 A SUMITOMO BAKELITE CO, JP 5847733 B2 TAKAYAMA SHUICHI [US]; TUNG YI-CHUNG [US]; HSIAO AMY YU-CHING [US]; JAN EDWARD [US]; UNIV MICHIGAN [US]; 3D BIOMATRIX INC [US] , US 2020/392453 Al CORNING INC [US]; Jaganathan, H., Gage, J., Leonard, F. et al., Three-Dimensional In Vitro Co-Culture Model of Breast Tumor using Magnetic Levitation Sci Rep 4, 6468 (2014)). In such methods, cells are maintained in suspension, thus inducing cell aggregation and growth in 3D multicellular structures called spheroids or organoids. Another breakthrough in cell culture arised with hydrogels that were developed to allow the cells to grow in a physiologically relevant microenvironment (CA 2652138 C PURDUE RESEARCH FOUNDATION [US], KR 101449906 Bl PUSAN NAT UNIV IND COOP FOUND [KR]; Pradhan S., Hassani I., Seeto WJ., Lipke EA., PEG-fibrinogen hydrogels for three-dimensional breast cancer cell culture, Journal of Biomedical Materials Research Part A, Volume 105, Issue 1 (2017); US 9,771,556 B2 WISCONSIN ALUMNI RES FOUND [US], In addition to a stiffness similar to the one found in human tissues, hydrogels such as matrigel or collagen provide the cells with components of the extracellular matrix like adhesion signalling molecules and growth factors necessary for the maintenance of the cell phenotype. An example of a method combining 3D cell culture with an hydrogel scaffold uses cell encapsulation where cells are enclosed into an alginate capsule together with extracellular matrix (Lu YC., Song W., An D. et al., Designing compartmentalized hydrogel microparticles for cell encapsulation and scalable 3D cell culture, Journal of Materials Chemistry B, Issue 3 (2015)). However, the alginate capsule surrounding the microtissues creates a constraint for the diffusion of big particle. Even if the study describes the dissolution of the alginate capsule, it is performed only for the recovery of the microtissues, but not for their culture without capsule. Therefore, the compatibility of this approach with the diffusion of large particles in culture was not described in this study.

Although 3D cell culture methods and hydrogel scaffolds increase the physiological relevance of in vitro assays thanks to a local microenvironment similar to tissues, they are limited by the lack of possibility to mix several tissue types together. Indeed, for such multi-tissue system, each tissue needs to be individually identified to extract and analyze biological information. Moreover, a selective compartmentalization is necessary to isolate tissues from each other to avoid irrelevant cell-cell interactions and fusion of tissues normally distant from each other (e.g. hepatic and cerebral tissues) while allowing distant communications between tissues. An approach describes the generation of multicellular aggregates on a micro-structured cell culture made of a hydrogel, and a kit comprising an array of microwells for the culture of multicellular aggregates (EP 3296018 Al ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL)). Even if this method allows a compartmentalization through the physical separation of multicellular aggregates growing on a hydrogel in the same culture, it is not possible to discriminate multicellular aggregates from different tissue types in co-culture.

Both the lack of tissue identification and selective tissue compartmentalization restrict the use of 3D cell culture and hydrogel scaffold to single tissue assays and impair the development of systemic assays, i.e. assays involving several types of tissues. An attempt to create a multitissue system used the encapsulation of one cell type, Human Umbilical Vein Endothelial cells (HUVEC), expressing a GFP reporter gene for their identification, and co-cultured directly at the top of a cell culture monolayer of a second cell type, Normal Human Lung Fibroblast (NHLF), expressing a RFP reporter gene (Lu YC., Song W., An D. et al., Designing compartmentalized hydrogel microparticles for cell encapsulation and scalable 3D cell culture, Journal of Materials Chemistry B, Issue 3 (2015)). However, the cell type identification is invasive because it is provided by a fluorescent probe expressed by the cells, meaning that any fluorescent cellular readout having the same color as the fluorescent probe cannot be used. This multi-tissue system thus faces a strong limitation to discriminate the co-cultured tissues while measuring readouts in each tissue of the co-culture. It does not provide a non-invasive tissue identification which is required for the measurement of a fluorescent readout having the same color as the color used for the identification of a cell type.

Biophysical approaches were used to increase the relevance of in vitro testing by mimicking the systemic organization of the human body on microfluidic chips (US 9,791,433 B2 TISSUSE GMBH [DE], US 9,725,687 B2 HARVARD COLLEGE [US], UNIV VANDERBILT [US], WO 2022/180595 Al UNIV DO MINHO [PT], INT IBERIAN NANOTECHNOLOGY LABORATORY INL [PT], JP 2019/534000 A UNIV WAKE FOREST HEALTH SCIENCES [US] ). This technology, called microphysiological systems, consists on tissues growing in separate chambers and communicating through a porous membrane with a liquid flow in microfluidic channels. The microfluidic flow mimics the blood circulation and allows communications between tissues in distant chambers. Other systemic methods were also developed following the same concept of microfluidic-assisted communications between tissues (WO 2020/087148 Al CENTRO NAC DE PESQUISA EM ENERGIA E MATERIAIS - CNPEM [BR], WO 2022/265352 Al UNIV YONSEI IACF [KR], CN 114045218 A DALIAN INST CHEM & PHYSICS CAS et al., US 10,023,832 B2 UNIV VANDERBILT [US]). These microfluidics approaches facilitate the study of complex physiological processes, and notably their mechanic features like the shearing stress of blood vessels, or the extravasation of immune cells from the blood circulation.

Despite the capacity to mimic systemic physiological environment, microphy si ologi cal systems are too sophisticated to be routinely used in bioanalytic and research laboratories. The complexity of microphy si ologi cal systems prevents them to be flexible enough to adapt to the diversity of questions that need to be addressed in vitro. Moreover, they are not compatible with high-throughput approaches since microphysiological systems does not allow the analysis of hundreds of different conditions simultaneously.

There is a need to develop a new method that combines the relevance and the flexibility of 3D cell culture and hydrogel scaffolds with the systemic capacity of microphysiological systems. Such method should allow the identification of tissues in a multi-tissue system while being flexible and providing a physiologically relevant microenvironment to the tissues. A first attempt was made in this direction with a method called “nested cell encapsulation” (WO 2011/047870 Al PLASTICELL LTD [GB], CHOO YEN [GB] et al.). This method was used to perform multiple parallel tissue culture experiments thanks to a specific label in each microcapsule allowing the identification of the specific series of culturing steps encapsulated cells were subjected to. However, the purpose of this invention was not to design a systemic assay but rather to make a tracking system for the fast identification of the best cell culturing paths. A method was much more relevant to address the need highlighted above: a multiplexing method allowing the measurement of multiple biological processes in several cell types in 3D encapsulated co-cultures (WO 2021/058557 Al UNIV GENEVE [CH]). The purpose of this document is to make systemic assays recapitulating distant communications between multiple tissues, and to simultaneously analyze the biological effects of drugs into each tissue of the system. To do so, tissues are encapsulated into color-coded alginate capsules to specifically identify each tissue type. Even if this method is relatively powerful to combine high-throughput, flexibility, and multiplexing with the systemic nature of the assay, the alginate capsule limits the diffusion of big molecules like antibodies, and totally prevent the release or the capture of viral vectors or lipid nanoparticles. WO 2021/058557 Al has a first limitation at the level of the alginate capsule surrounding the encapsulated cells. Indeed, the alginate capsule has a limited permeability (permeability cutoff), allowing only small molecules to cross it and to reach the encapsulated cells. An expert skilled in the art would decrease the percentage of alginate to increase the permeability cut-off, thus allowing bigger molecules to cross the alginate capsules. However, by using this approach, the structure of the alginate capsule is becoming loose, thus compromising the physical integrity of the capsule containing cells. It is thus impossible to decrease further the alginate concentration to allow molecules bigger than 200 kDa to cross the capsule. Moreover, using the lowest alginate concentration compatible with capsule integrity, only a fraction of big molecules, such as antibodies (150 kDa) can cross the alginate capsule, thus introducing biases for research and analyses using such materials in combination: encapsulated multicellular structures and big molecules (or biologies) (size > 150 kDa). In addition, WO 2021/058557A1 has a second limitation: the unability to identify individual multicellular structures from the same tissue within a co-culture. Even if the multiplexing capability of the method allows the discrimination of different tissues (heterotypic multicellular structures) of a co-culture based on color-coded capsules, the method does not allow the discrimination of different multicellular structures from the same tissue type (homotypic multicellular structures) in such co-culture. This additional layer of discrimination is important to increase the relevance of systemic assay because multicellular structures are complex and subjected to intra-tissue variability. It is well documented that spheroids and organoids, two predominant types of multicellular structures, are difficult to standardize because of their intrinsic cellular complexity (Zanoni M., Piccinini F., et al., 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained, Sci Rep 6, 19103 (2016) ; Zhao Z., Chen X., et al., Organoids, Nat Rev Methods Primers 2:94 (2022)). For example, it was demonstrated by RNA-sequencing that an important organoid-to-organoid variability in the gene expression profile exists within a culture of liver organoids (Gehling K., et al., RNA-sequencing of single cholangiocyte-derived organoids reveals high organoid-to-organoid variability, Life Science Alliance, 5: 12 (2022)). This example shows that an individual measurement of the biological effects occuring in each multicellular structure of a co-culture is necessary to capture the variability between homotypic multicellular structures, and hence to obtain statistically relevant results.

Ideally, in addition to all the features of the invention described in WO 2021/058557 Al, a systemic assay should also be compatible with the diffusion of big molecules (biologies), viral vectors, and carrier nanoparticles, and it should allow the individual and independent measurement of multiple biological effects in each multicellular structure of a same co-culture. This is one of the scope of the present invention described herein.

BRIEF DESCRIPTION OF THE INVENTION

One of the aims of the present invention is to provide a systemic phenotype screening method to predict the systemic effect of therapies in drug discovery. The principle of phenotypic screening is to identify potent candidate drugs by directly measuring their final biological effect, also known as phenotype, on cells, tissues, or organisms.

Briefly, one or several cells mixed with a fluorescent hydrogel matrix are encapsulated in alginate capsules. After the formation of multicellular structures representing one or several tissue types within the capsules, the alginate capsules are dissolved to release complexes comprising the multicellular structures embedded in the fluorescent hydrogel matrix. In addition to the capacity of being identified thanks to the specific color of the hydrogel, following the same principle described in WO 2021/058557 Al, these complexes have an increased permeability in comparison to the alginate capsule used in WO 2021/058557 Al.

As explained above, the invention described in WO 2021/058557A1 has a limitation at the level of the alginate capsule surrounding the encapsulated cells. Indeed, the alginate capsule has a limited permeability (permeability cut-off), allowing only small molecules to cross it and to reach the encapsulated cells. An expert skilled in the art would decrease the percentage of alginate to increase the permeability cut-off, thus allowing bigger molecules to cross the alginate capsules. However, by using this approach, the structure of the alginate capsule is becoming loose, thus compromising the physical integrity of the capsule containing cells. It is thus impossible to decrease further the alginate concentration to allow molecules bigger than 200 kDa to cross the capsule. Moreover, using the lowest alginate concentration compatible with capsule integrity, only a fraction of big molecules, such as antibodies (150 kDa) can cross the alginate capsule, thus introducing biases for research and analyses using such materials in combination: encapsulated multicellular aggregates and big molecules (or biologies) (size > 150 kDa). To solve this problem, Applicants have encapsulated cells mixed with a fluorescent hydrogel matrix in an alginate capsule. After the cells form a multicellular structure or aggregate inside the capsule, Applicants dissolved the capsule by depleting divalent cations from the medium/ solution to only keep the multicellular aggregate embedded in the fluorescent hydrogel matrix. Surprisingly the fluorescent hydrogel matrix keeps the capacity to identify the multicellular aggregate thanks to the specific color of the fluorescent matrix while drastically increasing the permeability cut-off. Applicants evidenced that antibodies, and even lentiviral particles (size > 1 MDa) can easily reach the multicellular structure or aggregate embedded in the matrix. Moreover, Applicants also proved that the multicellular structure can be identified by fluorescence microscopy.

In particular, one of the objects of the present invention is to provide an in vitro method to test drugs, chemicals, biologies, and/or biological particles, having the capacity to simultaneously measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the method comprises: a) a first step where one or several suspensions of cells representing one tissue type per cell suspension are independently mixed with a hydrogel matrix composed of a mixture of unlabelled biopolymers and, at least for one of said one or several suspensions of cells, one biopolymer labelled with a fluorophore conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the biopolymers by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures embedded in said mixture of biopolymers; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is embedded in a fluorescent hydrogel matrix; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the hydrogel matrix surrounding the multicellular structure; e) exposing said multi-tissue co-culture to said drugs, chemicals, biologies, and/or biological particles to be tested; f) analysis of the exposed multi-tissue co-culture is performed by staining the at least two different types of multicellular structures of step c) by either: i) performing an immunolabelling on the multi-tissue co-cultures with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe or; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for the bioluminescence, in which each of the at least two different types of multicellular structures is discriminated through the specific color code of the fluorescent hydrogel matrix in which it is embedded, multicellular structures from the same tissue of said at least two different types of multicellular structures are individually discriminated through the stable spatial positioning in the microcavities of the multi well plates and whereas the biological effect of interest is specifically measured in said each multicellular structure of said at least two different types of multicellular structures.

Another object of the present invention is to provide an in vitro drug testing kit for use in measuring the effects of one or more drugs, chemicals, biologies, and/or biological particles on multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures embedded in an hydrogel matrix representing at least two tissue types, wherein in case there are more than one tissue type, at least one type of multicellular structure is embedded in a specific color-coded fluorescent hydrogel.

Yet, another object of the present invention is to provide an in vitro method based on cell coating to test drugs, chemicals, biologies, and/or biological particles, having the capacity to simultaneously measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the method comprises: a) a first step where one or several suspensions of cells representing one tissue type per cell suspension are independently mixed with fluorescent cell-coating molecules conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the fluorescent cell-coating molecules by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures coated with fluorescent cell-coating molecules; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is coated with fluorescent cell-coating molecules; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the fluorescently- coated multicellular structure; e) exposing said multi-tissue co-culture to said drugs, chemicals, biologies, and/or biological particles to be tested; f) analysis of the exposed multi-tissue co-culture is performed by staining the at least two different types of multicellular structures of step c) by either: i) performing an immunolabelling on the multi-tissue co-cultures with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe or; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for the bioluminescence, in which each of the at least two different types of multicellular structures is discriminated through the specific color code of the fluorescent cell coating, multicellular structures from the same tissue of said at least two different types of multicellular structures are individually discriminated through the stable spatial positioning in the microcavities of the multiwell plates and whereas the biological effect of interest is specifically measured in said each multicellular structure of said at least two different types of multicellular structures.

It is another object of the present invention to provide an in vitro kit based on cell-coating for use in drug testing by measuring the effects of one or more drugs, chemicals, biologies, and/or biological particles on multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures coated with cell-coating molecules representing at least two tissue types, wherein at least one type of multicellular structure is coated with specific color-coded fluorescent cell-coating molecules.

The invention also provides an in vitro method based on fluorescent capsules to test drugs or chemicals, with the capacity to measure multiple biological processes in individual encapsulated multicellular structures representing several tissue types in co-culture, the method comprises: a) separately encapsulating one or more cell types to be analyzed in the same in vitro drug testing assay with a biopolymer of alginate, wherein in the case there are more than one cell type, all, or all but one, of the respective capsules are labelled, each with a different type of fluorophore; followed by culturing said encapsulated cells to form encapsulated multicellular structures; b) at least two different types of encapsulated multicellular structures are combined together to form a multi-tissue co-culture and where at least one of said two different types of encapsulated multicellular structures is encapsulated in a fluorescent capsule; c) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities; d) exposing said multi-tissue co-culture to drugs, and/or chemicals e) analysis of the multi-tissue co-culture is performed by staining at least two different types of encapsulated multicellular structures of step b) by either: i) performing an immunolabelling on the multi-tissue co-culture with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for bioluminescence, in which each of the at least two different types of tissues is discriminated through the specific color code of the fluorescent capsule in which it is encapsulated. Encapsulated multicellular structures from the same tissue of said at least two different types of tissues are individually discriminated through the stable spatial positioning in the microcavities of the multiwell plates and whereas the biological effect of interest is specifically measured in said each encapsulated multicellular structure of said at least two different types of tissues.

Another object of the invention is to provide an in vitro kit based on fluorescent capsules for use in drug testing by measuring the effects of one or more drugs or chemicals on multiple biological processes in individual capsules containing multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures encapsulated in alginate capsules representing at least two tissue types, wherein at least one type of capsule is labelled with specific color-coded fluorescent alginate.

Other objects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Quantification of the proliferation rate and the total cell number in two tissues in coculture. HEK-293 cells were embedded in non-fluorescent matrix while MCF-7 cells were embedded in fluorescent matrix. Both type of embedded cells formed multicellular structures and were mixed together to form a co-culture. Representative pictures were taken in 4 different channels, transmitted light, DAPI, Cy3, and Cy5, to show the multicellular structures in coculture, Hoechst staining, the fluorescent matrix, and Ki67 immunostaining, respectively. An image segmentation was performed to discriminate multicellular structures embedded in fluorescent matrix (MCF-7) from multicellular structures embedded in unlabeled matrix (HEK- 293), followed by the quantification of cell nuclei (total cell count) and the proliferating cells in each type of multicellular structure. The results of the quantification of the total cell number and the proliferating cells in each type of multicellular structures are shown in the graph at the bottom. Figure 2: Comparative lentiviral transduction of encapsulated multicellular structures and hydrogel matrix-embedded multicellular structures. HEK-293 cells were encapsulated in alginate capsules with or without hydrogel matrix. After the formation of multicellular structures within the capsules, the capsule was removed or not, to only keep naked multicellular structures (spheroids) or matrix-embedded multicellular structures. All the conditions are then transduced with a lentiviral expression vector carrying the gene for the expression of Renilla Luciferase. The activity of Renilla was then measured by bioluminescence for each condition to quantify the transduction efficacy, and thus the capacity of the lentiviral particles to reach and penetrate the cells of the multicellular structures. The quantification results are shown in the graph and they are normalized to the conditions with the naked HEK-293 spheroids for which the Renilla activity was taken to be 100%.

Figure 3: Individual measurement of the beating rate (functional readout) in cardiac multicellular structures in microcavities. Spatial positioning in microcavities of cardiac multicellular structures (beating cardiac organoids) allows to perform single-organoid beating analysis. Spatial positioning: Cardiac multicellular structures are distributed in 96-well plate with microcavities. Each round structure in which individual multicellular structures are positioned corresponds to a microcavity. Movies are recorded before and after 2h of treatment with Verapamil (scale bar = 200 pm). Beating profiles: The beating profile of six cardiac multicellular structures (representative profiles of multicellular structures 1 and 2 are shown) were extracted using MYOCYTER, a macro for the free image processing software “ImageJ”. Numerical data extraction: The bar graph represents the beating rates recorded over 30 seconds for each cardiac multicellular structure before and after treatment, and the bulk average beating rates of all the cardiac multicellular structures and the subpopulations Pop. 1 and Pop. 2.

Figure 4: Concept of the method. Two different types of multicellular structures embedded either in color-coded fluorescent or non-fluorescent matrix in co-culture in a well with microcavities. After drug treatment and image acquisition, the analysis discriminates each type of tissue thanks to the color code of the hydrogel matrices and each multicellular structure is individually identified thanks to the stable spatial positioning in microcavities. The method allows the individual measurement of several readouts in each multicellular structure from each type of tissue in co-culture. Figure 5: Proof of concept of the method. Two distinct types of multicellular structures generated from two different breast cancer cell lines, SK-BR-3 and HCC1395, were embedded in either CF750 fluorescent dye (Cy7, far far-red) or Atto-647N (Cy5, far-red), respectively, and co-cultured in a well with microcavities. Following treatment with FITC-labelled Trastuzumab, images were acquired over a period of 120 minutes. The analysis differentiates the binding of Trastuzumab to each tissue type at t=0 and 120 minutes, utilizing the color coding of the hydrogel matrices and allow the measurement of Trastuzumab to each multicellular structure thanks to the spatial positioning in microcavities. This method allows for the simultaneous and independent measurement of the binding of a drug (readout) on each tissue type and on each multicellular structure in co-culture.

DETAILED DESCRIPTION OF THE INVENTION

The advantages of a drug testing methodology that combines multiplexed detection of tissues and biological processes, and physical separation of different tissue types, includes the obvious simple economic measures of experiment cost, labor, and time. It additionally encompasses non-obvious scientific conceptual advances that are unique to the current art. This includes the ability to identify drugs with potentially disparate effects on cells from distinct human organs. For example, a hepatocyte-metabolized drug may have extremely potent anti -turn or proliferation effects and yet exhibit high toxicity to lung cells at very low concentrations, as well as induce unexpected inflammatory responses from immune cells. Therefore, the ability to simultaneously detect the modulation of multiple biological process in different cell types allows unparalleled high-content screening of drug activity. Thus, the present invention offers unique advantages over the prior art because it is possible to down select drug candidates that would have otherwise progressed into the next round of the development process in the absence of simultaneous evaluation of biological processes. An additional favourable feature of the invention is the possibility to identify, more efficaciously, drugs with multiple desirable features such as potent biological activity in the target cells of interest, and low toxicity or inflammation of cells from vital organs, thus favoring the selection of potent compounds otherwise removed from the drug discovery pipeline with the use of traditional in vitro drug testing methods.

The embedment of cells in hydrogel matrices to prevent most direct contact of cell types coming from different organs, such as hepatocytes or cardiac cells, with the majority of other cell types is also advantageous because these cells do not normally come into direct contact with most other cell types in vivo. For example, hepatocytes often metabolize circulating drugs, however these cells do not come into direct contact with neurons or pulmonary cells etc. However, the hydrogel matrices allow relevant physiological interactions between embedded tissues and immune cells since the later are naturally able to interact with cells surrounded by extracellular matrices in the human body. As previously described, most other cell culture systems do not include a means to separately analyze multiple biological processes in distinct cell types, and the current art is therefore uniquely positioned to detect the effects of chemical entities, tested in a drug screen, on cells from a variety of tissues that together aim to better recapitulate an entire biological system.

The use of fluorescent hydrogel matrices makes the drug testing methodology compatible with the assessment of big molecules or big particles that are increasingly used in therapy: biologies, lipid nanoparticles, viral vectors. Hydrogel matrices drastically increase the scope of in vitro testing applications when compared to alginate capsule that restricts drug testing to small molecules.

In addition to the color-based identification of the different tissues of the co-culture, the spatial positioning of the embedded multicellular structures in microcavities allows the individual identification of each multicellular structure. Instead of performing batch analysis on all the multicellular structures embedded in the same color-coded hydrogel matrices, spatial positioning introduces an additional layer of discrimination to perform single-multicellular structure level analysis for each tissue type. Thus, coupling color-based identification of tissues with spatial positioning confers the unique capacity to individually measure multiple biological processes in each multicellular structures of the same tissue type in a multi-tissue co-culture.

It is one of the objects of the present invention to provide an in vitro method to test drugs, chemicals, biologies, and biological particles such as viral vectors, with the capacity to measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture.

The method comprises: a) a first step where one or several suspensions of cells representing one tissue type per cell suspension are independently mixed with a hydrogel matrix composed of a mixture of unlabelled biopolymers and, at least for one of said one or several suspensions of cells, one biopolymer labelled with a fluorophore conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the biopolymers by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures embedded in said mixture of biopolymers; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is embedded in a fluorescent hydrogel matrix; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the hydrogel matrix surrounding the multicellular structure; e) Optionally one tissue is genetically engineered to express a fluorescent or bioluminescent reporter gene; f) Optionally the multi-tissue co-culture is exposed to one or more drugs/compounds.

Analysis of the multi-tissue co-culture is performed before and/or after exposure to said drug/compound/biologics/viral vectors/carrier nanoparticles by staining the tissues with either one or several of the different methods described below: a) Immunolabelling performed on the multi-tissue system with antibodies recognizing a specific target and coupled directly or indirectly with fluorophore or any measurable activity; b) Biological effects are specifically measured with fluorescent dyes or bioluminescent dyes; c) One or several tissues express one or several bioluminescent and/or fluorescent reporter genes to measure specific biological effects; d) a), b), and c).

Acquisition is performed by fluorescence microscopy and/or multi-plate reader. Each tissue is discriminated thanks to the specific color code of the fluorescent hydrogel matrix in which it is embedded, multicellular structures from the same tissue are individually discriminated thanks to the stable spatial positioning in the microcavities, and the biological effect is thus specifically measured in said tissue and individual multicellular structures. 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 the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

As used herein the terms “subject” or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

The term “Multiplexed”, refers to drug testing that is capable of simultaneously detecting multiple biological processes as well as multiple tissue types based via their associated chemical or biological tags. In particular, the term “multiplexing” refers to a methodology to simultaneously measure multiple biological parameters in the same biological sample.

The term “spheroid” refers to a 3D agglomeration of cells, namely a multicellular structure, in the approximate shape of the sphere.

The term “hepatocyte” refers to any primary hepatocytes or any cell lines derived from healthy or transformed hepatocytes and includes for example liver cancer (LC) cell lines.

“Capsule”, refers to a polymeric sheet or layer produced by the microfluidics device, typically comprising of alginate in the current invention that is used to encapsulate or encase the mixture of cells with the hydrogel matrix, with cell-coating molecules, or to encapsulate or encase cells. It is known in the art that a capsule is a protective barrier which encloses a cell unit. This term is commonly used in the art to refer to semi-permeable or impermeable structures; in the context of the present invention, microcapsules are semi-permeable and allow the passage of the components of growth media and other reagents for the formation of multicellular structures.

The term “microfluidic” refers to technics handling liquids with a micrometric precision to form microdroplets, such as, but not limited to, encapsulation and bioprinting.

Microencapsulation, or encapsulation, is the enclosing of a cell unit in a microcapsule.

The term “ultralow attachment multiwell plate” refers to a cell culture multiwell plate preventing the cells to attach to the bottom surface of the well, thus forcing the cells to grow in suspension and to form 3D multicellular structures.

The term “hanging drop method” refers to a method where a cell suspension droplet is hanging vertically to force the cells to grow in suspension and to form multicellular structures.

As used herein, the term “culture conditions” refers to the environment which cells are placed in or are exposed to in order to promote growth or differentiation of said cells. Thus, the term refers to the medium, temperature, atmospheric conditions, substrate, stirring conditions and the like which may affect the growth and/or differentiation of cells. More particularly, the term refers to specific agents which may be incorporated into culture media and which may influence the growth and/or differentiation of cells. This includes supernatants or lysates after the culture of primary cells, cell lines, or microbes, or biological fluids or extracts derived from living organisms.

A “cell”, as referred to herein, is defined as the smallest structural unit of an organism that is capable of independent functioning, or a single-celled organism, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable cell membrane or cell wall. The cell may be prokaryotic, eukaryotic, animal or plant, or archaebacterial. For example, the cell may be a eukaryotic cell. Cells may be natural or modified, such as by genetic manipulation or passaging in culture, to achieve desired properties. A stem cell is defined in more detail below, and is a totipotent, pluripotent or multipotent cell capable of giving rise to more than one differentiated cell type. Stem cells may be differentiated in vitro to give rise to differentiated cells, which may themselves be multipotent, or may be terminally differentiated. Cells differentiated in vitro are cells which have been created artificially by exposing stem cells to one or more agents which promote cell differentiation.

The term “cells” is used in its general and broad context and defines cells as being either a cell line, primary cells from an organism or human, or bacterial, fungal, or plant cells. The properties of encapsulated cell culture, such as specific pressure forces exerted on encapsulated cells or the formation of 3D tissue like structures, may favour cell differentiation or growth that is otherwise challenging to attain in vitro. For example, encapsulation allows the enhanced expansion of pluripotent neuronal stem cells or chondrocytes, and facilitates long term maintenance of hepatocytes, thus providing additional translational relevance to the proposed drug testing methodology.

A “group” of cell units (or cells) is a plurality of such units which are not linked together. For example, a cell unit is not a group of cells, but one or more cells clustered together in one single unit. Single cells, and individual cell units, may be pooled to form a group of cells or cell units. Groups can be split, by dividing the groups into two or more groups of cells or cell units.

The terms “drug testing” is a generic term that includes drug screening. According to the invention, drug testing can comprise of screening molecule libraries to identify new chemical entities with the desired biological effect, but can also include testing of known or previously approved drugs in vitro to identify which drugs or drug combinations are the most appropriate for a patient, or to characterize the mechanisms or on- and/or off-target effects of new or previously identified drugs.

The term “biological particles” refers to vesicular-like structures made of nanomaterials, such as viral vector, liposomes, or lipid nanoparticles, used to deliver therapeutic product into cells such as proteins and/or nucleic acids.

The term “extracellular matrix” refers to a scaffold made of proteins and oligosaccharides forming an hydrogel between cells of the same tissue and ensuring the cohesion of a tissue.

The term “tissue” refers to an organized multicellular structure ensuring a physiological function into the human body. Tissues are considered different when they originate from different organs like liver versus brain, and/or when their physiological state is different like healthy versus disease, wild-type versus mutant, and/or neoplasic versus healthy, and/or when their physiological function is different within the same organ like a tissue formed from neurons ex vivo versus a tissue formed from glial cells ex vivo, in the case of the brain.

The term “organoid” refers to an in vitro multicellular structure derived from stem cells, following a physiologically relevant organization in 3D, and comprising several cell types naturally found in their corresponding tissue counterparts in the human body.

The term “biopsies” refers to tissues directly sampled from patients, such as blood sample, liquid or solid samples from organs, or tissue explants.

The term “co-culture” refers to the combination of two or more different types of cells or tissues together into the same cell culture medium.

The term “multi-tissue co-culture” refers to the combination of two or more different types of tissues together in the same container. The term “hydrogel” refers to a scaffold of biopolymers forming an hydrated gel after their gelation.

The term “biopolymer” refers to a complex biological molecule formed by the association of several oligomers or monomers of peptidic or oligosaccharidic types and that may eventually have the capacity to form gels after their gelation.

The term “coating” refers to the process of covering partially or integrally the surface of cells with a molecule without penetrating inside the cells.

The term “cell-coating molecule” refers to a molecule with the capacity to stably interact directly or indirectly with the plasma membrane of cells and to stay outside of the cytosolic compartment of the cells. A non-exhaustive list of cell-coating molecules comprises components of the extracellular matrix, synthetic alternatives to extracellular matrix, plasma membrane dyes.

The term “immunolabelling” refers to the staining of cells or tissues with antibodies recognizing a molecular target and coupled with a measurable signal that can be a fluorophore, an enzymatic activity, or a bioluminescent probe.

The term “fluorophore” refers to molecule emitting fluorescence at a specific emission wavelength after being excited by a light source at a specific excitation wavelength.

The term “fluorescence” refers to the emission of fluorescent light by a fluorophore after the excitation by another light source.

The term “bioluminescence” refers to the emission of light from an enzymatical reaction.

The term “reporter gene” refers to a gene reporting a specific biological activity, response, mechanism, function, or process thanks to an expression product with a measurable activity.

The term “phenotype” refers to an observable and/or measurable biological activity, response, mechanism, process, morphology, function, or behavior. A “label” or “tag”, as used herein, is a means to identify a cell unit and/or determine a culture condition, or a sequence of culture conditions, to which the cell unit has been exposed. Thus, a label may be a group of labels, each added at a specific culturing step; or a label added at the beginning or the experiment which is modified according to, or tracked during, the culturing steps to which the cell unit is exposed; or simply a positional reference, which allows the culturing steps used to be deduced. A label or tag may also be a device that reports or records the location or the identity of a cell unit at any one time, or assigns a unique identifier to the cell unit. Examples of labels or tags are molecules of unique sequence, structure or mass; or fluorescent molecules or objects such as beads; or radiofrequency and other transponders; or objects with unique markings or shapes. Because different fluorochromes may have overlap in their light emission spectra, the selection of different tags must be performed so that there is minimal or no fluorescence overlap between the different fluorochromes, or that the measurements are compensated using staining control samples, so that the interpretation of the imaging results is not compromised.

An “identifying label” is a label which permits the nature of the cell unit to which it is attached to be determined. This allows the exposure of cell units to different culture conditions to be recorded, by addition of an identifying label at each exposure, and subsequently deconvoluted by analysis of the labels.

A cell is “exposed to culture conditions” when it is placed in contact with a medium, or grown under conditions which affect one or more cellular process(es) such as the growth, differentiation, or metabolic state of the cell.

Thus, if the culture conditions comprise culturing the cell in a medium, the cell is placed in the medium for a sufficient period of time for it to have an effect. Likewise, if the conditions are temperature conditions, the cells are cultured at the desired temperature. The “pooling” of one or more groups of cell units involves the admixture of the groups to create a single group or pool which comprises cell units of more than one background, that is, that have been exposed to more than one different sets of culture conditions. A pool may be subdivided further into groups, either randomly or non-randomly; such groups are not themselves “pools” for the present purposes, but may themselves be pooled by combination, for example after exposure to different sets of culture conditions.

“Cell growth” and “cell proliferation” are used interchangeably herein to denote multiplication of cell numbers without differentiation into different cell types or lineages. In other words, the terms denote increase of viable cell numbers. Preferably proliferation is not accompanied by appreciable changes in phenotype or genotype.

“Cell differentiation” is the development, from a cell type, of a different cell type. For example, a bipotent, pluripotent or totipotent cell may differentiate into a neural cell. Differentiation may be accompanied by proliferation, or may be independent thereof. The term ‘differentiation’ generally refers to the acquisition of a phenotype of a mature cell type from a less developmentally defined cell type, e. g. a neuron, or a lymphocyte, but does not preclude transdifferentiation, whereby one mature cell type may convert to another mature cell type e. g. a neuron to a lymphocyte, or a monocyte to a macrophage.

The term “plurality” means more than one. In the context of labels, it describes the fact that each encapsulation allows at least one more label to be added, such that a multiply-encapsulated cell unit can be labelled with at least one label per encapsulation. In the context of cell units within microcapsules, two, three, four, five, six or more cell units can be included in a single micro-capsule.

The terms “phenotypic screening” refers to a type of screening used in biological research and drug discovery to identify substances such as small molecules, peptides, antibody or RNAi that alter the phenotype of a cell or an organism in a desired manner.

Fluorescent reagents are commonly used in Life Sciences laboratories to measure the activities of biological processes. For instance, combination of the fluorescent reagents DiOC6 and propidium iodide allows the detection and quantification of living (green fluorescence) and dead cells (red fluorescence) in a cell population. Dihydroethidium is a fluorescent probe to measure the production of reactive oxygen species in cells, which is an indicator of cellular stress, by becoming red into the cells after being oxidized. Coumarin boronate or fluorescein- boronate can be used to measure the production of nitric oxides into the cells, which are involved in many physiological processes including inflammation, by emitting blue or green fluorescence, respectively. Fura-2 is a fluorescent probe to measure the intracellular concentration of calcium which fluctuates upon stimulation of signal transduction pathways.

Incorporation of such reagents in the present invention helps to expand the spectra of biological activities that can be analyzed. However, instead of reporter genes, fluorescent reagents indifferently report a biological activity in all cells of a cell population. Indeed, fluorescent reagents are soluble molecules that freely diffuse after addition in the cell culture medium, thus staining all cells. Fluorescent reagents can be used in combination with fluorescent or bioluminescent reporter genes to measure an additional biological activity, sometimes for which no reporter gene may exist. To do so, fluorescent reporter gene activities have to be measured first because they specifically report biological activities in the cell types where they are expressed. Addition of the fluorescent reagent can then be performed to stain all cells. Colors of fluorescent matrices can still be used to separately measure the fluorescence of the reagent, and thus to measure the related biological activity, in a specific hydrogel-embedded tissue type in the cell co-culture. Also, several fluorescent reagents can be simultaneously used until their fluorescence spectra do not overlap.

In particular, the invention provides for an in vitro method to test drugs, chemicals, biologies, and/or biological particles, having the capacity to simultaneously measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the method comprises: a) a first step where one or several suspensions of cells representing one tissue type per cell suspension are independently mixed with an hydrogel matrix composed of a mixture of unlabelled biopolymers and, at least for one of said one or several suspensions of cells, one biopolymer labelled with a fluorophore conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the biopolymers by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures embedded in said mixture of biopolymers; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is embedded in a fluorescent hydrogel matrix; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the hydrogel matrix surrounding the multicellular structure; e) exposing said multi-tissue co-culture to said drugs, chemicals, biologies, and/or biological particles to be tested; f) analysis of the exposed multi-tissue co-culture is performed by staining the at least two different types of multicellular structures of step c) by either: i) performing an immunolabelling on the multi-tissue co-cultures with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe or; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) Performing both i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for the bioluminescence, in which each of the at least two different types of multicellular structures is discriminated through the specific color code of the fluorescent hydrogel matrix in which it is embedded, multicellular structures from the same tissue of said at least two different types of multicellular structures are individually discriminated through the stable spatial positioning in the microcavities of the multi well plates and whereas the biological effect of interest is specifically measured in said each multicellular structure of said at least two different types of multicellular structures.

According to an embodiment of the invention, the method comprises a molding step b) that can be alternatively performed by technics not requiring the use of a dissolvable capsule for the formation of multicellular structures, and selected from bioprinting, ultralow attachment multiwell plates, or the hanging drop method.

According to another embodiment, one of said one or several suspensions of cells of said at least two different types of multicellular structures of step c) is genetically engineered to express a fluorescent or bioluminescent reporter gene. In accordance with said embodiment, the analysis of step f) further comprises: iii) measuring biological effects of interest specifically in said at least two different types of multicellular structures expressing bioluminescent and/or fluorescent reporter genes.

Thus this allows, the analysis in step f) of the multi-tissue co-cultures of step c) which is performed by staining the at least two different types of multicellular structures by steps i), and/or ii) and/or iii).

According to an embodiment, the in vitro method of the invention is designed for drug screening or testing, wherein said multiple fluorescent and/or bioluminescent cellular readouts, that are detected from either labelled antibodies, fluorescent/bioluminescent dyes, or reporter genes, selected for biological processes that are investigated during phenotypic drug discovery process.

For example, the gene reporter is selected because it is a specific target that has previously been identified via mechanistic biological studies, i.e. a receptor with important roles in the pathogenesis of particular cancers or other specific diseases.

The choice of fluorochromes is such that spectral overlap between and amongst the fluorescent hydrogel matrices and fluorescent readouts is minimized or negated using proper controls when configuring the detection equipment (microscope or microplate reader).

In other words, an object of the invention consists of fluorescent readouts emitted from multicellular structures embedded in color-coded fluorescent hydrogel matrices. The combination of these two different types of fluorescent labelling (cellular readouts and hydrogel matrix labelling) allows the simultaneous monitoring of multiple biological processes in one or more cell types.

The panel of cellular readouts that can be simultaneously monitored includes key regulators or biomarkers of commonly investigated features i.e. biological processes, which can be detected in a drug test. These features include cell toxicity, cell proliferation, inflammation, hormone response, xenobiotic response, genotoxic stress response, mitochondrial dysfunction, apoptosis, necrosis, metabolic fluxes and gradients, hypoxia, and antibiotic resistance. In particular, the invention comprises of a method to characterize the effects of drugs on multiple cellular processes in multiple tissue types, simultaneously within the same test. To achieve this, the method employs tissues embedded in a fluorescently-labelled hydrogel matrix coupled with the measurement of fluorescent and/or bioluminescent cellular readouts, such as immunolabelling, chemicals probes, or reporter genes, so that one or more cellular process can be monitored through their fluorescent activities which are indicative of a biological process activity. Cellular readouts that are measured in the assays report biological process activities, including but not limited to: cell proliferation, xenobiotic stress response, inflammation, tumor growth or inhibition, hormone response, hypoxia, genotoxic stress, cytotoxicity or detoxification responses.

Several cellular readouts can be measured in the same tissue type to simultaneously assess different biological processes. In the case of cellular readouts based on reporter genes, to multiplex the analysis of multiple reporter genes, cells are divided into the required number of separate batches of cells, and each batch is engineered to contain a single reporter gene corresponding to a single biological process. The separate batches of cells are either pooled together to be encapsulated in the same capsule, or they are each encapsulated separately, to measure the activity of multiple biological processes either in mixed co-culture, or in physically separated co-culture, respectively. Multiplexed analysis of cellular readouts that correspond to different cellular processes is intended to provide many benefits including increased ability to predict the best drug candidates and economy of reagents, consumables, and time required to perform in-depth characterization of drug candidates.

Each type of hydrogel-embedded multicellular structure contains one or more cell types that are normally found in a single human organ or distinct physiological site such as a tumor. The generation of the hydrogel-embedded multicellular structures involves the preparation of separate single cell suspensions of different cell types. The suspensions are then mixed with an hydrogel biopolymer which has been previously labelled with one of many fluorescent markers that allows the identification of the hydrogel-embedded multicellular structures by microscopy.

When cell encapsulation is used as a molding method, encapsulated cells are incubated to facilitate the formation of 3D tissue-like structures called spheroids or organoids. After the dissolution of the alginate capsule, spheroids/organoids embedded in different color-coded fluorescent hydrogel matrix can then be mixed to create a co-culture of different tissue types, which may include but are not limited to, liver spheroids, cardiac organoids, primary cell spheroids, tumor spheroids, patient-derived spheroids/organoids.

The use of fluorescent hydrogel-embedded 3D cell cultures (i) prevents cell to cell contact of different cell types which are not typically found in the same human organ, and (2) enables the simultaneous analysis of different cell types within the same well, for example the toxicity of a drug can be separately evaluated on hepatocytes and cancer cells within the same co-culture.

The aim of co-culturing multiple cell types within the same well is to recapitulate multicellular microenvironments, tissues, or inter-organ systems that occur in the human body, in order to increase the clinical relevance of this drug testing approach. For example, co-culture of tumor cells and hepatocytes in separate hydrogel-embedded multicellular structures aims to simulate common processes after the administration of drugs in vivo, where the liver first metabolizes the drug and then drug metabolites are the main effectors of anti-tumor responses in distal anatomical sites.

The invention is also time and resource saving because multiple effects can be investigated simultaneously, for example anti-tumor effects as well as liver toxicity. This is important because drug-induced liver injury (DILI) is one of the main reasons that many drug candidates do not progress through the development process. Moreover, this allow the simultaneous and unbiased measurement of the efficacy and safety of a drug to predict the benefit/risk ratio of a drug to make decisions on its future development.

One unique advantage of the present invention relies in the ability to measure multiple biological processes in a co-culture of mixed cell types, which increases the efficiency and accuracy (more translational relevance to in vivo models) of drug testing.

Shared advantages of the current invention with other 3D co-culture or hydrogel-embedded cell-based drug testing methodologies is to increase the clinical relevance of the drug test via the following features, which are common to existing technologies: Hydrogel matrices act as a physical separation of different cell types that do not normally contact each other. Also, in vivo 3D spheroid cell cultures behave more like in vivo tissue compared to cell culture monolayers.

An important advantage of the cell embedment in a fluorescent hydrogel matrix for the identification of cell types is the permeability. Instead of the alginate capsule which is filtering out molecules with a size above 150 kDa, the hydrogel matrix allows the diffusion of very big particles of the size of lentiviral vectors (> IMDa). The range of possible in vitro applications is largely extended, from in vitro testing restricted to small molecules with the encapsulation of cells into alginate capsules, to the possibility to test biologies and lipid-based nanoparticles or viral vectors with the embedment of cells into hydrogel matrices.

Another important advantage of the cell embedment into a fluorescent hydrogel matrix is to allow cell-cell contacts between embedded cells and not-embedded cells. For example, immune cells have the capacity to make cell-cell interactions with any type of cells in the human body, despite most of them are surrounded by an extracellular matrix. The same type of biopolymer is used to embed the multicellular structures in hydrogel matrix, making it compatible with the interactions of multicellular structures with immune cells. Applications depending on cell-cell- interactions, such as applications in the field of immuno-oncology, are thus becoming possible. For example, a multi-tissue co-culture can be made by co-culturing neoplasic multicellular structures embedded in fluorescent hydrogel matrix together with immune cells in suspension in the co-culture cell medium. Such multi-tissue co-culture opens the possibility to measure the interactions of immune cells with the neoplasic multicellular structures and the resulting biological effects.

Further, the invention allows an increase in the number of biological processes that can be investigated simultaneously, as well as an increase in the repertoire of biological processes i.e. the creation of a library of cellular readouts to quantitate a large portion of biological process that are commonly investigated by pharmaceutical companies.

The combination of the capacity to categorize the different tissues based on a color code with the capacity to measure biological processes in individual multicellular structures from the same tissue based on a spatial positioning solve multiple issues inherent to co-cultures made of complex tissues. Indeed, in a multi-tissue co-culture, several tissues from different origins are mixed together, it is thus absolutely required to discriminate tissues from each other for a relevant analysis of the biological processes occurring in each tissue type. The color-based identification of each tissue type thanks to the embedment of multicellular structures in a fluorescent hydrogel matrix solves the incapacity to identify tissues in a multi-tissue co-culture where several tissue types are mixed in the same culture well. After the color-based categorization of the multicellular structures in the several types of tissues of a co-culture, it remains the problem that only batch analysis can be performed on each category of tissues. However, each category of tissue contains multiple multicellular structures with intrinsic variabilities. A batch analysis gives the average activity of the measured biological processes on the whole population of multicellular structures from the same tissue, but individual and specific activities in each multicellular structure are missed. It is very important to extract this type of information since it allows a precise and unbiased identification and characterization of biological effects in a multi-tissue co-culture. The spatial positioning in microcavities allows a fragmentation of the tissue categories into individual multicellular structures to perform singlestructure level analysis for each tissue type of the co-culture. The resulting statistical analyses are improved since sub-populations effects can be extracted with a potential statistical significance for each type of sub-population. The relevance of each sub-populations can then be evaluated to explain the variability or the stability of the biological effects on the tissue type considered by the analysis.

Preferably, at least one of said one or several tissue types is an hepatic tissue.

Advantageously, the one or several tissue type is selected from the list comprising hepatic, cardiac, neural, renal, bone marrow, blood, pulmonary, gastrointestinal, gastric, pancreatic, neoplasic , skin, embryonic, stem cells, endothelial, immune, bone, endocrine, muscle, or adipose.

According to one embodiment, the multiple biological processes to be analyzed are selected from the list comprising: cell proliferation, inflammation, cytotoxicity, tissue stress responses, detoxification responses, hormone responses, xenobiotic responses, genotoxic stress responses, apoptosis, necrosis, lipid accumulation, mitochondrial dysfunction, metabolic fluxes and gradients, or hypoxia.

Preferably, one or several tissue types of said at least two are genetically engineered to contain at least one fluorescent reporter gene for each multiple biological process to be analyzed.

According to another embodiment, said one or more drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready -to-use microcavity microwell plate to identify the effects of said one or more drugs, chemicals, biologies, and/or biological particles on said multiple biological processes within said one or several tissue types.

Preferably, more than one drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready-to-use microcavity microwell plate to identify interactions between said more than one drugs, chemicals, biologies, and/or biological particles.

Another object of the invention is to provide for an in vitro method based on cell coating to test drugs, chemicals, biologies, and/or biological particles, having the capacity to simultaneously measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the method comprises: a) a first step where one or more suspensions of cells representing one tissue type per cell suspension are independently mixed with fluorescent cell-coating molecules conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the fluorescent cell-coating molecules by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures coated with fluorescent cell-coating molecules; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is coated with fluorescent cell-coating molecules; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the fluorescently- coated multicellular structure; e) exposing said multi-tissue co-culture to said drugs, chemicals, biologies, and/or biological particles to be tested; f) analysis of the exposed multi-tissue co-culture is performed by staining the at least two different types of multicellular structures of step c) by either: i) performing an immunolabelling on the multi-tissue co-cultures with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe or; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for the bioluminescence, in which each of the at least two different types of multicellular structures is discriminated through the specific color code of the fluorescent cell coating, and multicellular structures from the same tissue of said at least two different types of multicellular structures are individually discriminated through the stable spatial positioning in the microcavities of the multiwell plates and whereas the biological effect of interest is specifically measured in said each multicellular structure of said at least two different types of multicellular structures.

Preferably, the molding step b) is alternatively performed by technics not requiring the use of a dissolvable capsule for the formation of multicellular structures, and selected from bioprinting, ultralow attachment multiwell plates, or the hanging drop method.

According to an embodiment, one of said one or several suspensions of cells of said at least two different types of multicellular structures of step c) is genetically engineered to express a fluorescent or bioluminescent reporter gene. Preferably, the analysis of step f) further comprises: iv) measuring biological effects of interest specifically in said at least two different types of multicellular structures expressing bioluminescent and/or fluorescent reporter genes.

A yet further object of the invention is to provide an in vitro kit based on cell-coating for use in drug testing by measuring the effects of one or more drugs, chemicals, biologies, and/or biological particles on multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures coated with cell-coating molecules representing at least two tissue types, wherein at least one type of multicellular structure is coated with specific color-coded fluorescent cell-coating molecules.

Preferably, at least one of said at least two tissue types is a hepatic tissue.

According to one embodiment, the at least two tissue types are selected from the list comprising hepatic, cardiac, neural, renal, bone marrow, blood, pulmonary, gastrointestinal, gastric, pancreatic, neoplasic, skin, embryonic, stem cells, endothelial, immune, bone, endocrine, muscle, or adipose.

According to another embodiment, the multiple biological processes to be analyzed are selected from the list comprising: cell proliferation, inflammation, cytotoxicity, tissue stress responses, detoxification responses, hormone responses, xenobiotic responses, genotoxic stress responses, apoptosis, necrosis lipid accumulation, mitochondrial dysfunction, metabolic fluxes and gradients, or hypoxia.

According to a preferred embodiment, one or several tissue types are genetically engineered to contain at least one fluorescent reporter gene for each multiple biological process to be analyzed.

Preferably, said one or more drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready -to-use microcavity microwell plate to identify the effects of said one or more drugs, chemicals, biologies, and/or biological particles on said multiple biological processes within said at least two tissue types.

More preferably, more than one drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready-to-use microcavity microwell plate to identify interactions between said more than one drugs, chemicals, biologies, and/or biological particles.

According to another embodiment of the invention, the combination of the spatial positioning with the color-based identification of fluorescent matrix-embedded multicellular structure can also be applied for the combination of the spatial positioning with the color-based identification of cells encapsulated in alginate capsules. Indeed, the combination of spatial positioning with cells encapsulated in color-coded capsules allows the individual identification of the multicellular structures based on their color and based on their spatial positioning. However, the capsules are not permeable to biologies and biological particles, limiting this combination to drug testing on small molecules.

Thus it is another object of the invention to provide an in vitro method based on fluorescent capsules to test drugs or chemicals, with the capacity to measure multiple biological processes in individual encapsulated multicellular structures representing several tissue types in coculture, the method comprises: a) separately encapsulating one or more cell types to be analyzed in the same in vitro drug testing assay with a biopolymer of alginate, wherein in the case there are more than one cell type, all, or all but one, of the respective capsules are labelled, each with a different type of fluorophore; followed by culturing said encapsulated cells to form encapsulated multicellular structures; b) at least two different types of encapsulated multicellular structures are combined together to form a multi-tissue co-culture and where at least one of said two different types of encapsulated multicellular structures is encapsulated in a fluorescent capsule; c) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities; d) exposing said multi-tissue co-culture to drugs, and/or chemicals e) analysis of the multi-tissue co-culture is performed by staining at least two different types of encapsulated multicellular structures of step b) by either: i) performing an immunolabelling on the multi-tissue co-culture with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for bioluminescence, in which each of the at least two different types of tissues is discriminated through the specific color code of the fluorescent capsule in which it is encapsulated and whereas encapsulated multicellular structures from the same tissue of said at least two different types of tissues are individually discriminated through the stable spatial positioning in the microcavities of the multiwell plates where the biological effect of interest is specifically measured in said each encapsulated multicellular structure of said at least two different types of tissues.

According to one embodiment of the invention, one of said at least two encapsulated multicellular structures of said multi-tissue co-culture of step c) is genetically engineered to express a fluorescent or bioluminescent reporter gene.

Preferably the analysis of step e) further comprises: iv) measuring biological effects of interest specifically in said at least two different types of multicellular structures expressing bioluminescent and/or fluorescent reporter genes.

It is yet another object of the invention to provide for an in vitro kit based on fluorescent capsules for use in drug testing by measuring the effects of one or more drugs or chemicals on multiple biological processes in individual capsules containing multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures encapsulated in alginate capsules representing at least two tissue types, wherein at least one type of capsule is labelled with specific color-coded fluorescent alginate. Preferably, at least one of said at least two tissue types is a hepatic tissue.

According to one embodiment of the invention, the at least two tissue types are selected from the list comprising hepatic, cardiac, neural, renal, bone marrow, blood, pulmonary, gastrointestinal, gastric, pancreatic, neoplasic, skin, embryonic, stem cells, endothelial, immune, bone, endocrine, muscle, or adipose.

Preferably, the multiple biological processes to be analyzed are selected from the list comprising: cell proliferation, inflammation, cytotoxicity, tissue stress responses, detoxification responses, hormone responses, xenobiotic responses, genotoxic stress responses, apoptosis, necrosis lipid accumulation, mitochondrial dysfunction, metabolic fluxes and gradients, or hypoxia.

According to another embodiment, one or several tissue types are genetically engineered to contain at least one fluorescent reporter gene for each multiple biological process to be analyzed.

Preferably, said one or more drugs or chemicals are tested per well of said at least one ready- to-use microcavity microwell plate to identify the effects of said one or more drugs or chemicals on said multiple biological processes within said at least two tissue types.

More preferably, more than one drug or chemicals are tested per well of said at least one ready- to-use microcavity microwell plate to identify interactions between said more than one drug or chemicals.

One advantage of the in vitro drug testing kit is that it can be used for phenotypic (affects general biological processes with common cellular readouts for nearly all cells) or target-based (modulation of specific pathways that might only be present in some cells e.g. estrogen receptor, and have been previously identified as ideal therapeutic targets) drug discovery.

In accordance with one embodiment of the invention, one or more drugs are tested per well of said at least one ready-to-use microcavity microwell plate to identify the effects of said one or more drugs on biological processes within said hydrogel-embedded tissue types. In accordance with one another embodiment of the invention, more than one drug are tested per well of said at least one ready-to-use microcavity microwell plate to identify interactions between said drugs.

In particular, one or more drugs are tested per well to identify the effects of a drug on biological processes within encapsulated tissues of interest, or alternatively interactions between multiple drugs e.g. synergy or dysergy of the modulation of biological processes including but not limited to cell cytotoxicity or proliferation.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

Examples

Example 1:

Description :

Multicellular structures composed of human non-cancerous embryonic kidney cells (HEK293, Tablet) embedded in non-fluorescent matrix and multicellular structures composed of human breast cancer cells (MCF7, Tablet) embedded in fluorescent matrix are co-cultured. The proliferation status is independently determined in each cell type by measuring the Ki-67 protein expression level after an image segmentation based on matrix colors.

Material and methods:

Protocol for the encapsulation of cells:

Encapsulation of cells:

Aspirate the culture medium on the cells (see Table 1)

Wash once with PBS and aspirate the PBS

Add Trypsin on the cells and aspirate the excess of trypsin

Incubate 5 minutes at 37°C

Repeatedly pipette cell culture medium (DMEM with phenol red 10% Fetal Bovine Serum) over the cells to detach and then collect them into a tube

Count the cells and prepare a cell suspension with 5 million cells per ml

Pour the cell suspension through a 40 pm Corning filter into a 50 mL Falcon tube

Put 1 mL of the cell suspension (i.e. 5 million of cells) into a 1.5 mL Eppendorf tube Centrifuge cells at 1000 rpm for 5 minutes to pellet them and resuspend them in Sorbitol 300 pM (90 or 112.5 pL)

Keep the cells on ice

Table 1: Cell lines and cell culture conditions used for the experiment of the example 1.

Encapsulation of cells:

Encapsulation is performed with a patented device (WO/2013/113855) as described in Alessandri K, Feyeux M, Gurchenkov B, Delgado C, Trushko A, Krause KH, Vignjevic D, Nassoy P, Roux A. Lab Chip. 2016 Apr 26; 16(9): 1593-1604. Doi: 10.1039/c61c00133e.

Prepare the cells for encapsulation:

Note: Work on ice • For cells embedded in unlabeled matrix: Take 112.5 pL cells + 37.5 pL of Matrigel Basement Membrane Matrix, *LDEV-Free (Coming, ref 356234)

• For cells embedded in fluorescent labelled matrix: Take 90 pL cells + 20 pL of Matrigel Basement Membrane Matrix, *LDEV-Free (Coming, ref 356234) mixed with Laminin Red Fluorescent, Rhodamine (Cat#: LMN01-A) (10% m:m)(Resuspend in lOpL ddH2O cold to get 2 mg/mL)

Collect capsules into 15-cm petri dish containing calcium chloride solution

Transfer the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in DMEM medium

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in DMEM without phenol red 10% Fetal Bovine serum Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in DMEM without phenol red 10% Fetal Bovine serum Transfer the capsules in a fresh 10 cm dish

Incubate capsules at 37°C, 5% CO2.

Co-culture of capsules:

After two weeks in separated culture medium, encapsulated cells formed spheroids (multicellular structures) in the capsules :

Transfer 10 mL of medium with the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant

Resuspend in 3 mL medium

Pipet 1 mL of encapsulated HEK293 spheroids and pipet 2 mL of encapsulated MCF7 spheroids.

Resuspend in a well of a 6 well plate

Incubate 72h at 37°C, 5% CO2.

Protocol for the removing the alginate capsules:

Transfer the capsules into Eppendorf tubes

Centrifuge 1 min at 600 rpm

Wash once with PBS without calcium nor magnesium

Incubate 10 min at RT in PBS without calcium nor magnesium Check under the microscope that the capsule is removed and put the spheroids back into wells of a 6-well plate.

Immunostaining:

Solutions preparation:

Fixation solution (50 mL):

Dissolve in 50 mL PBS warmed at 60°C (Gibco, 910288 with Ca/Mg or Pan Biotech P04-36100 without Ca/Mg)

- 2g PF A (Sigma, 158127-100G)

Note 1 : It will dissolve completely in PBS without calcium.

Note 2: For PBS with calcium, it will stay turbid. Add few drops of NaOH IM following the pH. It will reach 11. Then put back in the back until dissolution. The solution is still turbid, however, when the pH is re-adjusted to pH 7.4 with HC1 37 %, it will be transparent (add one drop at the time as the pH will drop extremely fast). The pH of PFA-PBS without calcium is already at pH7.2-7.4

Filter solution and store in 15 mL falcon tubes with 5 mL at -20°C.

Basis solution (200 mL) :

200 mL of PBS (Gibco, 910288 with Ca/Mg or Pan Biotech P04-36100 without Ca/Mg)

- Add 400 pL TRITON X-100 ie 0.2% (Sigma Aldrich, 102386719)

Filter and keep at 4°C

Permeabilization solution (10 mL):

10 mL of Basis solution

- Add 0.5mL DMSO (Fisher Chemical, D/412/PB15) Filter and keep at 4°C

Blocking/Antibody solution (50 mL):

50 mL of Basis solution

- Add 1.5 g BSA i.e. 3%

Filter and keep at 4°C

Wash solution (100 mL):

100 mL of Basis solution Add 1 mg Heparin (Heparin Sodium salt from porcine intestine, Sigma Aldrich, ref

H3393-1 OKU)

Filter and keep at 4°C

Remove the supernatant from the wells

Add 400 pL of Fixation solution with calcium, 1H, RT

Remove the supernatant

Add 400 pL of Permeabilization solution, 1H, RT

Remove the supernatant

Add 400 pL of Blocking solution, 1H, RT

Remove the supernatant

Split the samples:

■ 1 : with Hoechst only

■ 2: with Hoechst and secondary antibodies

■ 3 : with primary and secondary antibodies as well as Hoechst

Add 400 pL of Blocking solution with 1/200 of primary antibodies, O/N, 4°C

Ki-67 (D3B5) Rabbit mAb (Cell signaling, ref 9129T)

Wash 3xl5min with Wash solution at RT

Add 400 pL of Blocking solution with 1/200 of each secondary antibodies and 10 pg/ mL Hoechst, 1H, RT

Cy™5 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, 711-175-152) Hoechst 33342 Solution (Stock: 12.3 mg/mL, Thermo Fisher Scientist, ref 62249) diluted at 1 mg/mL in BPS

Wash 3xl5min with Wash solution at RT

Resuspend in FluoroBrite

Coat p-Plate 96 Well Black (IBIDI, ref 89626) with 80 pL of Poly-D-Lysin (0, Img/mL) (Gibco, ref A38904-01) for 10 min at RT

Remove the excess and wash once with FluoroBrite

Dispense resuspended spheroids into 96-well plate Measurement of fluorescent intensities of multicellular structures (spheroids) in coculture

Image acquisition of the 96-well plate containing the multicellular structures in co-culture is performed with an automated confocal microscope (IXM-C, Molecular Devices™). A lOx objective is used and 10 z-steps are performed on 16 different fields per well and for each fluorescent channel.

After image acquisition, an image analysis is developed in two steps:

1- Creation of masks: o The identification of the multicellular structures using the fluorescent signal of the nuclei (DAPI channel) o Creation of a mask for the multicellular structures embedded into a fluorescent matrix (Cy3 channel) o Creation of a mask for the nuclei count (DAPI channel) o Creation of a mask for the proliferating cells (Cy5 channel)

2- Extraction of parameters: o Identification of multicellular structures present or absent from the Cy3 channel o The area occupied by the nuclei in each type of multicellular structure. o The area occupied by the proliferating cells in each type of multicellular structure.

The ratio of the two last parameters allows the quantification of the proliferation cells percentage per type of multicellular structure, i.e. by cell type.

Results:

In Figure 1, Applicants observe by confocal microscopy, the population of human non- cancerous embryonic kidney cells (HEK-293) embedded in non-fluorescent matrix (unlabeled multicellular structures) and human breast cancer cells (MCF-7) embedded in fluorescent matrix (fluorescent multicellular structures) in co-culture.

The image segmentation enables: the recognition of both populations; unlabeled and fluorescent multicellular structures, the identification of the total cell count, and the determination of proliferating cells. The image segmentation allowed to determine that 18% of the multicellular structures are human breast cancer cells and the other 82% are human non-cancerous embryonic kidney cells. In addition, 30% of cells present in the multicellular structure composed of human breast cancer cells proliferate, while 17% of cells present in the multicellular structure composed of human non-cancerous embryonic kidney cells proliferate. These results confirm that the method of embedding the cells in color-coded fluorescent matrix allowed the discrimination of each cell type of the co-culture to simultaneously measure several readouts, here the proliferation with Ki67 immunostaining and the cell number with Hoechst staining, in each type of multicellular structure.

Example 2:

Description :

Multicellular structures composed of human non-cancerous embryonic kidney cells (HEK-293, Table 2) embedded in non-fluore scent matrix or not, and encapsulated HEK-293 embedded in non-fluorescent matrix or not, were transduced with a lentivirus to allow the stable expression of the Renilla Luciferase. The expression level of Renilla was assessed in presence or in absence of biopolymer capsule with the Renilla Luciferase Assay Kit measuring bioluminescence signal on plate-reader.

Material and methods:

Protocol for the encapsulation of cells:

Encapsulation of cells:

Aspirate the culture medium on the cells (see Table 2)

Wash once with PBS and aspirate the PBS

Add Trypsin on the cells and aspirate the excess of trypsin

Incubate 5 minutes at 37°C

Count the cells and prepare a cell suspension with 5 million cells per ml

Pour the cell suspension through a 40 pm Corning filter into a 50 mL Falcon tube Put 1 mL of the cell suspension (i.e. 5 million of cells) into a 1.5 mL Eppendorf tube Centrifuge cells at 1000 rpm for 5 minutes to pellet them Keep the cells on ice

Prepare the cells for encapsulation:

Resuspend pellet in Sorbitol 300 pM: a. For cell encapsulation without extracellular matrix: Resuspend cells in 150 pL of Sorbitol solution b. For cells embedded in unlabeled matrix: Take 112.5 pL cells + 37.5 pL of Matrigel Basement Membrane Matrix, *LDEV-Free (Coming, ref 356234) and work on ice.

Collect capsules into 10cm petri dish containing calcium chloride solution

Transfer the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in DMEM medium

Centrifuge 2 min at 300 rpm

Incubate capsules for 7 days at 37°C, 5% CO2.

Protocol for the lentiviral production:

Thraw 3 plasmids:

PS-PAX2 (viral enveloppe)

PM-D2G (viral machinery) PLenti-Renilla

Polyethylenimine (PEI): cationic polymer that forms a complex of transfection with the plasmids.

Trypsin HEK-293 (confluent) and resuspend them in 8 mL.

Wash lx with DMEM-5% Charcoal-stripped serum and pipet 300 pL in 2x 10 cm dish containing 5 mL of DMEM-5% Charcoal-stripped serum

In an Eppendorf tube: pipet 400 pL of PBS (200 ul/transfection) and add 5 ug of total DNA with the ratio 4:2: 1 (Renilla: PAX:PM-D2G, 5.7ug:2.8ug: 1.4ug, 1.8uL:0.7uL:0.3uL)

Mix at ratio 1 :3 with PEI so add 30 pL/transfection. Pipet up and down and vortex. Incubate at room temperature for 15 minutes.

Resuspend and drop homogenously on the plate 200 pL of the mix.

Incubate at 37°C, 5% CO2 overnight.

In the morning exchange the medium with 5 mL of DMEM medium

In the evening, harvest the virus so remove the supernatant and store it in a falcon tube and let in the fridge

Add 5 mL of fresh DMEM medium

Redo those 3 last steps the following day.

HEK-293 infection (viral transduction):

In 96-well plate:

Pipet 100 pL of DMEM complete medium and 5 pL of capsules with or without extracellular matrix

For the conditions requiring decapsulation:

Transfer the capsules into Eppendorf tubes

Centrifuge 1 min at 600 rpm

Wash once with PBS without calcium

Incubate 10 min at RT in PBS without calcium

Check under the microscope that the capsule is removed.

Remove medium for all conditions

Add 100 pL of medium containing virus

Incubate for 3 days at 37°C, 5% CO2. Remove virus medium (infected condition) and replace with fresh medium Incubate capsules for 2 days at 37°C, 5% CO2.

Renilla expression measurement:

Cell Lysis:

For 96 well plate (100 pL /well):

Add 25 pL of lysis buffer (Renilla Luciferase Assay Kit ,Ref E194A, Promega)

Agitate 30 min at room temperature, 550 rpm on a thermomixer

Measure Renilla activity:

Prepare a mix : Dilute 1/50 Stop and Glow Substrate in Stop and Glow

Add 10 pL on the wells

Shake manually the plate

Read the bioluminescence with the Biotek Cytation Multireader

Results:

In Figure 2, Applicants observe that the Renilla is very poorly expressed in multicellular structures composed of encapsulated HEK-293 embedded in non-fluorescent matrix (4%) and in encapsulated HEK293 (16%), where the capsule is present in both cases. On the contrary, once the capsule is dissolved, Renilla is highly expressed; in multicellular structures composed of HEK-293 embedded in non-fluorescent matrix (43%) and in naked HEK293 multicellular structures (100%).

These results confirm that the method of embedding multicellular structures in fluorescent matrix is compatible with the diffusion of big molecules (biologies), like viral vectors. The presence of color-coded fluorescent matrix allows tissue identification if at least two tissue type are present in co-culture.

Example 3:

Description :

Cardiac multicellular structures were distributed into a 96-well plate with microcavities. Movies were recorded with a confocal microscope set up stream mode, before and after 2h of treatment with Verapamil. Verapamil is a calcium channel blocker medication used for the treatment of high blood pressure, and angina. The spatial positioning of individual cardiac multicellular structures allows to perform single-organoid beating analysis. Material and methods:

Generation of cardiac multicellular structures:

Generation of iPSC (induced Pluripotent Stem cells)-Cardiomyocytes

The generation of cardiomyocytes from iPSC was performed using the cell line DF19-9- 11.T.H (Healthy adult, no cardiomyopathy, see Table 3) maintained in mTeSR™ Plus medium (Stemcells Technologies).

The protocol used for cardiac differentiation and the different media composition is described in Mills et al., 2017, as well as in Mills et al., 2019 (https://doi.org/10.1073/pnas.170731611) At day 15 after the beginning of the differentiation process, iPSC-CMs start beating and are transferred to the Control Medium

At day 16 after the beginning of the differentiation process, iPSC-CMs present a regular beating and are used for encapsulation.

Encapsulation of cells

Aspirate the culture medium on the cells (see Table 3)

Wash once with PBS and aspirate the PBS

Add Accutase® Cell Detachment Solution (Thermo Fischer Scientific) and leave on cells

Incubate 5 minutes at 37°C

Repeatedly pipet Accutase® over the cells to detach, collect, and filter through a 100 pm Corning filter into a 50 mL Falcon tube

Count the cells and prepare a cell suspension with 4 million cells per ml

Centrifuge cells at 1000 rpm for 5 minutes to pellet them

Keep on ice from now on until the generation of capsules.

Resuspend the pellet in 112.5 pL of Sorbitol buffer 300 pM with 37.5 pL of Matrigel Basement Membrane Matrix (Coming, ref 356234) i.e., 25% final volume.

Table 3: Cell line and cell culture conditions for the experiment of the example 3

Collect capsules into 15-cm petri dish containing calcium chloride solution 100 mM with 0.1%

Tween

Transfer the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in a-MEM medium

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in Control Medium

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in Control Medium

Transfer the capsules in a fresh 15-cm dish

Incubate capsules for 14 days at 37°C, 5% CO2. iPSC-CM organoids maturation

For the maintenance of the capsules, change the medium of the capsules every 2-3 days

Then, transfer the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in Maturation Medium (Recipe described in document

WO 2015/040142 Al)

Incubate capsules for 5 days at 37°C, 5% CO2.

Change the medium of the capsules every 2-3 days

Preparation of the plate and drug treatment

Preparation of the plate

For the experiment, use Elplasia® 96-well Clear bottom plate (Corning, ref 4442)

Rince the well that will be used with 160 pL of Maturation Medium

Pipet up and down until all the air bubbles are removed from the microcavities Preparation of the assay

Transfer the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant leaving 1 mL

Resuspend the capsules and transfer into a 1.5 mL falcon tube

Centrifuge 2 min at 300 rpm

Pipet 40 pL of pelleted capsules into the 96-well

Pipet up and down to homogenize.

Incubate the plate overnight at 37°C, 5% CO2

Record movie of cardiac multicellular structures (see: Measurement of beating rate of multicellular structures in wells with microcavities)

Add 100 nM of Verapamil (from 10 mM DMSO stock). Intermediate dilutions were performed in Maturation Medium

After the addition of the drug, pipet gently 2x up and down staying at the surface of the medium to prevent multicellular structures displacement

Incubate the plate 2 hours at 37°C, 5% CO2

Measurement of beating rate of multicellular structures in wells with microcavities

Acquisition

Movies acquisition of the 96-well plate containing the multicellular structures is performed with an automated confocal microscope (IXM-C, Molecular Devices™). A lOx objective and the stream acquisition mode (Transmitted light, TL25 open without z-stack) are used. The recording is performed on 16 different fields per well with the following settings: 20 fps (Frame per second) for 30 seconds

Data analysis

After the acquisition, an analysis is developed in two steps:

3- Concatenate time lapse images

The movies acquisition generates 3 files corresponding to the 30 seconds movie divided into 3 In the software “Image!”, go to Image -> Stacks -> Tools -> Concatenate and order your open files into a single, T-stacked file

4- Select the ROI (Region Of Interest) and parameters analysis

For this analysis, we use MYOCYTER, a free macro for the free image processing software ImageJ. Open all the movies recorded before treatment

Create ROIs only on cardiac multicellular structures. Myocyter creates a folder to report the ROIs selected for every movie.

Open the movies recorded after treatment and recall the ROIs previously created Manually choose the ref frame for each ROI

Run the analysis with a detection at 4

5- Extraction of parameters (directly created by Myocyter):

We extracted the dataplots corresponding to the detected beatings over time

We also extracted the beating rate value for each cardiac multicellular structure before and after treatment.

Results:

In Figure 3, Applicants observe the unique spatial positioning of each cardiac multicellular structure in Corning® Elplasia® 96-well Clear bottom plate using transmitted light microscopy. From the movies that were recorded before and after treatment with Verapamil, single analyses were performed using MYOCYTER macro. Thus, one can independently observe the beating profiles (functional readout) of three representative cardiac multicellular structures in the same field, out of a total of six different cardiac multicellular structures measured in the same well. Before treatment, the beating rate is regular although different for each of the multicellular structure; 35 and 19 beatings per minute for the multicellular structure 1 and 2, respectively. After treatment with Verapamil, the beating rates drastically decreased with 0 and 7 beatings per minute for the multicellular structures 1 and 2, respectively. The graph at the bottom summarizes the results of the beating rates obtained for the six cardiac multicellular structures before and after treatment with Verapamil. The multicellular structures 1, 2, 3, and 4 (responders) showed a strong decrease of the beating rate after treatment with Verapamil while the multicellular structures 5 and 6 (not-responders) showed no effect. The average beating rate of all multicellular structures is not significantly different before and after treatment with Verapamil. However, after organizing the results in two sub-populations of multicellular structures, i.e. not-responders (pop. 1) vs responders (pop. 2), the responders showed a significant decrease of the beating rate after treatment with Verapamil while the not-responders sub-population showed no variation in the beating rate after treatment with Verapamil.

These results confirm the capacity of multicellular structure spatial positioning to individually measure readouts in different multicellular structures within the same well, allowing to capture variations of the biological effects of a drug in the same population of multicellular structures. This enables the creation of subpopulations, if necessary, to increase the statistical significance of a relevant biological effect, here the decrease of the cardiac beating rate by Verapamil, and to better observe potential variabilities of biological effects within the same sample, here a minority subpopulation of not-responders cardiac multicellular structures.

Example 4:

Schematic illustrating the concept of the method described herein. In this example, two different types of multicellular structures, the tissue 1 embedded in fluorescent matrix and the tissue 2 embedded in non-fluorescent matrix, are loaded in a well with microcavities and co-cultured. After drug treatment and image acquisition, an analysis is performed where the different types of multicellular structures are discriminated and identified thanks to the color code of the hydrogel matrix in which they are embedded. After the tissue type identification, each multicellular structure is individually identified thanks to the stable spatial positioning in microcavities, allowing to perform an individual measurement of several readouts (readouts 1 and 2) in each multicellular structure from each type of tissue in co-culture.

Example 5:

Description:

Multicellular structures composed of human breast cancer cell line overexpressing Her2 (SK- BR-3, Table 1) embedded in dye amine C750 matrix and multicellular structures composed of human triple-negative breast cancer cell line (HCC1395, Table 1) embedded in Atto647N matrix are co-cultured in wells containing microcavities and treated with FITC-labelled Trastuzumab. The selective binding of Trastuzumab to each type of multicellular structure is determined by measuring the average intensity of FITC-labelled Trastuzumab on multicellular structures after an image segmentation based on matrix colors.

Material and methods:

Protocol for the encapsulation of cells:

Encapsulation of cells:

Aspirate the culture medium on the cells (see Table 4) Wash once with PBS and aspirate the PBS

Add Trypsin on the cells and aspirate the excess of trypsin Incubate 5 minutes at 37°C

Count the cells and prepare a cell suspension with 5 million cells per ml

Keep the cells on ice

Table 4: Cell lines and cell culture conditions used for the experiment of the example 5.

Matrigel Labelling:

Use 300 pL of Matrigel Basement Membrane Matrix, *LDEV-Free (Corning, ref 356234), corresponding to 3 mg of proteins.

Add 90 pg of CF® dye amine C750 (Biotium, ref BIO92102) or ATTO 647N (Sigma- Aldrich, ref 95349), previously resuspended at 5 mg/mL in acetonitrile.

Homogenize well and incubate for 1 hour at 4°C.

Briefly spin the dye-coupled Matrigel in a pre-chilled centrifuge to remove air bubbles from the mixture.

Encapsulation of cells: Encapsulation is performed with a patented device (WO/2013/113855) as described in Alessandri K, Feyeux M, Gurchenkov B, Delgado C, Trushko A, Krause KH, Vignjevic D, Nassoy P, Roux A. Lab Chip. 2016 Apr 26; 16(9): 1593-1604. Doi: 10.1039/c61c00133e.

Prepare the cells for encapsulation:

Note: Work on ice

For cells embedding in fluorescently labelled matrix: Take 112.5 pL cells + 37.5 pL of dye- coupled Matrigel.

Collect capsules into 15-cm petri dish containing calcium chloride solution

Transfer the capsules in a 15 mL falcon tube

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in DMEM medium

Centrifuge 2 min at 300 rpm

Remove the supernatant and resuspend in McCoy's 5A Medium with 10% Fetal Bovine serum for SK-BR-3 or in RPMI 1640 Medium (ATCC modification) with 10% Fetal Bovine serum and ImM Strontium chloride hexahydrate for HCC1395.

Centrifuge 2 min at 300 rpm

Incubate capsules at 37°C, 5% CO2.

Alginate capsules removal:

Transfer 10 mL of medium with the capsules into a 15 mL Falcon tube.

Centrifuge for 2 minutes at 300 rpm.

Remove the supernatant.

Resuspend a 1 : 1 mix for each cell type into an Eppendorf tube and exchange the medium with FluoroBrite DMEM, 10% Fetal Bovine Serum, 1% GlutaMAX.

Resuspend in a well of a 96-well plate with microcavities (Coming, ref 4442).

Remove the medium from the well.

Wash once with PBS without calcium or magnesium.

Remove the PBS from the well. Resuspend in PBS without calcium or magnesium with 0.5M EDTA pH 8. Wait for 2 minutes at room temperature.

Remove the solution

Wash once with FluoroBrite DMEM

Resuspend in FluoroBrite DMEM with 10% Fetal Bovine Serum.

Drug treatment:

Add 20 nM of FITC-labelled Trastuzumab (Human ErbB2/Her2 Alexa Fluor® 488- conjugated Antibody, Biotechne, ref: FAB9589G)

Measurement of fluorescent intensities of multicellular structures (Spheroids) in coculture treated with FITC-labelled Trastuzumab

Image acquisition of the 96-well plate containing the multicellular structures in co-culture is performed with an automated confocal microscope (IXM-C, Molecular Devices™). A lOx objective is used, and 10 z-steps are performed on 9 different fields per well for each fluorescent channel. Images are acquired every 15 minutes over a period of 120 minutes.

After image acquisition, the analysis is developed in two steps:

1. Creation of Masks: o Identification of the types of multicellular structures using the fluorescent signal of the Matrigel (Cy5 and Cy7 channels). o Creation of a mask for spheroids bound to Trastuzumab (FITC channel). o Position-based identification of each multicellular structure from each cell type in microcavities.

2. Extraction of Parameters: o The average intensity of FITC in the multicellular structures masked in Cy5 or Cy7 channels is extracted for each time point and for each positioned multicellular structure.

Results:

In Figure 5, confocal microscopy reveals the differential binding profile of FITC-labelled Trastuzumab to the multicellular structures composed of the human breast cancer cell line SK- BR-3, embedded in a dye amine C750 matrix, and HCC1395 cells, embedded in an Atto647N matrix, when co-cultured and treated with FITC-labelled Trastuzumab. The binding selectivity is assessed by measuring the average intensity of FITC-labelled Trastuzumab on each of these structures, following image segmentation based on matrix colors and individual position-based identification.

Image segmentation allows for:

• Recognition of both cell populations labeled with different fluorescent matrigels.

• Analysis of the kinetics of FITC-labelled Trastuzumab binding to each of the cocultured multicellular structures with a precision at the single multicellular structure level thanks to the spatial positioning in microcavities.

• Identification of selective binding of FITC-labelled Trastuzumab to SK-BR-3 cells.

The image segmentation revealed that multicellular structures composed of HCC1395 cells do not bind with Trastuzumab. In contrast, the SK-BR-3 structures, which express the HER2 receptor, show progressive binding over time, with maximum binding observed at 120 minutes. This represents a 140% increase in binding compared to the control multicellular structures composed of HCC1395 cells. The quantification of the increase in binding of Trastuzumab to SK-BR-3 compared to HCC1395 was performed from multicellular structures co-cultured in the same well, thus avoiding any comparative bias to conclude on the selective binding of Trastuzumab.

Claims

1. An in vitro method to test drugs, chemicals, biologies, and/or biological particles, having the capacity to simultaneously measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the method comprises: a) a first step where one or more suspensions of cells representing one tissue type per cell suspension are independently mixed with a hydrogel matrix composed of a mixture of unlabelled biopolymers and, at least for one of said one or several suspensions of cells, one biopolymer labelled with a fluorophore conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the biopolymers by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures embedded in said mixture of biopolymers; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is embedded in a fluorescent hydrogel matrix; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the hydrogel matrix surrounding the multicellular structure; e) exposing said multi-tissue co-culture to said drugs, chemicals, biologies, and/or biological particles to be tested; f) analysis of the exposed multi-tissue co-culture is performed by staining the at least two different types of multicellular structures of step c) by either: i) performing an immunolabelling on the multi-tissue co-cultures with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe or; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for the bioluminescence, in which each of the at least two different types of multicellular structures is discriminated through the specific color code of the fluorescent hydrogel matrix in which it is embedded, multicellular structures from the same tissue of said at least two different types of multicellular structures are individually discriminated through the stable spatial positioning in the microcavities of the multi well plates and whereas the biological effect of interest is specifically measured in said each multicellular structure of said at least two different types of multicellular structures.

2. The in vitro method to test drugs, chemicals, biologies, and/or biological particles according to claim 1, wherein the molding step b) is alternatively performed by technics not requiring the use of a dissolvable capsule for the formation of multicellular structures, and selected from bioprinting, ultralow attachment multiwell plates, or the hanging drop method.

3. The in vitro method to test drugs, chemicals, biologies, and/or biological particles according to any one of the preceding claims, wherein one of said one or several suspensions of cells of said at least two different types of multicellular structures of step c) is genetically engineered to express a fluorescent or bioluminescent reporter gene.

4. The in vitro method to test drugs, chemicals, biologies, and/or biological particles according to claim 3, wherein analysis of step f) further comprises: iv) measuring biological effects of interest specifically in said at least two different types of multicellular structures expressing bioluminescent and/or fluorescent reporter genes.

5. An in vitro drug testing kit for use in measuring the effects of one or more drugs, chemicals, biologies, and/or biological particles on multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures embedded in an hydrogel matrix representing at least two tissue types, wherein at least one type of multicellular structure is embedded in a specific color-coded fluorescent hydrogel.

6. The in vitro drug testing kit for use according to claim 5, wherein at least one of said at least two tissue types is a hepatic tissue.

7. The in vitro drug testing kit for use according to any one of claims 5-6, wherein the at least two tissue types are selected from the list comprising hepatic, cardiac, neural, renal, bone marrow, blood, pulmonary, gastrointestinal, gastric, pancreatic, neoplasic, skin, embryonic, stem cells, endothelial, immune, bone, endocrine, muscle, or adipose.

8. The in vitro drug testing kit for use according to any one of claims 5-7, wherein the multiple biological processes to be analyzed are selected from the list comprising: cell proliferation, inflammation, cytotoxicity, tissue stress responses, detoxification responses, hormone responses, xenobiotic responses, genotoxic stress responses, apoptosis, necrosis lipid accumulation, mitochondrial dysfunction, metabolic fluxes and gradients, or hypoxia.

9. The in vitro drug testing kit for use according to any one of claims 5-8, wherein one or several of said at least two tissue types are genetically engineered to contain at least one fluorescent reporter gene for each multiple biological process to be analyzed.

10. The in vitro testing kit for use according to any one of claims 5-9, wherein said one or more drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready-to-use microcavity microwell plate to identify the effects of said one or more drugs, chemicals, biologies, and/or biological particles on said multiple biological processes within said at least two tissue types.

11. The in vitro testing kit for use according to any one of claims 5-10, wherein more than one drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready-to-use microcavity microwell plate to identify interactions between said more than one drugs, chemicals, biologies, and/or biological particles.

12. An in vitro method based on cell coating to test drugs, chemicals, biologies, and/or biological particles, having the capacity to simultaneously measure multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the method comprises: a) a first step where one or more suspensions of cells representing one tissue type per cell suspension are independently mixed with fluorescent cell-coating molecules conferring a specific color code to said at least one cell suspension; b) a molding step to form microdroplets with said one or several suspensions of cells mixed with the fluorescent cell-coating molecules by using micro-encapsulation into a dissolvable hydrogel capsule, followed by culturing said encapsulated microdroplets to form multicellular structures coated with fluorescent cell-coating molecules; c) at least two different types of multicellular structures originating from at least two different tissue types are combined together to form a multi-tissue co-culture and where at least one of said tissue type is coated with fluorescent cell-coating molecules; d) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities and dissolving the hydrogel capsule to only keep the fluorescently- coated multicellular structure; e) exposing said multi-tissue co-culture to said drugs, chemicals, biologies, and/or biological particles to be tested; f) analysis of the exposed multi-tissue co-culture is performed by staining the at least two different types of multicellular structures of step c) by either: i) performing an immunolabelling on the multi-tissue co-cultures with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe or; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for the bioluminescence, in which each of the at least two different types of multicellular structures is discriminated through the specific color code of the fluorescent cell coating, and multicellular structures from the same tissue of said at least two different types of multicellular structures are individually discriminated through the stable spatial positioning in the microcavities of the multiwell plates and whereas the biological effect of interest is specifically measured in said each multicellular structure of said at least two different types of multicellular structures.

13. The in vitro method based on cell coating to test drugs, chemicals, biologies, and/or biological particles according to claim 12, wherein the molding step b) is alternatively performed by technics not requiring the use of a dissolvable capsule for the formation of multicellular structures, and selected from bioprinting, ultralow attachment multiwell plates, or the hanging drop method.

14. The in vitro method based on cell coating to test drugs, chemicals, biologies, and/or biological particles according to any one of the preceding claims, wherein one of said one or several suspensions of cells of said at least two different types of multicellular structures of step c) is genetically engineered to express a fluorescent or bioluminescent reporter gene.

15. The in vitro method based on cell coating to test drugs, chemicals, biologies, and/or biological particles according to claim 14, wherein analysis of step f) further comprises: iv) measuring biological effects of interest specifically in said at least two different types of multicellular structures expressing bioluminescent and/or fluorescent reporter genes.

16. An in vitro kit based on cell-coating for use in drug testing by measuring the effects of one or more drugs, chemicals, biologies, and/or biological particles on multiple biological processes in individual multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures coated with cell-coating molecules representing at least two tissue types, wherein at least one type of multicellular structure is coated with specific color-coded fluorescent cell-coating molecules.

17. The in vitro kit based on cell coating for use according to claim 16, wherein at least one of said at least two tissue types is a hepatic tissue.

18. The in vitro kit based on cell coating for use according to any one of claims 16-17, wherein the at least two tissue types are selected from the list comprising hepatic, cardiac, neural, renal, bone marrow, blood, pulmonary, gastrointestinal, gastric, pancreatic, neoplasic, skin, embryonic, stem cells, endothelial, immune, bone, endocrine, muscle, or adipose.

19. The in vitro kit based on cell coating for use according to any one of claims 16-18, wherein the multiple biological processes to be analyzed are selected from the list comprising: cell proliferation, inflammation, cytotoxicity, tissue stress responses, detoxification responses, hormone responses, xenobiotic responses, genotoxic stress responses, apoptosis, necrosis lipid accumulation, mitochondrial dysfunction, metabolic fluxes and gradients, or hypoxia.

20. The in vitro kit based on cell coating for use according to any one of claims 16-19, wherein one or several tissue types are genetically engineered to contain at least one fluorescent reporter gene for each multiple biological process to be analyzed.

21. The in vitro kit based on cell coating for use according to any one of claims 16-20, wherein said one or more drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready-to-use microcavity microwell plate to identify the effects of said one or more drugs, chemicals, biologies, and/or biological particles on said multiple biological processes within said at least two tissue types.

22. The in vitro kit based on cell coating for use according to any one of claims 16-21, wherein more than one drugs, chemicals, biologies, and/or biological particles are tested per well of said at least one ready-to-use microcavity microwell plate to identify interactions between said more than one drugs, chemicals, biologies, and/or biological particles.

23. An in vitro method based on fluorescent capsules to test drugs or chemicals, with the capacity to measure multiple biological processes in individual encapsulated multicellular structures representing several tissue types in co-culture, the method comprises: a) separately encapsulating one or more cell types to be analyzed in the same in vitro drug testing assay with a biopolymer of alginate, wherein in the case there are more than one cell type, all, or all but one, of the respective capsules are labelled, each with a different type of fluorophore; followed by culturing said encapsulated cells to form encapsulated multicellular structures; b) at least two different types of encapsulated multicellular structures are combined together to form a multi-tissue co-culture and where at least one of said two different types of encapsulated multicellular structures is encapsulated in a fluorescent capsule; c) loading said multi-tissue co-culture in wells of multiwell plates comprising microcavities; d) exposing said multi-tissue co-culture to drugs, and/or chemicals e) analysis of the multi-tissue co-culture is performed by staining at least two different types of encapsulated multicellular structures of step b) by either: i) performing an immunolabelling on the multi-tissue co-culture with suitable antibodies recognizing a specific target and coupled directly or indirectly with a suitable fluorophore or a bioluminescent probe; ii) measuring specific biological effects of interest with fluorescent dyes or bioluminescent dyes; iii) performing both steps i) and ii); and wherein, the data acquisition is performed by using fluorescence microscopy and/or multiplate reader for bioluminescence, in which each of the at least two different types of tissues is discriminated through the specific color code of the fluorescent capsule in which it is encapsulated and whereas encapsulated multicellular structures from the same tissue of said at least two different types of tissues are individually discriminated through the stable spatial positioning in the microcavities of the multiwell plates where the biological effect of interest is specifically measured in said each encapsulated multicellular structure of said at least two different types of tissues.

24. The in vitro method based on fluorescent capsules to test drugs or chemicals according to claim 23, wherein one of said at least two encapsulated multicellular structures of said multitissue co-culture of step c) is genetically engineered to express a fluorescent or bioluminescent reporter gene.

25. The in vitro method based on fluorescent capsules to test drugs or chemicals according to claims 23-24, wherein analysis of step e) further comprises: iv) measuring biological effects of interest specifically in said at least two different types of multicellular structures expressing bioluminescent and/or fluorescent reporter genes.

26. An in vitro kit based on fluorescent capsules for use in drug testing by measuring the effects of one or more drugs or chemicals on multiple biological processes in individual capsules containing multicellular structures representing several tissue types in co-culture, the kit comprising at least one ready to use microcavity multiwell plate containing at least two types of multicellular structures encapsulated in alginate capsules representing at least two tissue types, wherein at least one type of capsule is labelled with specific color-coded fluorescent alginate.

27. The in vitro kit based on fluorescent capsules for use in drug testing according to claim 26, wherein at least one of said at least two tissue types is a hepatic tissue.

28. The in vitro kit based on fluorescent capsules for use in drug testing according to any one of claims 26-27, wherein the at least two tissue types are selected from the list comprising hepatic, cardiac, neural, renal, bone marrow, blood, pulmonary, gastrointestinal, gastric, pancreatic, neoplasic, skin, embryonic, stem cells, endothelial, immune, bone, endocrine, muscle, or adipose.

29. The in vitro kit based on fluorescent capsules for use in drug testing according to any one of claims 26-28, wherein the multiple biological processes to be analyzed are selected from the list comprising: cell proliferation, inflammation, cytotoxicity, tissue stress responses, detoxification responses, hormone responses, xenobiotic responses, genotoxic stress responses, apoptosis, necrosis lipid accumulation, mitochondrial dysfunction, metabolic fluxes and gradients, or hypoxia.

30. The in vitro kit based on fluorescent capsules for use in drug testing according to any one of claims 26-29, wherein one or several tissue types are genetically engineered to contain at least one fluorescent reporter gene for each multiple biological process to be analyzed.

31. The in vitro kit based on fluorescent capsules for use in drug testing according to any one of claims 26-30, wherein said one or more drugs or chemicals are tested per well of said at least one ready -to-use microcavity microwell plate to identify the effects of said one or more drugs or chemicals on said multiple biological processes within said at least two tissue types.

32. The in vitro kit based on fluorescent capsules for use in drug testing according to any one of claims 26-31, wherein more than one drug or chemicals are tested per well of said at least one ready-to-use microcavity microwell plate to identify interactions between said more than one drug or chemicals.