Production of tissue-like structures and uses thereof

Abstract

The present invention relates to a method to create an artificial tissue-like structure, said method relying on the use of a first layer A and a second ECM layer B, into or on which is then introduced living cells, which are then cultured in a viscous cell growth medium layer C containing high molecular weight viscous water-soluble polymers formed on top of layer B. The invention also relates to an artificial tissue-like structure obtained from the method as defined above, said structure containing at least a gelled layer B of extracellular matrix (ECM) proteins comprising living cells, covered by a viscous cell growth medium layer C containing molecular weight water-soluble polymers.

Description

[0001] PRODUCTION OF TISSUE-LIKE STRUCTURES AND USES THEREOF

[0002] BACKGROUND OF THE INVENTION

[0003] The ability to produce biological tissue in vitro holds tremendous potential for translational research, regenerative medicine, and drug testing, lessening the burden on animal testing and removing the long wait times for transplants.

[0004] Tissue formation follows a tightly regulated morphogenetic program that involves iterative interactions between cells and cell assemblies guiding cellular differentiation and patterning over a large scale. This poorly characterized emergent process allows single cells to self-organize into complex interconnected macro-scale structures (Sasai, 2013). The study of this process is of fundamental importance both for understanding of natural tissue morphogenesis and for development of novel biomedical technologies. This stimulated a new field of research called "synthetic morphogenesis" that focuses on building of artificial livings materials (Matejcic and Trepat, 2023).

[0005] In the last decades, tissue biofabrication has been approached by various strategies employing different building blocks and engineered microenvironment to control self-organization process (Brassard and Lutolf, 2019; Ouyang et al., 2020).

[0006] Cells are the essential component for biofabrication of living materials, and are used as single-cell suspensions or as pre-assembled building blocks such as multicellular aggregates / spheroids, more recently, organoids. To facilitate cell growth and the formation of tissue structures, these cells are supplemented with suitable matrices. These matrices can be composed of natural materials, such as extracellular matrix (ECM) proteins, polysaccharides, and proteoglycans, or synthetic polymers like polyethylene glycol (PEG), poly-L-lactic acid (PLLA), poly(lactic-co-glycolic acid) (PLGA), and peptides. These various matrices, composed of natural extracellular proteins and synthetic polymers, are collectively called extracellular matrix (ECM). ECM provides structural support and biochemical cues for cells to attach, grow, migrate and communicate. ECM is also a primary mechanical scaffold that controls tissue shape, architecture and function as a whole. For some applications, such as bone, cartilage, and skin regenerations, additional scaffolding is used to control tissue geometry and mechanical properties on the macroscopic scale. These macro-scaffolds are produced from biomaterials including ceramics, natural and synthetic polymers, and composites (Chan and Leong, 2008; O'Brien, 2011).

[0007] The tissue components are assembled together manually or using elaborate technologies such as microfluidics or 3D printing to create the ordered macrostructures of defined shape. Subsequent cell growth, differentiation, and patterning result in auto-organization of a complex tissue-like architecture within the predefined scaffold. The resulting "tissue mimics" may pursue different structural (size, shape, porosity), mechanical (toughness, stiffness / elasticity, plasticity), and functional (barrier / conductivity, metabolism, secretion) requirement depending on targeted applications.

[0008] Often, in tissue biofabrication, the building blocks are arranged to mimic a multilayer / stratified architecture of natural tissues. The supporting "scaffold" layers are covered with ECM layers (with or without cells), which, in their turn, are seeded with a surface layer of cells and topped with a growth medium (or air in some cases), the latter, itself, can be considered as a separate layer. This multilayer setup, with predefined gradients of biochemical and mechanical cues, guides cell autoorganization on a tissue level.

[0009] Typically, established cell lines or primary cells are used to fabricate the upper "cell" layer. Despite of the limited complexity of the resulting tissues, this approach has been the starting point of many synthetic morphogenesis studies and tissue engineering efforts. More recently, stem cells, including induced pluripotent stem cells (iPSCs) (Morgani et al., 2018), embryonic stem cells (ESCs) (Warmflash et al., 2014), or ESC-like populations (Anlas and Trivedi, 2021) have been implemented to produce more complex tissue composition.

[0010] The use of pre-assembled building blocks such as multicellular aggregates is another way to create a more elaborate tissue architecture. Thus, solid-core spheroids of various types of cells can coalescence via so-called "tissue liquidity" to form macroscopic aggregates (Mironov et al., 2009). However, spheroid-derived tissue constructs do not exhibit organ-like morphology and characteristic anatomical features. Moreover, they cannot be grown beyond the millimeter scale without additional perfusion platforms or an engineered vasculature (Skylar-Scott et al., 2019).

[0011] In the last decade, a class of self-organizing multicellular assemblies called "organoids" has emerged as a powerful tool to study the behavior of their tissue of origin (Eiraku et al., 2011; Sato et al., 2009). Given their close resemblance to native organs in histology and cell composition, organoids represent ideal modular units for the biofabrication of biomimetic organs (Brassard and Lutolf, 2019; Fujii and Sato, 2021; Takebe and Wells, 2019). However, because the size of organoids is also limited by the millimeter scale, they lack architectural features of native organs that would allow the emergence of higher-level functional characteristics (Brassard et al., 2021; Fujii and Sato, 2021; Takebe and Wells, 2019).

[0012] To produce larger tissues with more complex architectures, current biofabrication techniques predominantly rely on external control mechanisms. These techniques, such as microfluidics and 3D printing, involve placing tissue building blocks in precisely defined positions to create tissues with specific shapes, morphologies, and architectures. While this approach does yield fabricated structures with desired characteristics, they are inherently artificial and may not fully represent the natural self-organization process that occurs during tissue formation in living organisms. This challenge highlights the ongoing difficulty in effectively controlling the self-organization potential of cells across various scales, ranging from the cellular to macroscopic levels.

[0013] Thus, controlling the self-organization potential of mammalian cells across the scales from cellular to macroscopic remains challenging with existing technologies.

[0014] Yet, there is currently no single, foolproof technology capable of fabricating tissues of varying sizes, ranging from several micrometers to decimeters in scale, exclusively relying on the self-organization process of cells.

[0015] This underscores the ongoing research and development efforts in the field of tissue biofabrication to further refine and expand these capabilities.

[0016] DETAILLED DESCRIPTION OF THE INVENTION

[0017] The present inventors herein propose a robust scalable method for fabricating biological tissue-like structures (also sometimes called "tissuloids") that could be used for translational research, tissue engineering, and / or food engineering.

[0018] The method of the invention is based on the fabrication of a stratified / layered architecture comprising a support / scaffold layer, an ECM layer covered by or containing living cells, and, on the top, a viscous cell growth medium layer (Figure 1). The essential component of the top viscous layer is high molecular weight / size compound (HMWC) that serves to control biological and physicochemical properties of the entire multilayer assembly. As a matter of fact, the present results of the inventors show that varying HMWC type and concentration allows an easy control over the self-organization process of the tissue cells and ultimately shapes the composition, morphology, and function of the resulting tissue.

[0019] The present invention describes various non-limiting embodiments of tissue-like structures formed by auto-organization of cells using this method.

[0020] The present inventors have identified two major ways to control self-organization potential of cells across various scales:

[0021] - First, by assembling the tissue-like structures in layers of distinct compositions (typically, polymers, ECM proteins, cells, and HMWC-medium). This enables the initial confinement of the cells within designated locations and imparts the necessary anisotropy for orchestrating the formation of stratified tissue architecture by regulating cell interactions with the extracellular matrix and with each other, and by guiding their proliferation, orientation and displacement. Additionally, this design facilitates and directs the transfer of structural orders across different scales: from the initial organization of multi-cellular assemblies on micrometer scale to patterning / positioning of tissuelike compartments within the final cm / dm-sized stratified tissue architecture.

[0022] - Second, by adding HMWC to the top layer of the tissue-like structure: this permits the regulation and precise adjustment of both rheological and biochemical properties of the entire multilayer structure. Specifically, HMWC changes the osmolality and viscosity of the cell growth medium, modifies the viscoelastic properties of cells and ECM, and affects the molecular interactions involved in cell / tissue functions. As a result, varying HMWC type and concentration allows for the creation of various tissue morphologies and architectures, all originating from the same initial setup. Moreover, HMWC alters the rates of cell proliferation and cell death in a cell-type-specific manner, which can vary from "fine tuning" (the changes in the rates of cell proliferation and cell death do not exceed two folds) for some epithelial cells to yes-or-no response for some fibroblasts, which are unable to growth in the given conditions without HMWC. This influences not only the structure of the tissue-like structure but also its cellular composition and functionality, particularly when the tissue-like structure contains multiple cell types (e.g., derived from biopsies and patient-derived- organoid, PDO, cultures). METHOD OF THE INVENTION

[0023] Thus, in a first aspect, the present invention relates to a method to create an artificial tissue-like structure, said method comprising: i) Providing a support layer A, ii) Providing extracellular matrix (ECM) proteins, gelling the proteins in appropriate conditions onto the support layer A, thereby forming a ECM layer B on the top of the support layer A, iii) Immerging the layers A and B in a cell growth medium that is able to favor the growth of living cells under appropriate culture conditions, iv) Providing living cells that can be cultured in said growth medium and introducing them in -or placing them on the top of - the ECM layer B, v) optionally, incubating the structure under appropriate conditions to allow the cells to attach to - or to invade - the ECM layer B, preferably at 37°C for 3 hours, vi) Providing a cell growth medium containing high molecular weight viscous water-soluble polymers, and forming a viscous cell growth medium layer C on top of layer B, vii) optionally, culturing the obtained layered structure during at least 6 hours in appropriate conditions for the living cells to self-assemble into tissue-like structures.

[0024] The different layers required by the method of the invention will be now described in details.

[0025] Layer A = support / scaffold layer

[0026] The first layer of the tissue-like structure of the invention is called "support / scaffold layer". Importantly, this layer will determine the final size and shape of the tissue-like structures of the invention. It can be rather flat (2D-I ike surface) or have a 3D-shape.

[0027] Typical 2D-like surface examples include: (i) the bottom of a cell culture dish, of a multiwell-cluster plate, or of a fabricated chamber; (ii) the surface of a flat hydrogel pad, which is sufficiently stiff to perform scaffolding function; and (iii) a porous membrane such as Corning® Transwell® membrane in cell culture inserts. Typical 3D-shape examples include: (i) various 3D scaffolds produced by (micro)machining, by casting / molding, and / or by 3D printing; (ii) fibrous and network / meshwork-like media produced by electrospinning or other solvent evaporation techniques, or by mesh fabrics technologies including knitting and netting and additive manufacturing; (iii) continuous porous media such as sponges or 3D-foams produced by phase separation, foaming, porogen leaching and similar techniques; (iv) granular materials produced by microfluidic techniques, by using emulsion polymerization or similar approaches.

[0028] The examples below describe 2D-like surfaces of the bottom of a cell culture dish (example 1.3.), of a multiwell-cluster plate (examples 1.1., 1.2., 2.1. and 2.2.), and the surface a flat hydrogel pad (example 2.3.).

[0029] The "support / scaffold layer" can be made of materials of various porosity and stiffness (Young's modulus).

[0030] (i) "Stiff" materials include metals, ceramics, glass and various plastics such as polystyrene, polyacrylate, and cyclic olefin co-polymer to name a few.

[0031] (ii) Materials of "intermediate stiffness" include natural and synthetic polymers such as cellulose-based materials and other polysaccharides (chitin, chitosan etc), leather and leather-like materials, poly-lactic acid (PLLA) and poly-lactic-co-glycolic acid (PLGA), poly-caprolactone (PCL) and polydimethylsiloxane (PDMS), to name a few.

[0032] (iii) "Relatively soft" materials include natural and synthetic polymers such as agarose, alginate, collagen, gelatin-polyacrylate, polyacrylate and polyacrylamide and their copolymers with polyethylenglycole / poly(ethylene oxide) (PEG / PEO), to name a few.

[0033] In the examples below, "support / scaffold layers" were made of glass (Figure 9), polystyrene (all Figures except Figures 6 & 9), and agarose (Figure 6).

[0034] The "support / scaffold layer" described above serves as a scaffold for the second layer B) hereafter called "ECM layer". Depending on the targeted applications, the properties of the layers can be modified to vary the strength of interlayer contacts from "strong" (sticky, adherent) to "week" (low attachment, non-adherent), or "switchable" from "strong" to "week".

[0035] "Strong" binding of the "ECM layer" to the "support / scaffold layer" can be achieved through (i) the physical sorption of the "ECM layer" due to the bulk non-specific hydrophobic interactions, van der Waals force, metal coordination, hydrogen bonding, electrostatic interaction, or more specific ligand-driven supramolecular interactions. This can employ various natural and synthetic glues such as mussel adhesive protein (MAP)-inspired materials and supramolecular adhesive hydrogels (Zhao et al., 2022); (ii) chemical covalent bonding using crosslinkers; (iii) interpenetration of the two layer and "velcro"-like mechanism.

[0036] On the contrary, "weak" binding of the "ECM layer" to the "support / scaffold layer" can be achieved by: (i) Using low-attachment support surfaces such as polymer / plastics grafted with hydrophilic polyethyleneglycol, polyacrylate and polyacrylamide chains (e.g. Corning Ultra-Low-Attachment plasticware), (ii) "Refouling" coating of the "support / scaffold layer" with albumin proteins (e.g. BSA), amphiphilic molecules / natural and synthetic surfactants (e.g. phospholipids, neutral detergents like TWEEN® 20, block copolymers like Pluronic F127), as well as synthetic polymers as poly(2-hydroxyethyl methacrylate) (polyHEMA), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA), to name a few. (iii) Using low attachment "support / scaffold layer" such as made of agarose, alginate, collagen, gelatin-polyacrylate, polyacrylate and polyacrylamide and their co-polymers with polyethyleneglycol to name a few.

[0037] Repellent coating of multi-well plates is particularly useful for studies / applications when tissuelike structures are grown as a "free-floating" culture. Treatment of the plates with non-fouling agents can reduce the attachment of the "ECM layer" to the "support / scaffold layer" that results in tissue release because of cell contraction and matrix remodeling. In this context, preferred nonfouling agents that can be used to coat the layer A) are synthetic polymers such as poly(2- hydroxyethyl methacrylate) (polyHEMA), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA). Although various types of plates can be used, "non-treated virgin polystyrene" plates generally provide better coating with PVA / polyHEMA and reduce cell / tissue growth outside the ECM (see example 2. below).

[0038] Finally, "switchable" (or "reversible" binding) of the "ECM layer" to the "support / scaffold layer" can be achieved by: (i) Using the polymer / plastics surfaces grafted / coated with a thermosensitive polymer such as Pluronic or poly(N-isopropylacrylamide) (PIPAAm), whose adhesive properties change within the physiological range of temperature allowing "ECM layer" to attach and then be released upon the temperature shift (e.g. Nunc UpCell Surface plasticware), (ii) Using the polymer / plastics scaffold coated with light-responsive molecules, whose adhesive properties change upon illumination allowing "ECM layer" to attach and then be released by light. These examples are described in the literature (Bordbar-Khiabani and Gasik, 2022; Thummarati et al., 2023). The first step of the method of the invention is step i) requiring to provide a support layer A.

[0039] In a particular embodiment, said support layer A is a stiff surface, preferably chosen from:

[0040] (i) the bottom of a cell culture dish, of a multiwell-cluster plate, or of a fabricated chamber, more preferably made of metals, ceramics, glass, polystyrene, polyacrylate, or cyclic olefin copolymer; more preferably polystyrene or glass,

[0041] (ii) the surface of a flat hydrogel pad, more preferably made of natural and synthetic molecules such as cellulose-based materials, polysaccharides, poly-lactic acid (PLLA), poly-lactic-co-glycolic acid (PLGA), poly-caprolactone (PCL), polydimethylsiloxane (PDMS), agarose, alginate, collagen, gelatin- polyacrylate, polyacrylate, polyacrylamide, polyethylenglycole / poly(ethylene oxide) (PEG / PEO), or combinations thereof;

[0042] (iii) leather and leather-like materials, and

[0043] (iii) a granular, fibrous or porous membrane.

[0044] Preferably, said support layer A is a stiff surface that is capable of binding the ECM protein layer B, due to non-specific hydrophobic interactions, van der Waals force, metal coordination, hydrogen bonding, electrostatic interaction, ligand-driven supramolecular interactions, natural and synthetic glues, crosslinkers; or "velcro"-like mechanism.

[0045] In another particular embodiment, the support layer A used in the method of the invention is a low attachment layer made of:

[0046] Glass or plastic or polymers materials that are:

[0047] • grafted with hydrophilic polyethyleneglycol, polyacrylate and polyacrylamide chains,

[0048] • coated with albumin proteins, amphiphilic molecules, natural and synthetic surfactants, synthetic polymers such as poly(2-hydroxyethyl methacrylate) (polyHEMA), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA), or

[0049] • coated with non-fouling agents such as poly(2-hydroxyethyl methacrylate) (polyHEMA), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA),

[0050] Agarose, alginate, collagen, gelatin-polyacrylate, polyacrylate and polyacrylamide and their co-polymers with polyethyleneglycol, or combinations thereof, preferably agarose-gelatin composite, Glass or plastic or polymers materials that are grafted or coated with a thermosensitive polymer such as Pluronic or poly(N-isopropylacrylamide) (PIPAAm) or with light-responsive molecules.

[0051] The examples below describe the use of polystyrene (rather "strong" ECM binding, all Figures except Figures 5, 6 & 9), glass ("strong" to "intermediate" ECM binding, Figure 9), polystyrene coated with PVA ("weak" ECM binding, Figure 5), or agarose ("weak" ECM binding, Figure 6).

[0052] Preferably, said support layer A is a strong attachment layer made of polystyrene, or is a low attachment layer made of polystyrene coated with polyHEMA or PVA, or made of agarose.

[0053] In this case, i.e., when the support layer A is a low attachment layer as described above, the method of the invention preferably further comprises a final step of separating the tissue-like structure resulting from steps i) to vi) from said support layer A.

[0054] Although various types of plates can be used, "non-treated virgin polystyrene"- and glass-bottomed plates - provide better attachment of the "ECM layer" and reduce cell / tissue growth outside the ECM. Typical examples are glass-bottomed MatTek 6-well plate (#P06G-1.5-20-F), non-treated PS CytoOne 6-well plate (#CC7672-7506), and non-treated PS Falcon® 6-well plate (#351146).

[0055] After coating, plates can be used to form tissue-like structures of different sizes (usually of 05-020 mm initial dimeter) by varying the volumes of Matrigel and cell suspension. For example, a tissuelike structure of 015 mm initial diameter can be formed using 85 pl of Matrigel solution (5 mg / ml in 50-50 medium) and 85 pl of H6c7 cells (single cell suspension of 5xl05cells / ml in 50-50 medium) on a MW6 plate.

[0056] In a particular embodiment, before applying the "ECM layer", the "support / scaffold layer" is washed, incubated, and equilibrated with an appropriate medium compatible with cell growth.

[0057] Layer B = ECM proteins and cells

[0058] The second step of the method of the invention is step ii) requiring to provide extracellular matrix (ECM) proteins, to gel the proteins in appropriate conditions onto the support layer A, so as to form an "ECM layer" B on the top of the support layer A provided in step i).

[0059] The "ECM layer" B is preferably applied in liquid form to cover the "support / scaffold layer" and then allowed to gel / polymerize to produce a viscoelastic gel layer. More preferably, the constituents of the "ECM layer" are diluted in an appropriate medium which is compatible with cell growth. This layer provides structural support and biochemical signals / cues for cells to attach, grow, migrate and communicate.

[0060] Preferably, the "ECM layer" B so formed is a layer of at least 50 pm, at least 100pm, at least 200 pm, at least 400pm, or at least 450pm. Thus, preferably, in the method of the invention, step (ii) comprises providing extracellular matrix (ECM) proteins, gelling the proteins in appropriate conditions onto the support layer A, thereby forming an ECM layer B of at least 50 pm, at least 100pm, at least 200 pm, at least 400pm, or at least 450pm, on the top of the support layer A.

[0061] Preferably, the ECM proteins containing in this layer are chosen among:

[0062] (i) Natural ECM proteins such as collagens (and gelatins), laminins, fibronectins, vitronectins, fibrins, nidogens / entactins to name a few.

[0063] (ii) Glycoproteins and polysaccharides including proteoglycans (e.g. heparin sulfate, chondroitin sulfate, hyaluronan). The proteins and their fragments can be isolated from natural sources or be produced recombinantly or synthetically.

[0064] (iii) Complex protein mixtures such as basement membrane (BME, e.g. Matrigel, Geltrex, Cultrex), plasma, lymph, avian egg white etc.

[0065] (iv) Decellularized Extracellular Matrix (DECMs) produced by decellularization of a variety of animal-derived tissues and organs (e.g. bone, heart, liver, kidney, intestine, skin, lung, adipose tissue).

[0066] (v) Synthetic hydrogels based on chemically modified polyacrylate, PEG / PEO, PLLA, PLGA, and PCL polymers to name a few.

[0067] In the method of the invention, the ECM proteins used in step ii) are preferably chosen from:

[0068] Natural ECM proteins such as collagens, gelatins, laminins, fibronectins, vitronectins, fibrins, nidogens, and entactins,

[0069] Natural or synthetic glycoproteins and polysaccharides,

[0070] Complex protein mixtures such as basement membrane, plasma, lymph, or avian egg white, Decellularized Extracellular Matrix (DECMs) produced by decellularization of a variety of animal-derived tissues and organs,

[0071] Synthetic hydrogels based on polyacrylate, polyethylenglycole / poly(ethylene oxide) (PEG / PEO), poly-lactic acid (PLLA), poly-lactic-co-glycolic acid (PLGA), and poly-caprolactone (PCL) polymers mimicking natural ECM. It is noteworthy that, depending on their structure (degree of polymerization, branching etc), the latter polymers can form non-elastic solid (for layer A), elastic solid (for layer A), viscoelastic gel (for layer B), or viscous solution (for layer C).

[0072] These materials are preferably chemically modified to carry ligands that facilitate cell attachment, growth, and migration.

[0073] In the examples of the invention, Matrigel has been used to produce the "ECM layer". In a preferred embodiment of the invention, the ECM proteins used in step ii) are selected from the list consisting of laminin, collagen, entactin, heparan sulfate proteoglycan and combinations of two or more thereof. In a more preferred embodiment of the invention, the ECM proteins used in step ii) comprise a mixture of laminin, collagen and entactin. In a yet preferred embodiment the ECM proteins used in step ii) comprise or consist in a mixture of laminin, collagen, entactin and heparan sulfate proteoglycan.

[0074] Step iii) of the method of the invention requires to immerge the layers A and B obtained in the preceding steps in a cell growth medium that is able to favor the growth of living cells under appropriate culture conditions.

[0075] The cell growth medium to be used will depend on the living cells to be used in the following step iv). The skilled person well knows which growth medium should be used for any kind of living cells. Information thereon can be found in (Freshney, 2015).

[0076] The immersion step can last few minutes, until all the layers are correctly imbibed with the cell growth medium.

[0077] This step is preferably followed by an incubation step in a cell incubator so that the temperature of the two layers is appropriate (typically 37°C) when the cells are added in step iv).

[0078] Step iv) of the method of the invention requires to introduce living cells in or on top of the ECM layer B.

[0079] Thus, the layer B contains living cells and can also be called the "cell layer" of the tissue-like structure of the invention.

[0080] The cells can be put in contact with the surface of the "ECM layer" by adding a cell suspension on the pre-formed gel layer, allowing the cells to attach to the matrix. The cells can be used as singlecell suspensions or as pre-assembled building blocks such as multicellular aggregates / spheroids or organoids or ex vivo microtissues. Any type of eukaryotic cells can be used including epithelial cells, endothelial cells, fibroblasts, or stem cells, to name a few. The cells can be of established cell lines (immortalized cancerous and non-cancerous cells), primary cells isolated from different organs & tissues, bio-engineered / genetically modified cells, and iPS cells, to name a few. Single type of cells or a mixture of different types of cells can be used. Mixed cells are preferably used as pre-assembled building blocks such as multicellular aggregates / spheroids, organoids or ex vivo microtissues.

[0081] Thus, the living cells used in step iv) of the method of the invention are typically cell lines or primary cells, preferably eukaryotic cells including epithelial cells, endothelial cells, fibroblasts, non- embryonic stem cells, iPS cells, or a mixture thereof, more preferably a mixture of stromal cells comprising fibroblasts, myofibroblasts, adipocytes, fibrocytes, pericytes, mesenchymal stem cells, macrophages, mast cells, and / or lymphocytes, with epithelial cells including pancreas, prostate, mammary, kidney, lung, and bladder cells.

[0082] The examples of the invention describe the use of immortalized human pancreatic cell lines H6c7 & HPNE, immortalized human prostatic cell line RWPE1, primary human pancreatic epithelial cells, and primary human fibroblasts. More precisely, Figure 8 discloses the tissue-like structures obtained with iPS, MCS, ADSC-d2, ADSC-d6, CAF, FMA, hDF, hLF, HPASTEC, HPNE, H6C7, HEK, HuEp, MCFlOa, RPE1, RWPE1, HaCaT, HKPM, hTert-Kera, HPaMVEC, and HUVEC.

[0083] In a particular embodiment, the cells are added along with some of the ECM components. For example, a suspension of cells can be added to a liquid ECM suspension and centrifuged in appropriate conditions (i.e. at a temperature below the ECM gelling point) to allow cells to sediment and to form a layer on the interface between the "support / scaffold layer" and the "ECM layer". Then the ECM containing the cells is allowed to gel / polymerize. This embodiment is highlighted in example 2.3, describing the formation of tissue-like structures from H6c7 cells added to the Matrigel and centrifuged to sediment and to form a layer on the interface between the agarose "support / scaffold layer" and Matrigel "ECM layer".

[0084] In some embodiments, the "cell layer" is produced with two different types of cells. In a particular embodiment, the "cell layer" contains ECM components and type-1 cells; and other cells (type-2 cells) are further grown on the top of this cell layer. The type-1 & 2 can be different or the same. For example, type-1 cells can be stromal cells comprising one or more types of fibroblasts, myofibroblasts, adipocytes, fibrocytes, pericytes, mesenchymal stem cells, macrophages, mast cells, and lymphocytes, to name a few; whereas type-2 cells can be epithelial cells including pancreas, prostate, mammary, kidney, lung, and bladder cells, to name a few. Example 5 of the invention discloses a tissue-like structure containing a "cell layer" containing multiple cell populations from a pancreatic organoid culture.

[0085] In some embodiments, the medium containing the cell suspension used in the cell layer contains additives / compounds that promote cell attachment to the "ECM layer". For example, it is possible to use the ROCK inhibitor Y-27632, as proposed in Example 1.2.

[0086] In a particular embodiment of the method of the invention, the steps ii), iii) and iv) are concomitant so that the living cells are introduced in a liquid extracellular matrix (ECM) protein suspension, before the ECM proteins are gelled. Afterwards, the ECM suspension containing the living cells can be placed onto layer A and the structure can be centrifugated before the ECM proteins are gelled, so that the cells are eventually located at the interface between layers A and B before step vi) or vii).

[0087] In this case, the method of the invention therefore advantageously contains an additional step of centrifugating the structure between step iv) and step v) so that the cells invade the layer B and rapidly sediment at the interface with layer A.

[0088] The method of the invention may require an incubation step v), after step iv), to allow the cells to attach to or to invade the ECM layer B. For example, it is possible to incubate the structure obtained after step iv) under classical conditions enabling cells to growth and migrate correctly. These conditions depend on the cells, it is usually at 37°C. The incubation time can vary from 1 min to 48 hours depending on type of cells and objectives of manipulation. An incubation time of 3 hours can be typically used.

[0089] At the end of step v) or vi) of the method of the invention, the living cells in the resulting artificial tissue-like structure are preferably embedded within the ECM layer B and / or are located at the interface between the support layer A and the ECM layer B, and / or are located at the interface between the ECM layer B and the viscous liquid upper layer C.

[0090] Layer C = cell growth medium + viscous polymers

[0091] Once the cells are attached to the "ECM layer", the "viscous cell growth medium layer" C) can be added. This layer containing high molecular weight viscous water-soluble polymers serves to increase the viscosity & osmolality of the medium. There is no need to obtain gelling of the polymers, it is sufficient to provide a viscous solution containing the cell growth medium. This is step vi) of the method of the invention, requiring to provide high molecular weight viscous water-soluble polymers that will be mixed with the cell growth medium that is appropriate for the growth of the living cells used in step iv), in order to form a viscous medium layer C that will be placed on top of the layer B.

[0092] This viscous medium layer C preferably contains water-soluble polysaccharides, which may be linear or branched, native or modified by physical and chemical methods. More preferred polymer molecules have a rather high molecular weight / size of 70 kDa to 7000 kDa (preferentially between 100 kDa and 4000 kDa) and may be used in the concentrations varying from 0.1% to 5% by weight (preferentially from 0.1 to 3%, more preferentially from 0.1 to 2%, even more preferentially from 0.1 to 0.8%, yet preferably from 0.3 to 0.8%).

[0093] Preferred polymers to be used in this layer are: hyaluronic acid, dextran, guar gum, methyl-cellulose, hydroxyethyl-cellulose, hydroxyethyl-methyl-cellulose, and hydroxypropyl-methyl-cellulose. Yet preferably, the high molecular weight polymers used in step vi) are chosen from methyl-cellulose, hydroxyethyl-cellulose, hydroxyethyl-methyl-cellulose, hydroxypropyl-methyl-cellulose and carboxymethyl-cellulose.

[0094] In a preferred embodiment, the high molecular weight polymers used in step vi) have a molecular weight of 70 kDa to 7000 kDa are preferably chosen from methyl-cellulose, hydroxyethyl-cellulose, hydroxyethyl-methyl-cellulose, hydroxypropyl-methyl-cellulose and carboxymethyl-cellulose, and are used in the concentrations varying from 0.3 to 0.8% by weight.

[0095] In a yet preferred embodiment, step (ii) comprises providing extracellular matrix (ECM) proteins, gelling the proteins in appropriate conditions onto the support layer A, thereby forming an ECM layer B of at least 50 pm, at least 100pm, at least 200 pm, at least 400pm, or at least 450pm, on the top of the support layer A, and wherein the high molecular weight polymers used in step vi) have a molecular weight of 70 kDa to 7000 kDa are preferably chosen from methyl-cellulose, hydroxyethyl- cellulose, hydroxyethyl-methyl-cellulose, hydroxypropyl-methyl-cellulose and carboxymethyl- cellulose, and are used in the concentrations varying from 0.3 to 0.8% by weight.

[0096] It is also possible to use the following viscous polymers:

[0097] (i) Native natural polymers such as polynucleotides, polypeptides, proteoglycans and polysaccharides including cellulose, starch, guar gum, gum arabic, alginates, carrageenan, chitosan, glycosaminoglycan, hyaluronic acid, dextran, and xanthan gum, to name a few; (ii) Modified natural polymers such as methyl-cellulose, hydroxyethyl-cellulose, hydroxyethyl-methyl-cellulose, hydroxypropyl-methyl-cellulose carboxymethylcellulose, Ficoll, to name a few;

[0098] (iii) Synthetic polymers such as based on chemically modified polyacrylate, PEG / PEO, PLLA, PLGA, and PCL polymers, to name a few.

[0099] These molecules are hereafter designated as "polymers of the invention".

[0100] Typically, the polymers of the invention are solubilized in water before being put in contact with the other layers, preferably leading to a viscous aqueous solution having a viscosity of 1- 100 mPa-s (cP).

[0101] Importantly, this viscous medium layer C containing high molecular weight / size polymers serve to tune the biological and physicochemical properties of the entire multilayer structure.

[0102] As shown in the examples below, changing initial conditions allows the tissue-like structure to evolve into tissue-like structures of varying shape / architecture and properties.

[0103] At the end of step vi), the structure of the invention is rather a layered structure without any cell network or tubules, although in some conditions (some epithelial cell types, relatively high cell density) the initial cell patterns are started to form.

[0104] As evidenced in the experimental part of the application, the high molecular weight polymers can impact and modify the viscoelastic properties of the ECM layer B, more specifically, it was shown that the use of increased concentrations of polymers lead to an increase in stiffness and viscosity of the ECM layer B (example 6, figure 11), which favors the formation of tissuloids with larger surface areas. The person skilled in art may thus choose the high molecular weight polymers and adapt their concentration in the cell growth medium according to the desired result. It has been shown in particular that viscoelastic properties corresponding to a Young's modulus, preferably a static Young's modulus, of at least 500, at least 750, at least 1000 Pa, as measured by AFM, is particularly appropriate for the formation of tissuloids.

[0105] Preferably, in the method of the invention, the high molecular weight viscous water-soluble polymers are chosen and used at a concentration appropriate for increasing stiffness of the ECM layer B. Yet preferably, in the method of the invention, the high molecular weight viscous water- soluble polymers are used at a concentration appropriate for increasing stiffness of the ECM layer B up to a stiffness corresponding to a static Young's modulus, of at least 500, at least 750, at least 1000 Pa, as measured by AFM. In the context of the invention, the term "measured by AFM" refers to the use of Atomic Force Microscopy (AFM) to evaluate the mechanical properties of materials, particularly soft materials like cells and polymers.

[0106] Briefly, the atomic force microscope (AFM) operates using a microscopic physical probe, which consists of an extended, flexible cantilever with a tip that points downward from its free end toward the sample, which is mounted on a moving piezoelectric stage. The mechanical and force properties of the sample are determined by varying the tip / sample distance (i.e., movement along the z-axis) and observing the resulting bending of the cantilever due to its interaction with the sample. This bending is measured by reflecting a laser beam off the top of the cantilever onto a position-sensitive photodetector, which detects deflection changes directly related to the forces involved in the tipsample interaction.

[0107] The AFM generates a force-distance curve by plotting cantilever deflection as a function of tipsample separation (z-axis distance). Various contact models, such as the Hertz, Sneddon, or JKR models, can then be applied to these curves to calculate the viscoelastic characteristics of the material.

[0108] AFM measurements can be performed in different operational modes, with the static and dynamic (oscillatory) modes being the most commonly used today for measuring the viscoelastic properties of soft materials (with stiffness values ranging from 0 to several thousand pascals (Pa)).

[0109] Typically, force curves obtained in static mode allow for the measurement of the static Young's (elastic) modulus, while the dynamic (oscillatory) mode enables the measurement of the complex viscoelastic modulus, G*, which comprises G1(storage / elastic modulus) and G" (loss / viscous modulus). Details of these methods are described, for instance, in Kaman, J. (2015).

[0110] Step vii)

[0111] The method of the invention further contains a last step vii) of culturing the obtained structure during at least 1 hour, preferably at least 2 hours, more preferably at least 3 hours, 4 hours, 5 hours or even 6 hours in appropriate conditions for the living cells to self-assemble into tissue-like structures.

[0112] The incubation time in this step can vary from 1 hours to more than 3 months, depending on the type of cells used in step iv) and of the objectives of manipulation. It is typically from 3 hours to 21 days. At the end of step vii), the living cells have reached all the layers and have formed structures resembling those found in real tissues, such as acini, tubular structures, cell networks, and cell sheets. The structures are therefore called "artificial tissue-like structures".

[0113] Chemical compounds

[0114] In some embodiments, the ECM layer B and / or the viscous medium layer C also contain chemical / biological compounds / entities that serve to modify / control / direct cell behavior / function and fate. These compounds can be modulators / regulators of nucleic acid & protein functions regulating gene expression, proteostasis, metabolism, cell signaling as well as cell structure / morphology and dynamics.

[0115] Useful chemical compounds can be for example:

[0116] (i) Epigenetic regulators such as RARa agonists, inhibitors of DNA and protein methyltransferases, inhibitors of histone deacetylase (HDAC), and inhibitors of histone acetyltransferase, to name a few.

[0117] (ii) Chemical regulators of cell signaling such as regulators of G protein-coupled receptors; regulators of PKC and PKA kinases and downstream signaling; small GTPase regulators; inhibitors MLCK, ROCK, LIM, MRCK and PAK kinases as well as of major components of actomyosin network actin & myosin; small molecule regulators of WNT / P-Cat pathway; small molecule regulators of TGF- pathway; small molecule regulators of Shh pathway; and small molecule regulators of Hippo-YAP / TAZ pathway, to name a few.

[0118] (iii) Inhibitors of mitogen-activated protein kinases (MAPK) and apoptosis.

[0119] Useful biological compounds / entities can be:

[0120] (i) Various growth factors, hormones, and cytokines including proteins targeting WNT / P- Cat pathway (e.g. Wnt3a & Rspol), TGF- pathway (e.g. TGF-P, Activin, Noggin, Foil isatin), and Shh pathway, to name a few.

[0121] (ii) Various nucleic acids such as expression vectors, siRNAs, sgRNAs, and RNPs to name a few.

[0122] (iii) Viruses, bacteria, and other microorganisms. The examples below describe the use of ROCK inhibitor Y-27632, myosin inhibitor blebbistatin, inhibitor of TGF-P pathway LY2109761, and agonist of mechanosensitive channel Piezo-1 Yoda-1 (Figure 3A).

[0123] In some embodiments, specific probes / reporters can be added to the ECM layer B and / or the viscous medium layer C in order to visualize cells / tissues and their functions. These can be:

[0124] (i) Vital probes for cell constituents / compartments such as nucleic acids / nucleus, cytoplasm, cytoskeleton, mitochondria, lysosomes, and plasma membrane, to name a few.

[0125] (ii) Fluorogenic / chromogenic / luminogenic substrates for cell enzymes / activities such as proteases / caspases, esterases, glutathione transferases, phosphatases and cell reducing potential / metabolism, to name a few.

[0126] (iii) Fluorescent probes for membrane channels / transporters, membrane potential and tension.

[0127] As shown in the examples below, it is possible to monitor the cells / tissues, by supplementing the layers B or C with LysoTracker™ Deep Red dye (LTDR, Thermo Fisher Scientific, #L12492). Other fluorescence probes can be used the same way for live cell imaging including SiR-actin / SPY650- FastAct to label actin, CellEvent™ Caspase-3 / 7 Reagent (CE, Thermo Fisher Scientific, #R37111) to measure apoptosis and various SYTOX™ dyes (Thermo Fisher Scientific) to detect dead cells, PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich, #MIDI26-1KT Phanos Technologies), or Calcein AM to stain bulk cell volume in living cells / tissues.

[0128] TISSUE-LIKE STRUCTURE OF THE INVENTION AND USES THEREOF

[0129] The examples below describe various non-limiting embodiments of tissue-like constructs (also called "tissuloids") formed by auto-organization of cells and pre-assembled cell aggregates in particular multilayer settings. Their methods of production and their potential applications are also disclosed.

[0130] The tissue-like structures of the invention are preferably obtained by implementing the method mentioned above.

[0131] In a second aspect, the present invention therefore targets an artificial tissue-like structure obtained from the method as defined above, said structure containing at least a gelled layer B of extracellular matrix (ECM) proteins comprising living cells, covered by a viscous cell growth medium layer C containing molecular weight water-soluble polymers.

[0132] In a preferred embodiment, said tissue-like structure further comprises a support layer A as defined above as "stiff" support enabling "strong interactions" with the ECM layer B. This support layer A being located preferably under the layer B of extracellular matrix (ECM) proteins comprising living cells.

[0133] In a third aspect, the present invention targets an artificial tissue-like structure containing:

[0134] - optionally a support layer A,

[0135] - a layer B containing gelled extracellular matrix proteins and living cells, and

[0136] - on the top of layer B, a layer C consisting of viscous cell growth medium that contains high molecular weight water-soluble polymers.

[0137] In these artificial tissue-like structures, the layers A, B and C are preferably as described above.

[0138] In a fourth aspect, the present invention targets the artificial tissue-like structure obtained at the end of the method of the invention, i.e., after step vii), when the cells have formed a network and a tubular structure (cf. figure 6). This artificial tissue-like structure does not necessarily contain organized layers but still contains gelled extracellular matrix proteins, living cells and high molecular weight water-soluble polymers.

[0139] The tissue-like structures, where the cells and ECM are organized into textured materials with characteristic structural elements having shape textures of spheres, tubes, layers, sponges and a combination of them. The entire tissue-like structure is composed by these elements.

[0140] The tissue-like structures, where the cells and ECM are organized into textured materials, which in 2D projection reveal one of the following patterns: (i) the array of rounded clusters / ellipsoids / tori, (ii) arrangement of stripes / wires / tubules, (iii) bi-continuous network / meshwork, (iv) bi-continuous sheet / layer / perforated layer, ...or a combination of them. Typical examples of the tissue-like structures are shown in Figures 2, 6, and 8.

[0141] The tissue-like structures, where the cells and ECM are organized into textured materials with characteristic structural elements formed by multiple cells (>12) and ECM. The tissue-like structures, where the cells and ECM are organized into textured materials with structural texture elements having characteristic size from 0.1 to 10 mm, preferentially from 0.2 to 2 mm.

[0142] The tissue-like structures, where the sphere / rounded clusters / ellipsoids / tori structural texture element formed by cells and ECM can be solid / fil led with cel Is / spheroid-like or hollow with a cavity inside showing acinus-like morphology.

[0143] The tissue-like structures, where the tube / wire / stripe structural texture element formed by cells and ECM can be solid / filled with cells / wire-like or hollow with a cavity inside showing epithelium tube-like or endothelium vessel-like morphology. The tubes can vary in length and cross-sectional area, can be irregular and anisotropic with a multi-scale texture.

[0144] In a preferred embodiment, the living cells contained in the tissue-like structures of the invention are cell lines or primary cells, preferably eukaryotic cells including epithelial cells, endothelial cells, fibroblasts, non-embryonic stem cells, iPS cells, or a mixture thereof, more preferably a mixture of stromal cells comprising fibroblasts, myofibroblasts, adipocytes, fibrocytes, pericytes, mesenchymal stem cells, macrophages, mast cells, and / or lymphocytes, with epithelial cells including pancreas, prostate, mammary, kidney, lung, and bladder cells.

[0145] The artificial tissue-like structures of the invention are preferably pseudo-two-dimension (flattened three-dimension) or three-dimension artificial tissue-like structures. They can have a 2D projection area of about 0.5 to more than 5000 square millimeters, preferably of 150 to 5000 square millimeters for pseudo-2D tissue-like structures. They can have an occupied volume of 0.1 to more than 500 cubic millimeters, preferably of 0.5 to 500 cubic millimeters for 3D tissue-like structures.

[0146] One particular artificial tissue-like structure according to the invention contains four different layers: a "support / scaffold layer", an "ECM layer", a "cell layer", and a top "viscous cell growth medium layer" (cf. Figure 1).

[0147] During step vii), the structure is preferably kept incubated in appropriate conditions (humidified cell culture incubator with temperature and CO2 control, preferably at 37°C, 5% CO2 and 95% humidity) to allow cell growth, auto-organization, and tissue formation.

[0148] After step vii), the tissue-like structure is also preferably maintained in a humidified cell culture incubator with temperature and CO2 control, or frozen or kept at low temperature (4°C), until use thereof. In some embodiments, the tissue-like structure of the invention can remain attached to the "support / scaffold layer" and be used as such for further characterizations / studies / applications.

[0149] In other embodiments, the tissue-like structure of the invention can be detached from the "support / scaffold layer" due to cell / tissue activities or to externally applied forces. The detached tissue-like structures are then preferably transferred into another setting for downstream uses / characterizations / studies / applications.

[0150] Advantageously, these tissue-like structures of the invention can be used as an in vitro tool in translational researches to study artificial tissues or organs, and / or to reproduce diverse biological processes both normal (e.g. morphogenesis) and pathological (e.g. cancer-linked tissue remodeling). Tissue-like structures of the invention can also be used to provide 3D models for preclinical drug testing. Finally, the technology disclosed herein can be used in food engineering to produce comestible products (e.g. cultured meat and similar products).

[0151] In regenerative medicine / tissue engineering, one can furthermore envisage to use them for tissue repair, as organ or tissue to be grafted in a patient in need thereof.

[0152] It is important to note that tissue-like structures of the invention can potentially be produced entirely from the components that meet the safety requirements of Current Good Manufacturing Practices (CGMPs). Indeed, components such as collagen and gelatins (ECM layer) as well as methylcellulose, guar gum, chitosan, hyaluronic acid, and xanthan gum (HMWC) are already used / authorized in tissue and food engineering and can therefore be used safely in these applications.

[0153] As in vitro tool, apart from the analysis done in the present examples, a number of different analysis can be performed on the structures of the invention, including: (i) immunofluorescence microscopy with / without quantification of cell morphology (for example, by using the automated Celllnsight NXT High Content Screening Platform (Thermo Fisher Scientific) and instrument software HCS Studio 6.5.0- "Morphology.V4") ; (ii) viability assays such as ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay (Lonza #LT07-121), CellTiter-Glo® 3D Cell Viability Assay (Promega #G9681), RealTime-Glo™ MT Cell Viability Assay (Promega #G9711), RealTime-Glo Annexin V Apoptosis & Necrosis assay (Promega #JA1011), and AlamarBlue HS Cell Viability Reagent (Thermo Fisher Scientific #A50100); (iii) RNA extraction and RNAseq analysis.

[0154] Celllnsight NXT High Content Screening Platform can be used for imaging in bright field and fluorescence modes. ChemDoc can also be used for imaging both in white light and fluorescence modes. The dynamics of tissue formation can be followed by lensless microscopy using Iprasense cytonote 6W machine (the use of glass-bottomed plate MatTek is recommended).

[0155] The tissues can be lysed in appropriate buffer (e.g. M-PERTM Mammalian Extraction Buffer (Thermo Fischer Scientific, #78503) supplemented with HaltTM Phosphatase (Thermo Fischer Scientific, #78420) and HaltTM Protease (Thermo Fischer Scientific, #78437) Inhibitor Cocktails) to produce the cellular extracts for enzyme activity analysis (i.e. kinases, PamGene). The tissues can be processed with Laemmli buffer for western blot analysis. Tissues can be fixed with 4% PFA and processed for immunofluorescence analysis or histochemistry staining with hematoxylin / eosin, Methylene Blue, or Giemsa dyes.

[0156] In a final aspect, the present invention targets a platform to produce the artificial tissue-like structure of the invention, said platform reducing to practice the steps of the method of the invention in an automatic manner.

[0157] FIGURE LEGENDS

[0158] Figure 1. Layered setup for tissue formation.

[0159] Figure 2. Different types of artificial tissue-like structures formed in 96-well plate. The growth medium contained: (A) 0% methylcellulose; (B) 0.2% methylcellulose; (C) 0.4% methylcellulose; (D) 0.8% methylcellulose. The tissue-like structures were stained with Calcein-AM and analyzed by fluorescence microscopy. Each image shows the entire well (05 mm).

[0160] Figure 3. Tissue-like structures of the invention formed in 6-well plate (A) and in Petri dish (B). (A) Matrigel pads ("'0.45 mm thick) were made manually in non-treated PS Falcon® 6-well plate (86 mm X 128 mm). The diameters of the pads were 2 cm for wells 1 to 5 and 1 cm for well 6. The tissue growth medium contained 0.3% methylcellulose supplemented with 10 pM Y27632 (well 2), 1 pM blebbistatin (well 3), 1 pM of TGF-P receptor type l / ll inhibitor LY2109761 (well 4), or 0.5 pM of mechanosensitive channel Piezo-1 agonist Yoda-1 (well 5). (B) Matrigel pad ("'0.45 mm thick, ~04 cm) was made manually in non-treated PS Sarstedt Petri dish (092 mm). The growth medium contained 0.3% methylcellulose. The tissue-like structures were stained with Giemsa dye and imaged on ChemDoc using "gel" mode (white light).

[0161] Figure 4. Scaling up the tissue size. The tissue-like structures were grown on the Matrigel pads of indicated diameters in the medium containing 0.3% methylcellulose. Figure 5. Different types of tissue-like structures formed in 24-well plate coated with PVA. Nontreated PS Falcon® 24-well plate was coated using 500 pl / well of 2% aqueous PVA solution. ECM pad was made by dispensing 25 pl / well drop of Matrigel solution in the center of the well. The tissue-like structures were assembled in the growth medium containing: (A) 0% methylcellulose; (B) 0.2% methylcellulose; (C) 0.3% methylcellulose; (D) 0.5% methylcellulose. The tissue-like structures were stained with Calcein-AM (false colored green) and LysoTracker™ Deep Red (LTDR, false colored red) dyes and analyzed by fluorescence microscopy on Axioimager microscope using 1.25x objective (Scale bar 1 mm).

[0162] Figure 6. Different types of tissue-like structures formed on the agarose layer in 24-well plate. The ~2.5 mm-thick agarose layer was made with 500 pl / well of 1% agarose in non-treated PS Falcon® 24-well plate. The solidified agarose was covered with 100 pl-Matrigel layer ("'0.5 mm-thick). Once the Matrigel is gelled at 37°C for lh, H6c7 cells were added on the top of the Matrigel pad and allow to attach for three hours in cell culture incubator. The tissue-like structures were assembled in the growth medium containing 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.8% final concentrations of MeC. The representative tissue-like structures are shown for conditions: (A) 0.1% methylcellulose; (B) 0.4% methylcellulose; (C) 0.3% methylcellulose; (D) 0.5% methylcellulose. The phase contrast images were taken on Axioimager microscope using 1.25x objective (scale bar 1 mm).

[0163] Figure 7. Example of a large tissue-like structure formed by pancreatic epithelium H6c7 cells in a 6-well plate. The "weak binding" conditions described for agarose-coated 6-well plate were used. The self-organized tissuloid detached from agarose and grew as free-floating structure. The medium was changed every two days maintaining tissuloid alive for three months. For fluorescence labeling, the medium was supplemented with 0.5 pM LysoTracker™ Deep Red dye to label live cells, 0.5 pM CellEvent™ Caspase-3 / 7 Reagent to measure apoptotic cells, and 0.5 pM SYTOX™ Orange dye to detect dead cells (Mittler et al., 2017). Figure 7A shows the image of whole well obtained using ChemDoc scanner. Figure 7B shows the fluorescence image of the whole tissue-like structure obtained by stitching multiple images using Adobe Photoshop. The image is false colored red for LTDR, green for CellEvent™, and orange for SYTOX™ Orange probes. Schematic representation of pancreas organ is given for comparison in Figure 7C.

[0164] Figure 8. Examples of tissue-like structures formed by different types of cells (iPS, MCS, ADSC-d2, ADSC-d6, CAF, FMA, hDF, hLF, HPASTEC, HPNE, H6C7, HEK, HuEp, MCFlOa, RPE1, RWPE1, HaCaT, HKPM, hTert-Kera, HPaMVEC, and HUVEC) in 96-well plates. The typical conditions described for 96-well plate were used, except that the growth medium was specific for a given cell type. The growth medium was supplemented with methylcellulose to obtain the final concentration (from left to right): 0%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%. The tissue-like structures were stained with Calcein- AM and analyzed by bright field and fluorescence microscopy. Each condition represents the binarized image of the entire well (05 mm).

[0165] Figure 9. Examples of epithelium H6c7 tissue-like structures formed with different types of HMW compounds (HMWC) in 96-well plates. The typical conditions described for 96-well plate were used. The growth medium was supplemented with an indicated HMWC to obtain the final concentration (from left to right): 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, and 0.8%. DEX = Dextran, Mw Ca 70,000; HAH = hyaluronic acid-high MW; GG = GUM GUAR; HEM = Hydroxyethylmethylcellulose, 600-1500 cP at 2 %; HPM40 = Hydroxypropylmethylcellulose, 40-60 cP at 2 %; HPM = Hydroxypropylmethylcellulose, 2600-5600 cP at 2 %; MC400 = METHYL CELLULOSE, 400 cP; MC4K = METHYL CELLULOSE, VISCOSITY 4,000 CP; MC1.5K = Methyl cellulose, viscosity 1,500 cP.

[0166] Figure 10. Comparison of pancreatic organoids (A) vs tissue-like structures of the invention (B) formed from the same (initially) number of cells in nine days. Pancreatic organoid culture was established from patient biopsies according to the standard protocols (Broutier et al., 2016; Hogenson et al., 2022). After multiple passages (>5), the single cell suspension was prepared, and the cells were seeded on the top of the Matrigel pads preformed in 6-well plate as described in "Examples" section below. For standard organoid culture, the cells were covered with 3 ml of organoid medium containing 5% Matrigel (A). For tissuloid culture, the cells were covered with 3 ml of organoid medium without Matrigel, containing 0.4% methylcellulose (B). Lensless microscopy images, scale bar 500 pm.

[0167] Figure 11. Effect of methylcellulose concentration on viscoelastic properties of Matrigel pads measured by atomic force microscopy in static (Al) and indentation (Bl, Cl) regimes. The corresponding surface areas of the tissuloids obtained in these conditions are shown on the right of the corresponding figures (A2-C2).

[0168] Figure 12. The differentially expressed genes (DEGs), which are identified by RNA-seq analysis only in the presence of the precasted ECM layer B and, thus, specific to the "tissuloid culture conditions". The DEGs are clustered according to their patterns of expression in response to four concentrations of methylcellulose. (A) Four major groups of DEGs sharing the scaled expression correlation patterns. Box plots are presented to illustrate the expression patterns of each gene within the group, providing a clearer understanding of how well the groupings align with the expression data. (B) The DEG heatmap for the main DE genes ( | Iog2 fold change | >1, adjusted P- value < 0.05 ) from the groups identified by clustered expression patterns in (A). The top ten DEGs from each group are listed on the right.

[0169] EXAMPLES

[0170] Example 1 : Strong binding

[0171] Formation of tissue-like structures in conditions of rather strong binding of the "ECM layer" to the "support / scaffold layer": the tissue-like structures remain attached to the "support / scaffold layer".

[0172] 1.1. Tissue-like structures formation in clustered in 96-well plate.

[0173] A minimal platform for tissue-like structures assembly and subsequent studies / applications is a clustered 96-well plate. This format is useful for screening studies when optimizing the conditions of tissue formation, analyzing the effect of various treatments and characterizing the expression of multiples markers. Various plastic & glass-bottomed plates can be used, the plate bottom serving as the "support / scaffold layer". The plates used in the example were 96-well black PhenoPlates (Perkin Elmer) with optically clear plastic (cyclic olefin) bottom.

[0174] Usually, the plate is pre-chilled at 4°C in the fridge and then is placed on the pre-chilled "Corning™ XT Starter Ice-free Cooler" for Matrigel dispensing. The Matrigel solution is prepared according to the manufacturer's recommendations (Corning) and is diluted to the concentrations of 3 mg / mL and above using an appropriate ice-cold buffer / medium.

[0175] In the present example, Matrigel was prepared at concentration of 5 mg / mL by diluting cold Matrigel stock solution with the ice-cold cell medium. The latter was composed of 50% of [keratinocyte serum-free medium (KSFM) supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE)] and 50% of [Dulbecco's Modified Eagle Medium (DMEM) supplemented with GlutaMAX™, pyruvate and 10% fetal bovine serum (FBS)], thereafter called "50-50 medium". Aliquots of Matrigel solution (50pl per well in the present example; usually > 40pl) were dispensed into pre-chilled 96-well black PhenoPlate (Perkin Elmer) by using a positive-displacement pipette (caution: avoid bubble formation). The plate was placed for 15 min at 4°C in the fridge to settle / distribute homogeneously Matrigel, and then was transferred into the incubator for 1 hour at 37°C to allow gelling the "ECM layer".

[0176] The suspension of cells in appropriate growth medium (concentration can vary; usually, from 104to 2xl06cells / ml) is also prepared following standard protocols.

[0177] In the present example, 40 pl / well aliquots of H6c7 human pancreatic epithelial cells (single cell suspension of 5xl04cells / ml in 50-50 medium) were added on the top of the Matrigel "ECM layer". The plate was transferred to the incubator and was incubated at 37°C for 3 hours (the incubation time can vary from 30 min to 24 hours depending on type of cells and experiments) allowing cells to attach and form the initial "cell layer". After that, 210 pl / well aliquots of methyl-cellulose solutions in 50-50 medium (MeC, methyl-cellulose viscosity: 1,500 cP, #M0387 Sigma-Aldrich in the present example; but other HMWC can be used) were carefully added on the top of the "cell layer" to the final concentration of 0%, 0.2%, 0.4%, and 0.8% of MeC. The final concentration took into account the dilution of the MeC aliquot in the total volume added to the well (300 pl = 50 pl of Matrigel + 40 pl of cell suspension + 210 pl of MeC solution). For fluorescence monitoring of cells / tissues, the MeC solution was supplemented with LysoTracker™ Deep Red dye (LTDR, Thermo Fisher Scientific, #L12492) to obtain a final concentration of 0.5 pM. Other fluorescence probes can be used the same way for live cell imaging including CellEvent™ Caspase-3 / 7 Reagent (CE, Thermo Fisher Scientific, #R37111) to measure apoptosis and various SYTOX™ dyes (Thermo Fisher Scientific) to detect dead cells.

[0178] The cells were grown in the incubator for several days. To follow the tissue assembly, bright field images were taken regularly (each one or two days) using Axioimager Z1 Apotome fluorescence microscope (Zeiss). The lOx and 20x objectives were used to visualize individual cells and their aggregates, whereas 1.25x objective was preferred to image the whole tissue-like structures (two images per well). When LTDR probe was used to label cells, fluorescent images were acquired with a Cy5 filter set.

[0179] In the present example, the measurements of H6c7 cells were continued for two weeks without changing the medium (Figure 2). In these conditions, the majority of the cells remained viable throughout the experiment. For the end-point analysis, the cells were treated with a live cellimaging dye calcein acetoxymethylester (Calcein AM, final concentration of 1 pM) along with a membrane channel blocker Verapamil (final concentration of 2.5 pM) for 1 hour in cell incubator. The imaging was performed as described above with additional acquisition of calcein AM signal using a GFP filter set.

[0180] Depending on the MeC concentration, the cells formed quite different patterns: (Figure 2A) arrays of spheres / droplets at 0% MeC; (Figure 2B) stripes / branches at 0.2% MeC; (Figure 2C) network / meshwork at 0.4% MeC; and (Figure 2D) continuous layer at 0.8% MeC.

[0181] Image analysis using an appropriate software (i.e. ImageJ, Fiji, Adobe Photoshop) allowed for tissue pattern classification / characterization by measuring quantitative parameters such as (i) occupied surface area (all patterns); (ii) fractal dimension (all patterns); (iii) particle size distribution (spheres- droplets); (iv) mesh parameters (network-meshwork).

[0182] 1.2. Tissue-like structures formation in a clustered 6-well plate

[0183] A clustered 6-well plate is another format for tissue formation (Figure 3A). It is particularly useful for studies / applications when larger (size, mass) tissue-like structures and longer times of cultivation are required such as analysis of tissue patterning, lensless microscopy, immunohistochemistry, and preparation of cell extracts for biochemical analysis (i.e. enzyme activity profiling and western blotting). Although various types of plates can be used, "non-treated virgin polystyrene"- and glass-bottomed plates - provide better attachment of the "ECM layer" and reduce cell / tissues growth outside the ECM. Typical examples are glass-bottomed MatTek 6-well plate (#P06G-1.5-20-F), non-treated PS CytoOne 6-well plate (#CC7672-7506), and non-treated PS Falcon® 6-well plate (#351146). The protocol for tissue-like structures formation is given for the latter one.

[0184] PS Falcon® 6-well plate was pre-chilled at 4°C in the fridge and then was placed on the pre-chilled "Corning™ XT Starter Ice-free Cooler" for Matrigel dispensing. A sheet of paper with a printed pattern of 6 circles of 2 cm diameter was inserted between the plate and the cooler to guide Matrigel dispense. Corning Matrigel solution was diluted to the concentrations of 5 mg / mL with ice- cold 50-50 medium (see above). The 140 pl drops of Matrigel solution were dispensed into the center of the wells (according to the printed pattern), and were spread to cover the entire circle area using a pipette tip (caution: avoid bubble formation). The plate was placed for 30 min at 4°C in the fridge to settle / distribute homogeneously Matrigel, and then was transferred into the incubator for 1 hour at 37°C to allow gelling the "ECM layer".

[0185] The suspension of cells in the appropriate growth medium (the concentration can vary; usually, from 104to 2xl06cells / ml) is prepared following standard protocols. In the present example, 140 pl / well 1 aliquots of H6c7 human pancreatic epithelial cells (single cell suspension of 5xl05cells / ml in 50-50 medium) were added on the top of the Matrigel "ECM layer". The plate was transferred to the incubator and was incubated at 37°C for 3 hours allowing cells to attach and form the initial "cell layer". Depending on type of cells and experiments, the incubation time can vary from 30 min to 24 hours, and ROCK inhibitor Y-27632 can be added to a final concentration of 1 pM to promote cell attachment to the "ECM layer". After that, 3 ml / well aliquots of methyl-cellulose solutions in 50-50 medium (MeC, methyl-cellulose viscosity: 1,500 cP, #M0387 Sigma-Aldrich in the present example; but other HMWC can be used) were carefully added on the top of the "cell layer" to the final concentration of 0.3% of MeC (Figure 3A).

[0186] For fluorescence monitoring of cells / tissues, the MeC solution was supplemented with LysoTracker™ Deep Red dye (LTDR, Thermo Fisher Scientific, #L12492) to obtain a final concentration of 0.5 pM.

[0187] The cells were grown in the incubator for several days. To follow the tissue assembly, bright field images were taken regularly (each one or two days) using Axioimager Z1 Apotome fluorescence microscope (Zeiss). The lOx and 20x objectives were used to visualize individual cells and their aggregates, whereas 1.25x objective was preferred to image the whole tissue-like structures (two images per well). When LTDR probe was used to label cells, fluorescent images were acquired with a Cy5 filter set.

[0188] Figure 3A shows the cell growth medium when layer C) containing 0.3% methylcellulose was supplemented with 10 pM Y27632 (well 2), 1 pM blebbistatin (well 3), 1 pM of TGF-P receptor type l / ll inhibitor LY2109761 (well 4), or 0.5 pM of mechanosensitive channel Piezo-1 agonist Yoda-1 (well 5).

[0189] Both Y27632 and blebbistatin facilitate the formation of network / meshwork tissue-like structures and decrease (compared to control) the mean mesh size. Compared to blebbistatin, Y27632 dose not show any cytotoxicity (to concentration up to 20 pM) and, therefore, can be used to improve / increase the robustness of the production of network / meshwork tissue-like structures. The inhibition of TGF-P receptors inhibits the tissuloid growth, while the stimulation of mechanosensitive channel Piezo-1 by Yoda-1 fine tunes the meshwork structure.

[0190] 1.3. Tissue-like structures formation in Petri dishes

[0191] Nunc™ Glass Bottom 035 mm Dishes (#150680, with bottom glass surface of 027 mm) can be used in the same way as glass-bottomed MatTek 6-well plate, essentially for time-lapse lensless microscopy of tissue assembly. After time-lapse recording, the tissue-like structures can be fixed with 4% PFA in PBS, stained with Giemsa and imaged on ChemDoc using the "gel" mode (white light).

[0192] Sarstedt Non-Treated PS Petri Dishes (#821473, 092 mm x hl6 mm) were used to prepare larger- size tissue-like structures (Figure 3B). The protocol was essentially the same as described above for clustered 6-well plates, except the size of circular pattern and the volumes of Matrigel / cells / medium used (Figure 4). In the present example, 535 pl of Matrigel solution (5 mg / ml in 50-50 medium) were dispensed into the center of the dish and were spread to cover the 040 mm circle area using a pipette tip (Figure 3B).

[0193] After settling and gelling of the "ECM layer" (see above), 535 pl of H6c7 cells (single cell suspension of 5xl05cells / ml in 50-50 medium) were added on the top of the Matrigel layer. The dish was transferred to the incubator and was incubated at 37°C for 3 hours allowing cells to attach and to form the initial "cell layer". After that, 12 ml of 0.3% methyl-cellulose solutions in 50-50 medium (MeC, methyl-cellulose viscosity: 1,500 cP, Sigma-Aldrich #M0387) were carefully added and the tissue-like structure was grown in the incubator for several days (> one week).

[0194] For analysis, the tissue-like structure was fixed with 4% PFA in PBS, stained with Giemsa and imaged on ChemDoc using "gel" mode (white light).

[0195] Notably, increasing the area of the tissuloid does not change its fine structure / shape texture. Indeed, the similar tissue-like patterns with the same characteristic structural texture element (mean mesh of ~ 0.3 mm2 area) are formed on the Matrigel pads of 1 cm, 2 cm, and 4 cm in diameter, which have the same thickness (0.42 mm) (Figure 4). Therefore, under the same culture conditions (medium composition, thickness of the deposited Matrigel pad), the final tissuloid morphology depends only on the concentration of methylcellulose and does not depend on the tissuloid area. This property can be useful for the production of the tissuloids of a bigger size.

[0196] Example 2 : weak binding

[0197] Formation of tissue-like structures in clustered multi-well plates & Petri dishes in conditions of weak binding of the "ECM layer" to the "support / scaffold layer": the tissuelike structures are released from the "support / scaffold layer" 2.1. 24-well plate-PVA or polyHEMA coating

[0198] Repellent coating of multi-well plates is useful for studies / applications when tissue-like structures are grown as a "free-floating" culture (Figure 5). Treatment of the plates with non-fouling agents can reduce the attachment of the "ECM layer" to the "support / scaffold layer" that results in tissuelike structures release because of cell contraction and matrix remodeling.

[0199] The present examples describe the use of polyvinyl alcohol (PVA, Sigma-Aldrich #341584) or poly(2- hydroxyethyl methacrylate) (polyHEMA, Sigma-Aldrich #529257) as non-fouling agents to coat the support / scaffold layer A).

[0200] Although various types of plates can be used, "non-treated virgin polystyrene" plates generally provide better coating with PVA / polyHEMA and reduce cell / tissue growth outside the ECM. The protocol for tissue formation is here given for non-treated PS Falcon® 24-well plate (#351147).

[0201] For PVA coating, the plate was incubated with 500 pl / well of 2% aqueous PVA solution (sterilized by autoclaving) for 24 h at 37°C. After that, the PVA solution was removed and the plate was air-dried under the laminar flow hood for 1 h. Then, the wells were rinsed twice with 1 ml of sterile water, were air-dried under the laminar flow hood for 1 h, and the plate was placed into a cell incubator for three hours at 37°C to finish the coating.

[0202] For polyHEMA coating, a 100 pl aliquot of 1.2% solution of polyHEMA in 95% ethanol was added to each well and was air-dried under the laminar flow hood. The wells were rinsed twice with 1 ml of PBS and twice with 1 ml of water, were air-dried under the laminar flow hood for 1 h, and the plate was placed into a cell incubator for three hours at 37°C to finish the coating.

[0203] The coated 6-well plate (PVA or polyHEMA) was pre-chilled for 1 h at 4°C in the fridge and then was placed on the pre-chilled "Corning™ XT Starter Ice-free Cooler" for Matrigel dispensing. The 25 pl / well drops of Matrigel solution (5 mg / ml in 50-50 medium) were dispensed into the wells. The plate was placed for 30 min at 4°C in the fridge to allow spreading the Matrigel drops, and then was transferred into the incubator for 1 hour at 37°C to allow gelling. After that, the 25 pl / well aliquots of H6c7 cells (single cell suspension of 5xl04cells / ml in 50-50 medium) were added on the top of the Matrigel "ECM layer". The plate was transferred to the incubator and was incubated at 37°C for 3 hours allowing cells to attach and to form the initial "cell layer". After that, 500 pl / well aliquots of methyl-cellulose solution in 50-50 medium (MeC, methyl-cellulose viscosity: 1,500 cP, Sigma-Aldrich #M0387) were carefully added and the tissue-like structures were grown in the incubator for several days (> one week). Starting from ~day 2-4 in culture, the activity of the growing cells leads to detachment of the "ECM layer" from the plate bottom thus producing free-floating tissue-like structures.

[0204] The tissue-like structures were stained with Calcein-AM (false colored green) and LysoTracker™ Deep Red (LTDR, false colored red) dyes and analyzed by fluorescence microscopy on Axioimager microscope using 1.25x objective (Scale bar 1 mm).

[0205] The tissue-like structures were assembled in the growth medium containing: (Figure 5A) 0% methylcellulose; (Figure 5B) 0.2% methylcellulose; (Figure 5C) 0.3% methylcellulose; (Figure 5D) 0.5% methylcellulose.

[0206] Compared to the « strong binding » supports described in previous examples, the «week binding » supports allow tissuloids to detach and to continue maturation into 3D free-floating tissue-like objects. The final morphology (and, plausibly, the functional status) of the 3D tissue-like structures can be adjusted depending on the targeted application by changing of methylcellulose concentration.

[0207] Quite similar results have been obtained using polyHEMA coating protocol (the results are not shown).

[0208] 2.2. 6-well plate-PVA or polyHEMA coating

[0209] For studies / applications when larger tissue-like structures and longer times of cultivation are required, a clustered 6-well plate can be repellent-coated using the same protocol as for 24-well plate and adjusting the amounts and volumes of the reagents according to the increased well capacity.

[0210] Thus, for non-treated PS Falcon® 6-well plate (#351146), 2 ml of 2% PVA solution, or 500 pl of 1.2% polyHEMA were used for repellent-coating. The volumes of washing solutions were adjusted accordingly.

[0211] A tissue-like structure of 015 mm initial dimeter was formed using 85 pl of Matrigel solution (5 mg / ml in 50-50 medium) and 85 pl of H6c7 cells (single cell suspension of 5xl05cells / ml in 50-50 medium). The preferential volume of MeC solution in 50-50 medium was 3 ml / well. The cells were grown in the incubator for several days. The self-organized tissuloid detached from agarose and grown as free-floating structure. Starting from three week the tissuloid medium was changed every two days by carefully replacing the old medium by 2 ml of fresh 50-50 medium. Under these conditions, the tissuloid was maintained alive for more than a month.

[0212] For fluorescence monitoring of functional status of the tissuloid, the cells can labeled with 0.5 pM LysoTracker™ Deep Red dye (LTDR, Thermo Fisher Scientific, #L12492) to label live cells, 0.5 pM CellEvent™ Caspase-3 / 7 Reagent (CE, Thermo Fisher Scientific, #R37111) to measure apoptotic cells, and 0.5 pM SYTOX™ Orange dye (Thermo Fisher Scientific) to detect dead cells (Mittler et al., 2017) (see example below: 2.4. 48-well & 6-well plates-agarose).

[0213] The tissuloids generated this way have a large size >1 cm comparable to the size of embryonic organs and are amenable to various kinds of physiological tests and organ-on-a-chip applications (see example below: 2.4. 48-well & 6-well plates-agarose).

[0214] The results are shown on Figure 7.

[0215] 2.3. 24-well plate-agarose

[0216] The "support / scaffold layer" can also be made from a hydrogel. When a non-modified agarose hydrogel is used, it provides a suitable repellent surface for the "ECM layer". Various types of agarose taken at different concentrations can be used (typical range 0.5-4.0%). In a given example, 1% UltraPure Agarose (Thermo Fisher Scientific, ## 16500100) was used (Figure 6).

[0217] The solution of 1% agarose in MilliQ. water was sterilized by autoclaving at 121°C for 20 min. The hot (~50°C) solution was dispensed into a 24-well plate (non-treated PS Falcon® 24-well plate (#351147), other types of plates can also be used) and was allowed to gel at room temperature under the laminar flow hood (the agarose volume may vary depending on application, 1 ml / well in the present example).

[0218] The agarose layer was covered with 1.75 ml / well DMEM (without serum) and the plate was kept at 37°C in a cell incubator for at least 8 hours allowing the gel to equilibrate with the medium. The medium was removed and the procedure was repeated once with DMEM and twice with 50-50 medium.

[0219] Before the experiment, the medium was removed and the plate was pre-chilled at 4°C for 2 h in the fridge. The Matrigel solution was prepared at concentration of 5 mg / mL by diluting cold Matrigel stock solution with the ice-cold 50-50 medium. Aliquots of Matrigel solution (100 pl per well in the present example; usually > 60pl) were dispensed in the center of agarose pad by using a positivedisplacement pipette. The plate was placed into the incubator for 1 hour at 37°C to allow gelling the "ECM layer".

[0220] The 100 pl / well aliquots of H6c7 cell suspension in 50-50 medium (5xl04cells / ml in this example) were slowly added on the top of the Matrigel "ECM layer". The plate is transferred to the incubator and was incubated at 37°C for 3 hours allowing cells to attach and form the initial "cell layer".

[0221] In one modification of the present protocol, the cells were added along with the Matrigel to form a mixed "cell-ECM layer". To this end, cell suspension in cold 50-50 medium was used to dilute Matrigel stock solution to obtain the final concentrations of 5 mg / ml Matrigel and 5xl04cells / ml. The resulted mix was dispensed on the agarose pads (100 pl per well) and the plate was placed into the incubator for 1 hour at 37°C to allow gelling the "cell-ECM layer". Before placing the plate into the incubator, it can be centrifuged at 4°C / 300 g for 10 min, to allow the cells to sediment and to form a layer on the interface between the Matrigel and agarose.

[0222] After the "cell layer" (or the "cell-ECM layer") was formed, 1 ml / well aliquots of methyl-cellulose solutions in 50-50 medium (MeC, methyl-cellulose viscosity: 1,500 cP, Sigma-Aldrich #M0387) were slowly added on the top of the "cell layer" (or of the "cell-ECM layer") to the final concentrations of 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.8% of MeC. The final concentration took into account the dilution of the MeC aliquot in the total volume added to the well (2.2 ml = 1 ml of agarose + 0.1 ml Matrigel + 0.1 ml of cell suspension + 1 ml of MeC solution). For fluorescence monitoring of cells / tissues, the MeC solution was supplemented with LysoTracker™ Deep Red dye (LTDR, Thermo Fisher Scientific, #L12492) to obtain a final concentration of 0.5 pM.

[0223] The cells were grown in the incubator for several days. To follow the tissue assembly, bright field images were taken regularly (each one or two days) using Axioimager Z1 Apotome fluorescence microscope (Zeiss). The lOx and 20x objectives were used to visualize individual cells and their aggregates, whereas 1.25x objective was preferred to image the whole tissue-like structure (two images per well). Fluorescent images were acquired with a Cy5 filter set.

[0224] Starting from ~day 2-4 in culture, the activity of the growing cells provoked detachment of the "ECM layer" from the plate bottom thus producing free-floating tissue-like structures. 2.4. 48-well & 6-well plates-agarose

[0225] For studies / applications where smaller / larger tissue-like structures are required, clustered 48-well or 6-well plates can be agarose-coated using the same protocol and adjusting the amounts and volumes of the reagents according to the decreased / increased well capacity. The volumes of washing solutions should be adjusted accordingly. For example, for non-treated PS Falcon® 48-well plate (#351178), one can use: 300 pl of 1% agarose, 50 pl of Matrigel solution, 50 pl of cell suspension, and 150 pl of MeC solution.

[0226] For non-treated PS Falcon® 6-well plate (#351146), one can use 4 ml of 1% agarose, 500 pl of Matrigel solution, 500 pl of cell suspension, and 2 ml of MeC solution. In the present example (Figure 7), a tissue-like structure was formed using 500 pl of Matrigel solution (5 mg / ml in 50-50 medium) and 500 pl of H6c7 cells (single cell suspension of 5xl05cells / ml in 50-50 medium). After the "cell layer" (or the "cell-ECM layer") was formed, 2 ml of methylcellulose solution in 50-50 medium were slowly added on the top of the "cell layer" (or of the "cell-ECM layer") to obtain the final methylcellulose concentration of 0.3%. The cells were grown in the incubator for several days. After ~one week, the self-organized tissuloid detached from agarose and grew as free-floating structure. Starting from three week the tissuloid medium was changed every two days by carefully replacing the old medium by 2 ml of fresh 50-50 medium. Under these conditions, the tissuloid was maintained alive for three months.

[0227] For fluorescence monitoring of functional status of the tissuloid, the tissuloid medium was supplemented with 0.5 pM LysoTracker™ Deep Red dye (LTDR, Thermo Fisher Scientific, #L12492) to label live cells, 0.5 pM CellEvent™ Caspase-3 / 7 Reagent (CE, Thermo Fisher Scientific, #R37111) to measure apoptotic cells, and 0.5 pM SYTOX™ Orange dye (Thermo Fisher Scientific) to detect dead cells (Mittler et al., 2017).

[0228] The images were taken using Axioimager Z1 Apotome fluorescence microscope (Zeiss). The lOx and 20x objectives were used to visualize individual cells and their aggregates, whereas 1.25x objective was preferred to image the whole tissue-like structure (multiple images were taken and stitched with Adobe Photoshop to obtain the whole tissuloid image on Figure 7). For LTDR, CellEvent™, and SYTOX™ Orange probes, the fluorescent images were acquired with Cy5, FITC, and TxR filter sets respectively.

[0229] These results show that using the method of the invention large >1 cm tissue-like structures can be produced and maintained in culture for a long period (at least 3 months). Notably, the tissuloids generated this way have the size comparable to the size of embryonic organs and are amenable to various kinds of physiological tests and organ-on-a-chip applications.

[0230] Example 3 : tissue-like structures with different cells

[0231] The method of the invention can be used to produce tissue-like structures from different types of cells. In the present example, 21 different types of cells (iPS, MCS, ADSC-d2, ADSC-d6, CAF, FMA, hDF, hLF, HPASTEC, HPNE, H6C7, HEK, HuEp, MCFlOa, RPE1, RWPE1, HaCaT, HKPM, hTert-Kera, HPaMVEC, and HUVEC) were tested for tissuloid formation in 96-well.

[0232] The protocol was primarily followed as outlined in the "1.1. Tissue-like structures formation in clustered 96-well plate" section, with the modification that, for each cell type, a cell type-specific medium (as recommended by the respective cell source, such as ATCC, https: / / www.atcc.org / ) was utilized instead of the 50-50 medium. Henceforth, this specific cell type medium will be referred to as the "cell medium".

[0233] Thus, Matrigel was prepared at concentration of 5 mg / mL by diluting cold Matrigel stock solution with the ice-cold "cell medium". Aliquots of Matrigel solution (50pl of per well) were dispensed into a pre-chilled 96-well black PhenoPlate (Perkin Elmer) by using a positive-displacement pipette. The plate was placed for 15 min at 4°C in the fridge to settle / distribute homogeneously Matrigel, and then was transferred into the incubator for 1 hour at 37°C to allow gelling the "ECM layer".

[0234] Single cell suspension was prepared in the "cell medium" (5xl04- 105cells / ml) and 40 pl aliquots were dispensed on the top of the Matrigel "ECM layer". The plate was transferred to the incubator and was incubated at 37°C for 3 hours allowing cells to attach and form the initial "cell layer". After that, 210 pl / well aliquots of methyl-cellulose solutions in the "cell medium" (MeC, methylcellulose viscosity: 1,500 cP, #M0387 Sigma-Aldrich) were carefully added on the top of the "cell layer" to the final concentration of (from left to right): 0%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%. The final concentration took into account the dilution of the MeC aliquot in the total volume added to the well (300 pl = 50 pl of Matrigel + 40 pl of cell suspension + 210 pl of MeC solution).

[0235] The tissue-like structures were grown for 9-15 days, stained with Calcein-AM lpM final concentration) and analyzed by bright field and fluorescence microscopy.

[0236] The results shown in Figure 8 reveled that all types of cells are able to form tissue-like structures under these conditions. Notably, methylcellulose alters the rates of cell proliferation and cell death in a cell-type-specific manner, which can vary from "fine tuning" (the changes in the rates of cell proliferation and cell death do not exceed two folds) for some epithelial cells / keratinocytes to almost yes-or-no response for some fibroblasts (CAF, FMA), which are unable to growth efficiently on the Matrigel without methylcellulose.

[0237] Therefore, in contrast to other methods, such as standard organoid cultures (Broutier et al., 2016; Hogenson et al., 2022), which frequently preclude the effective integration and growth of multiple cell types within a self-assembled tissue-like aggregate, the present approach provides a straightforward means to optimize co-culture conditions by adjusting the HMWC concentration. This opens the way to control not only the structure of the tissue-like structure but also its cellular composition and functionality, that is particularly important when working with biopsies and patient-derived-organoids.

[0238] Example 4: use of different HMWC

[0239] Various HMWC can be used to produce tissue-like structures. In the present example, 8 different HMWC (DEX = Dextran, Mw Ca 70,000; HAH = hyaluronic acid-high MW; GG = GUM GUAR; HEM = Hydroxyethylmethylcellulose, 600-1500 cP at 2 %; HPM40 = Hydroxypropylmethylcellulose, 40-60 cP at 2 %; HPM = Hydroxypropylmethylcellulose, 2600-5600 cP at 2 %; MC400 = METHYL CELLULOSE, 400 cP; MC4K = METHYL CELLULOSE, VISCOSITY 4,000 CP; MC1.5K = Methyl cellulose, viscosity 1,500 cP) were tested for their capacity to induce epithelium H6c7 tissue-like structures formation in 96- well.

[0240] The protocol was primarily followed as outlined in the "1.1. Tissue-like structures formation in clustered 96-well plate" section, with the modification that one of the indicated molecules was used as HMWC instead of methylcellulose. The growth medium was supplemented with an HMWC to obtain the final concentration (from left to right): 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, and 0.8%.

[0241] As shown on Figure 9, all HMWC (except DEX-70kD) were able to support the formation of tissuelike structures. Notably, among them such compounds as methylcellulose, guar gum, hyaluronic acid, and xanthan gum are already used / authorized in tissue and food engineering and can therefore be used safely in these applications. Example 5: tissue-like structures containing multiple cells populations

[0242] The method of the invention can be used to produce tissue-like structures from heterogeneous (multiple types) cell populations such as single cell suspensions produced from biopsies and patient- derived-organoids. In the present example, pancreatic organoids and tissuloids were grown from the same (initially) number of cells. Pancreatic organoid culture was established from patient biopsies according to the standard protocols (Broutier et al., 2016; Hogenson et al., 2022). After multiple passages (>5), the single cell suspension was prepared, and the cells were seeded on the top of the Matrigel pads preformed in 6-well plate as described in "Examples" section below. For standard organoid culture, the cells were covered with 3 ml of "organoid medium" containing 5% Matrigel (Broutier et al., 2016; Hogenson et al., 2022). For tissuloid culture, the cells were covered with 3 ml of "organoid medium" without Matrigel, containing 0.4% methylcellulose.

[0243] The growth and self-assembly of cells were followed by lensless microscopy (Figure 10). Compared to the organoids culture that showed the formation of small (<500 pm) dispersed acini-like organoid structures (Figure 10A), the tissuloid culture reveal the continuous tissue-like structures with significant outgrowth of fibroblast-like cells (Figure 10B).

[0244] Example 6: Effect of polymer concentration on viscoelastic properties of the ECM layer B

[0245] The circular Matrigel pads of 2 cm in diameter were formed in tissue culture dishes 0 40 mm (TPP Techno Plastic Products 93040, AFM-compatible) following the protocol described in Example 1.1. After that, 3 ml / well aliquots of methyl-cellulose solutions in 50-50 medium were added to the final concentrations of 0, 0.2, 0.3, 0.4, 0.6, and 0.8%. The plates were incubated in the cell culture incubator at 37°C for 24 hours before AFM measurements. AFM rheological measurements of the Matrigel pads are represented in figure 11. The quantification of the rheological properties of the gels was performed by Indentation -type atomic force microscopy, using a Nanowizard II AFM (JPK instruments, Berlin). The AFM was equipped with a heating platform, so the samples were incubated at 37°C for the duration of the experiment. The samples were probed with a MLCT tip C (Bruker, Santa Barbara) with a nominal spring constant of 0.01 N / m.

[0246] The static experiments were carried out by indentation at a speed of 0.5 pm / s with a set force of between 0.15 nN for the softest material and 0.5 nN for the stiffest material. The Young's moduli were obtained by fitting the indent curve with a Sneddon model assuming the Poisson's ratio to be 0,5.

[0247] Frequency dependence was quantified by oscillating the tip at a frequency between 1 Hz and 100 Hz with an amplitude of 20 nm once the force set point was reached. The measure of phase shift and amplitude attenuation was used to calculate the storage and loss moduli, using JPK Processing software.

[0248] For the sake of clarity, AFM rheological measurements refer to the use of Atomic Force Microscopy (AFM) to evaluate the mechanical properties of materials, particularly soft materials such as cells and polymers.

[0249] Briefly, the atomic force microscope (AFM) operates using a microscopic physical probe, which consists of an extended, flexible cantilever with a tip that points downward from its free end toward the sample, which is mounted on a moving piezoelectric stage. The mechanical and force properties of the sample are determined by varying the tip / sample distance (i.e., movement along the z-axis) and observing the resulting bending of the cantilever due to its interaction with the sample. This bending is measured by reflecting a laser beam off the top of the cantilever onto a position-sensitive photodetector, which detects deflection changes directly related to the forces involved in the tipsample interaction.

[0250] The AFM generates a force-distance curve by plotting cantilever deflection as a function of tipsample separation (z-axis distance). Various contact models, such as the Hertz, Sneddon, or JKR models, can then be applied to these curves to calculate the viscoelastic characteristics of the material.

[0251] AFM measurements can be performed in different operational modes, with the static and dynamic (oscillatory) modes being the most commonly used today for measuring the viscoelastic properties of soft materials (with stiffness values ranging from 0 to several thousand pascals (Pa)). Static AFM mode, also known as contact mode, is a fundamental operating mode where the AFM probe maintains continuous physical contact with the sample by pressing the AFM tip into a sample surface. The most common configuration of static mode is to use a constant force or deflection feedback regime. Dynamic AFM mode refers to a number of AFM modes in which the cantilever oscillates at a certain frequency.

[0252] Typically, force curves obtained in static mode allow for the measurement of the static Young's (elastic) modulus, while the dynamic (oscillatory) mode enables the measurement of the complex viscoelastic modulus, G*, which comprises G' (storage / elastic modulus) and G" (loss / viscous modulus). Details of these methods are described, for instance, in Kaman, J. (2015).

[0253] For the sake of clarity, static Young's modulus is a mechanical property that measures the stiffness of a solid material under static loading conditions. It is defined as the ratio of stress to strain in the linear elastic region of a material when subjected to a slowly applied force.

[0254] For the sake of clarity, storage modulus, denoted as G", is a measure of a material's ability to store energy elastically when subjected to deformation. It represents the elastic portion of a material's viscoelastic behavior, describing its solid-state characteristics. There can be several G' values in storage modulus data due to the different experimental conditions and material properties being measured.

[0255] For the sake of clarity, loss modulus, denoted as G", is a measure of the energy dissipated as heat in a material when it is subjected to stress, representing the viscous response of the material.

[0256] As shown in figure 11, increasing concentrations of high molecular weight polymers, here methylcellulose, impact the viscoelastic properties of the "ECM layer" B, as evidenced by the noticeable increase of static Young's modulus as well as the complex viscoelastic modulus, G*, comprising G' (storage / elastic modulus) and G"(loss / viscous modulus). As is known in the art, an increasing static Young's modulus is indicative that the material which elastic properties are being measured is becoming stiffer, meaning it requires more stress / force to produce the same amount of strain.

[0257] Example 7: Effect of different culture conditions on RNA expression of H6C7 cells

[0258] The H6C7 cells were cultured in the presence of four concentrations of methylcellulose (0, 0.2, 0.3, 0.5%) following the protocol described in Example 1.1. In control experiments, the H6C7 cells were cultured without the Matrigel as a 2D monolayer in a 96-well plate (black PhenoPlatesPerkin Elmer) with or without 0;5% of methylcellulose. After 7 days of culture, the Matrigel in the wells was digested to recover the live cells using 2% dispase (Dispase Type II, Gibco, Thermo Fisher Scientific, 17105041) and 5% collagenase in KSFM / DMEM medium. After Matrigel digestion, the cells were dissociated from one another using 100% accutase (Accutase cell detachment solution, Sigma Aldrich, SCR005). RNA was then extracted from the cells using the RNeasy Plus Mini Kit (50) from Qiagen (74134). In control experiments, the H6C7 cells were cultured without Matrigel as a 2D monolayer in a 96-well plate (black PhenoPlates, PerkinElmer) with or without 0.5% methylcellulose, and RNA was extracted directly using the RNeasy Plus Mini Kit. The quantity of the extracted RNA was measured using NanoDrop. The quality of the RNA extraction was assessed using the Agilent RNA 6000 Pico Kit (5067-1513) and the Agilent 2100 Bioanalyzer from Agilent Technologies. Finally, the RNA samples were sent for RNA-Seq analysis to the GenomEast platform at IGBMC, lllkirch. The data enrichment analysis was performed by the same platform.

[0259] Results are shown in figure 12. These results show a different pattern of gene expression depending on culture conditions, in particular a modification of gene expression with increasing concentrations of methylcellulose. Notably, the majority (~90%) of significantly differentially expressed genes (DEGs) observed under tissuloid conditions are not detected in control experiments, where H6C7 cells were cultured without Matrigel as a 2D monolayer, with or without 0.5% methylcellulose. This highlights the critical role of both Matrigel and the polymer (methylcellulose) layers in the formation of tissue-like structures characterized by specific gene expression patterns. Furthermore, the addition of methylcellulose alone, akin to macromolecular crowding conditions, is neither sufficient to produce tissue-like morphologies nor to induce the associated changes in gene expression. The characteristic pathways identified by the DEG enrichment analysis under tissuloid conditions include ribosome and protein synthesis, proteasome-mediated protein degradation, oxidative phosphorylation, apoptosis, immune response, TGF-beta and BMP signaling, beta-catenin signaling, and a shift in ECM proteolytic remodeling from metalloproteases to serine proteases.

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Claims

CLAIMS1. A method to create an artificial tissue-like structure, said method comprising: i) Providing a support layer A, ii) Providing extracellular matrix (ECM) proteins, gelling the proteins in appropriate conditions onto the support layer A, thereby forming an ECM layer B on the top of the support layer A, iii) Immerging the layers A and B in a cell growth medium that is able to favor the growth of living cells under appropriate culture conditions, iv) Providing living cells that can be cultured in said growth medium and introducing them in - or placing them on the top of - the ECM layer B, v) optionally, incubating the structure under appropriate conditions to allow the cells to attach to - or to invade - the ECM layer B, vi) Providing a cell growth medium containing high molecular weight viscous water-soluble polymers, and forming a viscous cell growth medium layer C on top of layer B, vii) optionally, culturing the obtained layered structure during at least 6 hours in appropriate conditions for the living cells to self-assemble into tissue-like structures.

2. The method of claim 1, further comprising a step of centrifugating the structure between step iv) and step v) so that the cells invade the layer B and rapidly sediment at the interface with layer A.

3. The method of claim 1 or 2, wherein the support layer A used in step i) is a stiff surface, preferably chosen from:(i) the bottom of a cell culture dish, of a multiwell-cluster plate, or of a fabricated chamber, more preferably made of metals, ceramics, glass, polystyrene, polyacrylate, or cyclic olefin copolymer; more preferably polystyrene or glass,(ii) the surface of a flat hydrogel pad, more preferably made of natural and synthetic polymers such as cellulose-based materials, polysaccharides, poly-lactic acid (PLLA), poly-lactic-co-glycolic acid (PLGA), poly-caprolactone (PCL), polydimethylsiloxane (PDMS), agarose, alginate, collagen, gelatin- polyacrylate, polyacrylate, polyacrylamide, polyethyleneglycol / poly(ethylene oxide) (PEG / PEO), or combinations thereof;(iii) leather and leather-like materials, and(iii) a granular, fibrous or porous membrane.

4. The method of claim 1, 2 or 3, wherein the support layer A used in step i) is a low attachment layer made of:Glass or plastic or polymers materials that are:• grafted with hydrophilic polyethyleneglycol, polyacrylate and polyacrylamide chains,• coated with albumin proteins, amphiphilic molecules, natural and synthetic surfactants, synthetic polymers such as poly(2-hydroxyethyl methacrylate) (polyHEMA), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA), or• coated with non-fouling agents such as poly(2-hydroxyethyl methacrylate) (polyHEMA), polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA),Agarose, alginate, collagen, gelatin-polyacrylate, polyacrylate and polyacrylamide and their co-polymers with polyethyleneglycol, or combinations thereof, preferably agarose-gelatin composite,Glass or plastic or polymers materials that are grafted or coated with a thermosensitive polymer such as Pluronic or poly(N-isopropylacrylamide) (PIPAAm) or with light-responsive molecules.

5. The method of any of claims 1-4, wherein the support layer A used in step i) is a low attachment layer according to claim 4, and the method further comprises a final step of separating the tissuelike structure resulting from steps i) to step vi) or vii) from said support layer A.

6. The method of any of claims 1-5, wherein the ECM proteins used in step ii) are chosen from:Natural ECM proteins such as collagens, gelatins, laminins, fibronectins, vitronectins, fibrins, nidogens, and entactins,Natural or synthetic glycoproteins and polysaccharides,Complex protein mixtures such as basement membrane, plasma, lymph, or avian egg white,Decellularized Extracellular Matrix (DECMs) produced by decellularization of a variety of animal-derived tissues and organs,Synthetic hydrogels based on polyacrylate, polyethyleneglycol / poly(ethylene oxide) (PEG / PEO), poly-lactic acid (PLLA), poly-lactic-co-glycolic acid (PLGA), and poly-caprolactone (PCL) polymers mimicking natural ECM.

7. The method of any of claims 1-6, wherein the ECM proteins used in step ii) comprise a mixture of laminin, collagen and entactin.

8. The method of any of claims 1-7, wherein the ECM proteins used in step ii) comprise or consist in a mixture of laminin, collagen, entactin and heparan sulfate proteoglycan.

9. The method of any of claims 1-8, wherein step (ii) comprises providing extracellular matrix (ECM) proteins, gelling the proteins in appropriate conditions onto the support layer A, thereby forming an ECM layer B of at least 50 pm, at least 100pm, at least 200 pm, at least 400pm, or at least 450pm, on the top of the support layer A.

10. The method of any of claims 1-9, wherein the high molecular weight polymers used in step vi) have a molecular weight of 70 kDa to 7000 kDa and are preferably chosen from :Native natural polymers such as polynucleotides, polypeptides, proteoglycans;Polysaccharides including cellulose, starch, guar gum, gum arabic, alginates, carrageenan, chitosan, glycosaminoglycan, hyaluronic acid, dextran, and xanthan gum;Modified natural polymers such as methyl-cellulose, hydroxyethyl-cellulose, hydroxyethyl- methyl-cellulose, hydroxypropyl-methyl-cellulose carboxymethyl-cellulose, Ficoll;Synthetic polymers such as based on chemically modified polyacrylate, polyethyleneglycol / poly(ethylene oxide) (PEG / PEO), poly-lactic acid (PLLA), poly-lactic-co- glycolic acid (PLGA), and poly-caprolactone (PCL) polymers.

11. The method of any of claims 1-10, wherein the high molecular weight viscous water-soluble polymers are used at a concentration appropriate for increasing stiffness of the ECM layer B up to a stiffness corresponding to a static Young's modulus, of at least 500, at least 750, at least 1000 Pa, as measured by AFM.

12. The method of any of claims 1-11, wherein the high molecular weight polymers used in step vi) have a molecular weight of 70 kDa to 7000 kDa are preferably chosen from methyl-cellulose, hydroxyethyl-cellulose, hydroxyethyl-methyl-cellulose, hydroxypropyl-methyl-cellulose carboxymethyl-cellulose.

13. The method of any of claims 1-12, wherein the high molecular weight polymers used in step vi) are used in the concentrations varying from 0.3 to 0.8% by weight.

14. The method of any of claims 1-13, wherein step (ii) comprises providing extracellular matrix (ECM) proteins, gelling the proteins in appropriate conditions onto the support layer A, thereby forming an ECM layer B of at least 50 pm, at least 100pm, at least 200 pm, at least 400pm, or at least 450pm, on the top of the support layer A, and wherein the high molecular weight polymers used in step vi) have a molecular weight of 70 kDa to 7000 kDa are preferably chosen from methylcellulose, hydroxyethyl-cellulose, hydroxyethyl-methyl-cellulose, hydroxypropyl-methyl-cellulose and carboxymethyl-cellulose, and are used in the concentrations varying from 0.3 to 0.8% by weight.

15. An artificial tissue-like structure obtained from the method of claims 1-14, containing at least a gelled layer B of extracellular matrix (ECM) proteins comprising living cells, covered by a viscous cell growth medium layer C containing molecular weight water-soluble polymers.

16. An artificial tissue-like structure containing:- optionally a support layer A;- a layer B containing gelled extracellular matrix proteins and living cells and,- on the top of layer B, a layer C consisting of viscous cell growth medium that contains high molecular weight water-soluble polymers.

17. The artificial tissue-like structure of claim 15 or 16, wherein said living cells are cell lines or primary cells, preferably eukaryotic cells including epithelial cells, endothelial cells, fibroblasts, non- embryonic stem cells, iPS cells, or a mixture thereof, more preferably a mixture of stromal cells comprising fibroblasts, myofibroblasts, adipocytes, fibrocytes, pericytes, mesenchymal stem cells, macrophages, mast cells, and / or lymphocytes, with epithelial cells including pancreas, prostate, mammary, kidney, lung, and bladder cells.

18. The artificial tissue-like structure of any of claims 15-17, wherein it is a two-dimensions or a three-dimensions artificial tissue-like structure.

19. A platform to produce the artificial tissue-like structure of any of claim 15-18, said platform reducing to practice the steps of the method as defined in claims 1-14 in an automatic manner.

20. Use of the product as defined in any of claims 15 to 18, as a non-medical comestible food product preferably as cultured meat.

21. Use of the product as defined in any of claims 15 to 18, as an in vitro tool in translational researches to study artificial tissues or organs, to reproduce normal or pathological biological processes, or for preclinical drug testing.

22. The product as defined in any of claims 15 to 18, for use as an organ or tissue to be grafted in a patient in need thereof.

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