Extracellular matrix substitutes in cellular microcompartments

Abstract

The present invention relates to the field of three-dimensional cell culture, in particular to cell microcompartments free of extracellular matrix of animal origin and / or derived from cancer cell lines, for the production of GMP-grade cells and tissues.

Description

[Technical Field]

[0001] The present invention relates to the field of three-dimensional cell culture, in particular to cell microcompartments free of extracellular matrix of animal origin and / or derived from cancer cell lines, for the production of GMP-grade cells and tissues. [Background technology]

[0002] Prof. Yamanaka's discovery of induced pluripotent stem cells (iPS or iPSC) gave new impetus to the field of cell culture. Clinical trials were soon initiated with the aim of treating diseases that were often rare and untreatable with conventional medicines.

[0003] Traditionally, cells, including induced pluripotent stem cells, have been cultured in two dimensions (2D). Due to the limitations of 2D cell culture, three-dimensional (3D) culture systems have been developed in recent years, which can partially overcome the drawbacks of 2D culture.

[0004] Such systems are advantageously closer to the natural in vivo system and can be used in many applications, especially cell therapy. The cells cultured in these systems can be of any type. The cultured cells can include both differentiated cells, progenitor cells, and stem cells with different phenotypes.

[0005] A particularly suitable technique is that described in WO 2018 / 096277, which describes three-dimensional microcompartments for stem cell culture, comprising, inter alia, cells, a Matrigel®-like extracellular matrix layer and an outer layer of hydrogel.

[0006] Although this is a very promising technology, it still has certain drawbacks that make it suitable for use in clinical settings. To achieve this, both the cells and the three-dimensional culture system from which they are derived must comply with Good Manufacturing Practices (GMP) regulations. However, most three-dimensional culture systems, such as cell microcompartments, contain extracellular matrices of animal origin and / or cancer cell line-derived extracellular matrices, such as Matrigel®, also known as Engelbreth-Holm-Swarm (EHS) matrix, which do not comply with such regulations.

[0007] Therefore, Matrigel® is derived from a mouse sarcoma, and its widespread use in cell culture, particularly iPS cell culture, is questionable due to the high likelihood of introducing xenogeneic contaminants into cells obtained from cultures containing Matrigel®. Furthermore, the complexity of the mixture and the variability in biochemical and mechanical properties between and within batches mean that cell culture experiments lack reproducibility.

[0008] To consider the emergence of cell therapy based on this technology or the production of animal or plant cells for human or animal consumption, it is necessary to develop substitutes, i.e., alternatives that allow the elimination of the presence of such an extracellular matrix while preserving the possibility for cells to adhere and grow satisfactorily.

[0009] During these studies, the inventors surprisingly discovered the use of a hydrogel with a high Young's modulus intended to form the outer envelope or outer layer of a cell microcompartment in combination with a second hydrogel with a lower Young's modulus than the outer layer, intended to form a layer or mesh in the inner part of the microcompartment capable of accommodating and growing at least one cell. Advantageously, the present invention makes it possible to obtain large amounts of cells, as well as rapid growth and low mutation rates. Such results therefore make it possible to envision their use in the production of three-dimensional cell microcompartments for human and veterinary use.

[0010] These studies certainly describe the use of synthetic matrices, such as in three dimensions. However, cell microcompartments containing two hydrogels, each with a different Young's modulus, have not been described, particularly when the Young's modulus of the outer hydrogel is strictly greater than that of the hydrogel constituting the inner layer or mesh. Such structures, however, are complex to implement, so that the outer layer forms the outer envelope of the cell microcompartment and is sufficiently rigid to thereby protect the cellular contents. Meanwhile, the inner portion of the cell microcompartment, which is bounded by the outer envelope of the microcompartment, including at least the hydrogel layer or mesh, possesses specific rheological properties and mechanical strength capable of providing cells with an environment conducive to their dispersion, proliferation, and migration.

[0011] Furthermore, to meet this need for cell microcompartments that do not contain non-GMP extracellular matrices such as Matrigel® and that have rheological properties suitable for cell protection and growth within the microcompartment, the present invention proposes a three-dimensional culture system based on a three-dimensional cell microcompartment that contains at least two hydrogels with different Young's moduli, in particular the Young's modulus of the hydrogel in the outer layer being strictly greater than the Young's modulus of the hydrogel contained in the inner part and constituting the layer or mesh within the microcompartment. Summary of the Invention

[0012] Accordingly, the present invention provides novel three-dimensional cellular microcompartments comprising: an outer layer of hydrogel; an inner part comprising at least one cell and at least one layer or mesh of a hydrogel of plant or synthetic origin, It concerns three-dimensional cellular microcompartments in which the Young's modulus of the hydrogel in the inner portion is strictly smaller than the Young's modulus of the hydrogel in the outer layer.

[0013] The present invention therefore relates to the use of two particular hydrogels, the first intended to form an outer layer and the second intended to form a layer or mesh within an inner portion, said inner portion being bounded by an outer layer of hydrogel, it being understood that the Young's modulus of the hydrogel of the inner portion is strictly less than the Young's modulus of the hydrogel of the outer layer.

[0014] On the one hand, the outer layer of the hydrogel forms a protective outer envelope and thus constitutes a capsule, and on the other hand, the layer or mesh of the inner part is less rigid and more loose, allowing cell growth, given the particularly favorable rheological properties of the hydrogel of said layer or mesh of the inner part.

[0015] Thus, the microcompartment according to the invention is composed of two different hydrogels, the hydrogel constituting the outer layer having a Young's modulus strictly greater than that of the hydrogel constituting the inner layer or mesh, thereby improving the protection of the cells and cell aggregates present within the cell microcompartment.

[0016] In fact, the outer layer advantageously provides a first protective envelope. This effect is enhanced by at least one hydrogel layer or mesh in the inner part, which provides an additional protective mesh for the cells and aggregates present in the microcompartments, allowing cell migration and expansion and loose enough to maintain their pluripotent characteristics. In contrast, known prior art structures consisting of a single layer of hydrogel are either too stiff, preventing cell migration and expansion and inducing cell apoptosis, or too loose, making them incompatible with bioreactor culture. Indeed, the mechanical constraints of bioreactor culture, especially those imposed by the high shear forces that occur during culture, require suitable cell microcompartments.

[0017] Thus, an outer layer of hydrogel with a high Young's modulus forms the outer envelope of the microcompartments or capsules according to the invention and makes it possible to protect the contents of the capsule from the external environment, especially when the capsules are cultured in a bioreactor. This is further enhanced by the presence of a hydrogel layer or mesh in the inner part of the microcompartment. Advantageously, the hydrogel layer or mesh in the inner part is at least partially juxtaposed to the inner surface of the outer layer.

[0018] According to a preferred object of the invention, the Young's modulus of the hydrogel of the mesh or of the layer in the inner part is between 0.01 and 200 kPa, more preferentially between 0.1 and 60 kPa, even more preferentially between 0.1 and 5 kPa.

[0019] Preferentially, the Young's modulus of the hydrogel of the outer layer is greater than 10 kPa, more preferentially greater than 60 kPa, and even more preferentially greater than 100 kPa.

[0020] As an example, if the Young's modulus of the hydrogel present in the inner part is 10 kPa, preferentially 60 kPa, more preferentially 100 kPa, then the Young's modulus of the hydrogel of the outer layer is necessarily strictly greater than 10 kPa, preferentially greater than 60 kPa, more preferentially greater than 100 kPa.

[0021] According to a particularly preferred embodiment of the invention, the hydrogel of the outer layer and / or the inner part constituting the microcompartment consists or consists exclusively of alginate.

[0022] Alginate is a monomer of β-D-mannuronic acid (M units) and α-L-guluronic acid (G units) with (1-4) linkages, which vary in amount and sequential distribution along the polymer chain. Divalent cations, such as Ca2+, bind cooperatively between the G blocks of adjacent alginate chains, creating interchain ionic crosslinks that cause gelation of aqueous alginate solutions.

[0023] If the hydrogel contained in the inner part is alginate, the concentration of the alginate solution intended to form an alginate layer or mesh inside the microcompartment is preferentially 0.25-2%, more preferentially 0.25-1%, even more preferentially 0.5% (±0.1%). If the concentration of the alginate solution intended to form an alginate layer or mesh in the inner part of the microcompartment is 0.5%, the viscosity of the alginate is preferentially 3 mPa / s.

[0024] Advantageously, the inner part also contains at least one culture medium.

[0025] According to a preferred object of the invention, the hydrogel layer or mesh of the inner part is arranged between the outer layer of hydrogel and said at least one layer of cells. More preferentially, the inner part comprises at least one layer of cells.

[0026] According to another preferred object, the molecular weight of the hydrogel in the inner part is strictly lower than that of the hydrogel in the outer layer. More preferentially, the alginate in the inner part has a molecular weight of 75 kDa or less, and / or the alginate in the outer layer has a molecular weight of 150 to 250 kDa, which allows, in particular, to have a sufficiently rigid envelope to protect the capsule contents and a looser layer or mesh in the inner part that can act as an extracellular matrix substitute for the cells present in the capsule and enhance the protective effect. The low molecular weight alginate with short chains allows for less polymerization and a gel with less viscosity, resulting in a looser hydrogel in which cells can migrate, grow, and form cell aggregates or even one or more cysts.

[0027] According to another object, the hydrogel layer or mesh of the inner part may contain other components. Thus, said layer or mesh of hydrogel preferentially contains at least one peptide sequence, more preferentially a peptide sequence of interest capable of interacting with the cells constituting the cell layer present in the microcompartment according to the invention, thereby improving the adhesion and survival of the cells in the microcompartment. By way of example, the peptide sequence may be a peptide or a protein, preferentially derived from an extracellular matrix protein selected from collagen, fibronectin, laminin, etc. More preferentially, the peptide sequence is an RGD motif or a YIGSR motif.

[0028] The RGD motif allows cell adhesion through the interaction of the RGD peptide sequence with integrins on cells. RGD motif refers to a peptide having an RGD sequence that may contain one or two other residues before and after the RGD motif to enhance cellular detection of the RGD motif.

[0029] The YIGSR motif allows cell adhesion through interaction of the YIGSR peptide sequence with laminin receptors on cells. By YIGSR motif is meant a peptide having the YIGSR sequence, which may contain one or two other residues before and after the YIGSR motif to enhance cellular detection of the YIGSR motif.

[0030] Advantageously, the hydrogen layer or mesh of the inner part also comprises at least a second hydrogel different from the first hydrogel of the inner part, more preferentially this being chosen from fibrin, laminin, collagen, fibronectin, entactin and hyaluronic acid.

[0031] According to another object of the invention, the microcompartments are preferentially closed. The microcompartments according to the invention may also be of different shapes, preferentially ovoid, cylindrical, spheroidal, spherical or teardrop-shaped.

[0032] Furthermore, the microcompartment according to the invention is composed of three main components: an outer layer of hydrogel with a high Young's modulus that forms the envelope of the microcompartment; a layer or mesh of hydrogel with a low Young's modulus in the inner part of the cell microcompartment, said layer or mesh having a Young's modulus strictly lower than that of the hydrogel intended to form the outer layer, said layer or mesh intended to function as a substitute for the extracellular matrix, in particular to replace Matrigel®; and finally, said inner part contains at least one cell.

[0033] The cells present in the inner part advantageously constitute a layer of cells, where the cells may be of any cell type, preferentially the cells are not cells derived from a human embryo or cells requiring the destruction of a human embryo, more preferentially the cells are selected from human, animal and plant eukaryotic cells, even more preferentially pluripotent cells, progenitor cells, differentiating cells and differentiated cells.

[0034] When a microcompartment comprises the aforementioned three components, the microcompartment preferentially comprises at least one layer of cells and at least one lumen. When a microcompartment comprises at least one lumen, the layer of cells, the hydrogel layer or mesh of the inner portion, and the outer layer are preferentially organized continuously around the lumen.

[0035] Preferentially, the inner part of the microcompartment comprises at least one three-dimensional cell aggregate and / or cell microtissue.

[0036] According to another aspect, the present invention also relates to a micro-compartment assembly, comprising at least one micro-compartment according to any one of the preceding embodiments.

[0037] The microcompartments according to the invention or the microcompartment assemblies according to the invention are also particularly suitable for use in cell culture, in particular three-dimensional cell culture, which allows for the mass production of cells of interest, microtissues of interest or even organoids of interest, suitable for use in, for example, cell therapy. The present invention also relates to the microcompartments according to the invention or the microcompartment assemblies according to the invention for use as a pharmaceutical.

[0038] According to one variant, the present invention also relates to the use of microcompartments according to the invention or microcompartment assemblies according to the invention for producing microtissues. It is understood that in this particular embodiment, the microtissues are not transplanted into humans or animals. By way of example, they can be used as ex-vivo models.

[0039] On the one hand, the microcompartments according to the invention can be produced in different ways, but according to a particular aspect, the invention provides a method for preparing cellular microcompartments according to the invention, comprising the following steps: a. mixing cells, optionally pre-incubated in culture medium; b. encapsulating the mixture of step (a) in a hydrogel intended to form the outer layer; c. Cultivating the capsules obtained in step (b) in a culture medium, preferentially in a bioreactor, preferentially for at least 1 day, even more preferentially for 3 to 50 days; d. Optionally, recovering the resulting cell micro-compartments; wherein in step (a) and / or step (b) a hydrogel solution of plant or synthetic origin is added, the Young's modulus of said hydrogel solution of plant or synthetic origin being strictly smaller than the Young's modulus of the hydrogel used to form the outer hydrogel layer.

[0040] According to a particular object of the present invention, step (b) comprises the following sub-steps: - contacting the mixture from step (a) with a hydrogel solution (i) intended to form an outer layer to form at least one droplet; - collecting the droplets in a calcium bath that allows the hydrogel solution to harden and form the outer layer of each microcompartment.

[0041] In a particularly preferred method, step b) is carried out by simultaneously co-injecting the hydrogel solution (i) intended to form the outer layer, the mixture (ii) from step a) optionally comprising a hydrogel solution of plant or synthetic origin having a Young's modulus strictly smaller than that of the hydrogel used to form the outer layer, and the intermediate solution (iii) optionally comprising a hydrogel solution of plant or synthetic origin having a Young's modulus strictly smaller than that of the hydrogel used to form the outer layer, said co-injection being carried out concentrically via a microfluidic or millifluidic injector forming at the injector outlet a jet composed of the mixture of said solutions, said jet breaking up into droplets.

[0042] Advantageously, the final opening diameter of the microfluidic injectors is between 50 and 800 μm, preferentially between 80 and 240 μm, and the flow rate of each of the solutions is between 0.1 and 2000 mL / h, preferentially between 10 and 2000 mL / h, more preferentially between 10 and 150 mL, and even more preferentially between 11 and 100 mL / h.

[0043] According to a variant of the method according to the invention, step b) is carried out by simultaneously co-injecting the hydrogel solution (i) intended to form the outer layer, the mixture (ii) from step a) optionally comprising a hydrogel solution of plant or synthetic origin having a Young's modulus strictly smaller than that of the hydrogel used to form the outer layer, and the intermediate solution (iii) optionally comprising a hydrogel solution of plant or synthetic origin having a Young's modulus strictly smaller than that of the hydrogel used to form the outer layer, said co-injection being carried out concentrically via a microfluidic or millifluidic injector comprising a tip that is in contact with the calcium solution and forms at the injector outlet a jet of the mixture of the solutions, said jet forming a tube.

[0044] When the injector has a tip and the tip is in contact with the calcium solution, the final opening diameter of the microfluidic injector is preferentially between 50 and 1000 μm, more preferentially between 80 and 300 μm, and the flow rate of each of the solutions is between 1 and 100 mL / h.

[0045] According to a final aspect, the present invention relates to a kit comprising a hydrogel solution (i) intended to form the outer layer of the microcompartments, and a hydrogel solution (ii) of vegetable or synthetic origin, preferentially alginate, whose Young's modulus is strictly smaller than that of the hydrogel used to form the outer layer, and optionally an intermediate solution (iii), preferentially a sorbitol solution.

[0046] Finally, the present invention also relates to the use of a kit intended to prepare microcompartments according to the invention.

[0047] Other features and advantages will become apparent from the following detailed description of the invention, the examples, and the drawings. [Brief explanation of the drawings]

[0048] [Figure 1] 1 shows a cellular microcompartment 1 according to certain embodiments of the present invention, comprising an outer layer 2 of hydrogel and an inner portion 3 bounded by the outer layer 2. The inner portion 3 comprises a hydrogel layer or mesh 30 having a Young's modulus strictly less than that of the hydrogel of the outer layer 2, a layer 31 of cells, and a lumen 32. The layer or mesh 30 is disposed between the inner surface 20 of the outer layer of hydrogel 2 and the layer 31 of cells. [Figure 2] This shows one embodiment of the present invention, in which a 0.5% alginate solution is mixed with sorbitol and introduced into the first injection line "IS." Two other solutions, containing either 2% alginate "A" to form the outer layer or cells suspended in culture medium "CS," reside in the other two injection lines. The three lines are then co-injected with various solutions using a microfluidic injector to form a jet that breaks into droplets in a CaCl bath, which hardens the outer layer from the 2% alginate solution, thereby forming a capsule. [Figure 3] Panel A shows a second embodiment of the invention, in which a 0.5% alginate solution is mixed with sorbitol and introduced into the first injection line "IS." Two other solutions, containing either 2% alginate "A" or cells suspended in culture medium "CS," are present in the other two injection lines. The three lines are then co-injected using a microfluidic injector with its tip in contact with a CaCl bath, forming a jet that takes the form of a tube in the CaCl bath, which hardens the outer layer from the 2% alginate solution, thereby forming a tube. Panel B shows 4x magnification images taken with an EVOS microscope of cells in tubes containing 0.5% alginate at D4 and D5. [Figure 4]FIG. 1 shows the percentage of empty capsules containing no cells or cysts in capsules without exogenous extracellular matrix (outside the scope of the present invention), Matrigel®-based capsules (outside the scope of the present invention), and 0.5% alginate-based capsules as a matrix substitute (invention). [Figure 5A] 4× image taken using a transmitted light microscope of cells in capsules without exogenous extracellular matrix (outside the scope of the invention) at DO. [Figure 5B] 4× magnification image taken using a transmitted light microscope of cells in Matrigel®-based capsules (outside the scope of the invention) at DO. [Figure 5C] Figure 1 shows an image taken with a transmitted light microscope at 4x magnification of cells in 0.5% alginate-based capsules (invention) at DO. [Figure 6A] 4× image taken using a transmitted light microscope of cells in capsules without exogenous extracellular matrix (outside the scope of the invention) at D5. [Figure 6B] 4× magnification image taken using transmitted light microscopy of cells in Matrigel®-based capsules (outside the scope of the invention) at D5. [Figure 6C] 4× magnification image taken using a transmitted light microscope of cells in 0.5% alginate-based capsules (invention) at D5. [Figure 7] Figure 1 shows the expansion and pluripotency at D5 of cells present in capsules without exogenous extracellular matrix (outside the scope of the present invention), Matrigel®-based capsules (outside the scope of the present invention), and 0.5% alginate-based capsules (invention). [Figure 8]1 shows a three-dimensional cellular microcompartment 1 according to one embodiment of the present invention, comprising an outer layer of hydrogel 2 forming a hollow cavity 3, which is the inner portion of the microcompartment 1 according to the present invention. The hollow cavity 3 comprises a hydrogel mesh 4 in contact with the outer layer 2 and a plurality of cells organized in 3D to form a cyst 5, which comprises a lumen 6 at its center. [Figure 9] D5 amplification of cells present in 0.5% alginate-based capsules is shown, where the 0.5% alginate-based capsules are functionalized with at least one YIGSR motif, which further contains a spacer consisting of 6, 9, or 12 glycine residues. [Figure 10] We demonstrate pluripotency at D5 of cells present in 0.5% alginate-based capsules, which are functionalized with at least one YIGSR motif, which further contains a spacer consisting of 6, 9, or 12 glycine residues. [Figure 11] The results of D5 amplification of differentiated cells, ie, endoderm (Panel A) and mesoderm (Panel B) cells present in 0.5% alginate capsules, are shown. DETAILED DESCRIPTION OF THE INVENTION

[0049] definition By "hydrogel of plant or synthetic origin" is meant a hydrogel that is not of animal origin and / or is not derived from a cancer cell line, such as Matrigel®.

[0050] For the purposes of the present invention, a "hydrogel with a high Young's modulus" means a hydrogel having a Young's modulus strictly greater than that of the hydrogel constituting the layer or mesh in the inner part of the cell microcompartment according to the invention, and the Young's modulus of the hydrogel of the outer layer is strictly greater than that of the hydrogel present in the inner part constituting the layer or mesh intended to replace an extracellular matrix of animal origin, such as Matrigel®.

[0051] Conversely, "low Young's modulus" means a hydrogel that has a Young's modulus strictly lower than that of the hydrogel intended to form the outer layer of the microcompartment according to the invention.

[0052] For purposes of the present invention, "high molecular weight hydrogel" refers to a higher molecular weight hydrogel that constitutes the outer layer of a cell microcompartment according to the present invention, as opposed to a lower molecular weight hydrogel that constitutes the inner layer or mesh of the cell microcompartment, and the molecular weight of the hydrogel in the outer layer is greater than the molecular weight of the hydrogel present in the inner layer.

[0053] Conversely, for purposes of the present invention, a "low molecular weight hydrogel" refers to a hydrogel of lower molecular weight, i.e., a hydrogel of lower molecular weight than the hydrogel that constitutes the outer layer of a cell microcompartment, which hydrogel constitutes the layer or mesh of the inner portion of a cell microcompartment according to the present invention, and wherein the molecular weight of the hydrogel present in the inner portion is lower than the molecular weight of the hydrogel of the outer layer.

[0054] For the purposes of the present invention, "microcompartment" or "capsule" also refers to a partially or completely closed three-dimensional structure containing a plurality of cells. It is formed from a matrix of polymer chains, such as alginate, swollen by a liquid, and preferentially by water. The structure therefore consists of a hardened outer layer of hydrogel forming a hollow cavity or inner portion containing at least one cell, preferentially a plurality of cells, and a layer or mesh of hydrogel suitable for cell culture and growth.

[0055] For purposes of the present invention, "droplet" also refers to a three-dimensional structure formed from at least one liquid solution containing components of a non-hardened hydrogel (such as polymerized precursors, non-crosslinked or partially crosslinked polymer chains) of hydrogel precursor elements. Droplets also constitute a transitional state between the coinfusion of various components and microcompartments according to the present invention.

[0056] For purposes of the present invention, a "differentiated" cell refers to a cell that has a particular phenotype, as opposed to an undifferentiated pluripotent stem cell or a progenitor cell that has undergone differentiation.

[0057] For the purposes of the present invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even if this is not specified, the cells, stem cells, progenitor cells, and tissues according to the present invention consist of or are obtained from human cells or immunologically humanized non-human mammalian cells.

[0058] For purposes of the present invention, the term "mutant cell" refers to a cell that has at least one mutation.

[0059] For the purposes of the present invention, "progenitor cells" refers to stem cells that have already committed to cell differentiation but have not yet differentiated.

[0060] For the purposes of the present invention, "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4, NANOG, and SOX2, and surface markers such as SSEA4 / 5, Tra-1-60, and Tra-1-81. Embryonic stem cells used in the context of the present invention can be obtained without destroying the embryo from which they are derived, for example, using the techniques described in Chang et al. (Cell Stem cell, 2008, 2(2):113-117). Optionally, embryonic stem cells from humans can be excluded.

[0061] For purposes of the present invention, "pluripotent stem cells" or "pluripotent cells" refer to cells that have the potential to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. These may be, inter alia, induced pluripotent stem cells (iPSCs, or in the case of human induced pluripotent stem cells, hiPSCs), embryonic stem cells, or MUSE cells ("multilineage-differentiating stress enduring").

[0062] For the purposes of the present invention, "induced pluripotent stem cells" refers to pluripotent stem cells that have been induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4, and SSEA4 / 5, among others. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007, 318(5858):1917-1920), Takahashi et al. (Cell, 207, 131(5):861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1):101-106).

[0063] For the purposes of the present invention, a "layer of cells" or a "cell base layer" is understood to mean a plurality of cells forming a layer or base that can be structured around a lumen, which can be, for example, a cell tissue or microtissue or a three-dimensional grouping culture. The thickness of the cell layer can be variable. This cell layer is organized three-dimensionally in microcompartments.

[0064] For the purposes of this invention, "tissue" or "biological tissue" has the general meaning of tissue in biology, i.e., an intermediate level of organization between cells and organs. Tissues are collections of similar cells and of the same origin (most commonly derived from a common cell line, although they can also result from the association of different cell lines) grouped into clusters, networks, or bundles (fibers). Tissues form functional collections, i.e., their cells contribute to the same function. Biological tissues regenerate regularly and assemble together to form organs.

[0065] For purposes of the present invention, "lumen" means the volume of aqueous solution topologically surrounded by a cell, the contents of which are preferentially not in diffusional equilibrium with the volume of convective liquid present outside the microcompartment.

[0066] Microcompartments according to the present invention The object of the present invention is a three-dimensional cellular microcompartment comprising: an outer layer of hydrogel; - comprising at least one cell and at least one layer or mesh of a hydrogel of plant or synthetic origin, It is a three-dimensional cellular microcompartment in which the Young's modulus of the hydrogel in the inner layer or mesh is strictly less than the Young's modulus of the hydrogel in the outer layer.

[0067] In the context of the present invention, the inner portion is bounded by an outer layer of a hydrogel, said hydrogel having a Young's modulus strictly greater than that of the hydrogel forming the layer or mesh of the inner portion, thereby replacing extracellular matrices of animal origin, such as Matrigel®. Thus, the present invention relates to a three-dimensional cellular microcompartment comprising two different hydrogels, the first forming the outer layer forming the hollow cavity, and the second forming the layer or mesh present in the inner portion of the microcompartment, i.e., the hollow cavity.

[0068] On the one hand, the outer layer of the hydrogel forms a protective outer envelope and thus constitutes a capsule, and on the other hand, the less rigid and more loose layer or mesh of the inner part allows cell growth, given that its rheological properties are particularly suitable for cell growth and survival. To achieve this, in the context of the present invention, the Young's modulus of the hydrogel of the inner part is strictly smaller than that of the hydrogel of the outer layer.

[0069] Such microcompartments according to the invention are composed of two different hydrogels, the hydrogel constituting the outer layer having a Young's modulus strictly greater than that of the hydrogel constituting the inner layer or mesh, thereby improving the protection of the cells and / or cell aggregates present in the microcompartment, particularly in the context of culture in a bioreactor, resulting in high shear forces.

[0070] Advantageously, the outer layer provides a first protective envelope that is reinforced by the addition of at least one layer or mesh of hydrogel, thereby providing an additional protective mesh for the cells and aggregates present in the microcompartments, allowing cell migration and proliferation and being loose enough to maintain their pluripotent characteristics. Conversely, known prior art structures consisting of a single layer of hydrogel are either too stiff, preventing cell migration and proliferation and inducing apoptosis, or conversely, are not stiff enough and therefore not compatible with culture in a bioreactor.

[0071] The outer layer of hydrogel therefore forms the outer envelope of the microcompartments or capsules according to the invention and protects their contents from the external environment, especially when the capsules are cultured in a bioreactor, which is further enhanced by adding a layer or mesh of hydrogel to the interior portion of the microcompartment.

[0072] Advantageously, the hydrogel layer or mesh of the inner portion is at least partially apposed to the inner surface of the outer layer.

[0073] According to a preferred object of the present invention, the aforementioned advantages are further enhanced if the Young's modulus of the hydrogel of the inner part is between 0.01 and 200 kPa, more preferentially between 0.1 and 60 kPa, even more preferentially between 0.1 and 5 kPa. Preferentially, the Young's modulus of the hydrogel of the outer layer is greater than 10 kPa, more preferentially greater than 60 kPa, even more preferentially greater than 100 kPa.

[0074] Preferentially, the hydrogel of the inner layer or mesh is intertwined with the hydrogel of the outer layer, particularly on the inner surface of the outer layer. Also, the separation between the two hydrogels may not be completely clear, despite their different Young's moduli. As a result, at least a portion of the hydrogel of the inner layer or mesh can be intertwined with the inner surface of the outer layer.

[0075] The Young's modulus of the hydrogel in the inner portion is strictly less than that of the hydrogel in the outer layer, so that the outer layer is stiffer than the layer or mesh in the inner portion, and the hydrogel in the layer or mesh in the inner portion is looser and more slack, facilitating cell growth, especially due to the presence of short chains.

[0076] Preferentially, the hydrogel used is biocompatible, i.e. non-toxic to cells. The hydrogel should allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartments and ensure their survival. According to a particularly preferred embodiment, the outer layer of the hydrogel comprises at least alginate. It may consist exclusively of alginate.

[0077] The alginate may in particular be sodium alginate composed of 80% α-L-guluronate and 20% β-D-mannuronate and having a Young's modulus greater than 10 kPa, preferentially greater than 60 kPa, more preferentially greater than 100 kPa.

[0078] According to another object, the alginate advantageously has an average molecular weight between 100 and 400 kDa, more preferentially the outer layer has a molecular weight between 150 and 250 kDa. When the hydrogel of the outer layer is alginate, the concentration of the alginate solution intended to form said outer layer of the microcompartments is preferentially between 0.5 and 5% by weight, more preferentially the concentration is equal to 2% by weight (±0.5% by weight).

[0079] When the concentration of the alginate solution intended to form the outer layer of the microcompartments is equal to 2%, the viscosity of the alginate is preferentially equal to 144 mPa / s.

[0080] Advantageously, the outer layer of the hydrogel is devoid of cells.

[0081] The outer layer of the hydrogel therefore makes it possible to protect the cells from the external environment and to limit their differentiation in case of uncontrolled proliferation and differentiation of the cells.

[0082] According to a particularly preferred embodiment, the layer or mesh of the inner part of the hydrogel also comprises at least alginate, which may consist exclusively of alginate, in particular sodium alginate composed of 80% α-L-guluronate and 20% β-D-mannuronate and having a Young's modulus of 0.01 kPa to 200 kPa, preferentially 0.1 kPa to 60 kPa, more preferentially 0.1 kPa to 5 kPa.

[0083] According to another object, the alginate has an average molecular weight of at most 75 kDa. When the hydrogel of the outer layer is alginate, the concentration of the alginate solution intended to form the outer layer of the microcompartments is preferentially 0.25 to 2%, more preferentially 0.25 to 1%, and even more preferentially 0.5%. When the concentration of the alginate solution intended to form the outer layer of the microcompartments is 0.5%, the viscosity of the alginate is preferentially 3 mPa / s.

[0084] The inner layer or mesh with a low Young's modulus has viscoelastic and viscoplastic properties that are particularly advantageous for obtaining an extracellular matrix substitute that cells can accommodate and grow satisfactorily in. Furthermore, alginate hydrogels are rheofluidic, i.e., their viscosity decreases with increasing shear rate, which is particularly advantageous when passing through a microfluidic injector. Finally, alginates with a low Young's modulus also exhibit faster relaxation properties, also known as "fast relaxation," which allows cells to migrate, change shape, expand, proliferate, and mechanically remodel the alginate-based matrix.

[0085] Also, the hydrogel of the outer layer and / or of the inner part layer or mesh is very preferentially alginate.

[0086] Despite the fact that mammalian cells cannot interact with alginate, particularly since it allows minimal protein adsorption, alginate can be used as an extracellular matrix substitute, such as Matrigel®. Alginate has the following advantages: it is well characterized, easy to sterilize and store, can be optionally chemically modified, and confers interesting mechanical properties. Furthermore, alginate is particularly suitable in the context of the present invention, resulting in good growth, low cell death, and good proliferation.

[0087] In accordance with another preferred object of the present invention, the cell microcompartment comprises cells, an outer layer of hydrogel, and a layer or mesh within the inner portion of a hydrogel of lower molecular weight than the hydrogel of the outer layer.

[0088] Advantageously, a microcompartment according to any one of the preceding embodiments also comprises at least one layer of cells and / or at least one culture medium. If the microcompartment according to the invention comprises a layer or mesh in the inner part of the hydrogel, this is disposed between the outer layer of the hydrogel and the layer of cells. It is understood that the microcompartment may also comprise cells suspended in culture medium or optionally contained in a hydrogel.

[0089] According to one variant, the invention also relates to a cell microcompartment, comprising: at least one layer of cells; an outer layer of hydrogel; a layer or mesh within the inner portion of the hydrogel disposed between the outer layer of the hydrogel and the layer of cells, The invention relates to a cellular microcompartment characterized in that the inner portion comprises a hydrogel having a molecular weight strictly lower than that of the hydrogel of the outer layer.

[0090] Advantageously, the inventors observed better growth and the presence of nearly circular cysts at a concentration of 0.5%. At 2%, the cysts were substantially elongated, and the microcompartments became less cystic as the alginate concentration decreased. The higher the alginate concentration, the greater the physical constraints in the capsule, making the gel denser, and thus leaving less room for cells to grow and move around. In this case, the cysts were substantially elongated.

[0091] Thus, depending on the desired purpose or cell type, the alginate constituting the layer or mesh of the inner part of the microcompartment can be present at a total concentration of 0.25-2% by weight, which allows for good expansion and good pluripotency, and thus its use in three-dimensional cell culture.

[0092] The layer or mesh of the inner part of the hydrogel, preferentially of alginate, may comprise other components, and therefore preferentially comprises at least one peptide sequence, more preferentially a peptide sequence of interest, capable of interacting with the cells that constitute the layer of cells present in the microcompartments according to the invention. For example, the peptide sequence may be a peptide or a protein.

[0093] According to a particularly preferred purpose, the peptide sequence is a YIGSR motif and / or an RGD motif. The YIGSR motif is a peptide derived from the β1 chain of laminin with a tyrosine-isoleucine-glycine-serine-arginine sequence, which facilitates cell adhesion to the matrix. The RGD motif is a peptide with an arginine-glycine-asparagine sequence, which also facilitates cell adhesion to the matrix.

[0094] Therefore, to further improve the properties of this hydrogel-based extracellular matrix substitute, the layer or mesh of the inner part of the hydrogel preferentially contains at least one peptide sequence, i.e., a YIGSR motif or an RGD motif. Efficiency is further enhanced when this YIGSR motif or RGD motif contains a spacer located between the motif of interest (YIGSR or RGD) and the hydrogel of the inner part. Preferentially, the spacer is a peptide sequence containing n glycine residues, thereby improving the cell accessibility of the sequence of interest while enhancing polymerization of the hydrogel of the inner part. More preferentially, the spacer contains at least 3 glycine residues, preferentially 3 to 30 glycine residues, and even more preferentially 6 to 15 glycine residues. Effectiveness is particularly enhanced when the spacer contains 12 glycine residues, especially for cell amplification.

[0095] According to another aspect, the present invention also relates to a peptide sequence comprising a spacer consisting of n glycine (G) residues and at least one YIGSR motif or at least one RGD motif, wherein the peptide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0096] Thus, the hydrogel mesh of plant or synthetic origin of the cell microcompartment according to the invention preferentially comprises at least one peptide sequence containing n glycine residues and at least one YIGSR motif or at least one RGD motif. The hydrogel mesh of plant or synthetic origin of the cell microcompartment according to the invention is then functionalized with said peptide sequence containing n glycine residues and at least said YIGSR motif or at least said RGD motif, making it possible to improve the accessibility of the motifs of interest to the cells while improving the polymerization of the hydrogel in the inner part in order to improve cell survival and proliferation.

[0097] According to one variant, such YIGSR and / or RGD motifs, with or without a spacer, can also be used to functionalize the hydrogel of the outer layer and / or the hydrogel of the inner part.

[0098] According to another preferred object of the invention, the layer or mesh of the inner part of the hydrogel comprises at least a second hydrogel different from the first hydrogel of the layer or mesh of the inner part, more preferentially chosen from fibrin, laminin, fibronectin, entactin, hyaluronic acid and collagen.

[0099] If the layer or mesh of the inner part of the hydrogel contains fibrin, the fibrin is preferentially obtained from the polymerization of fibrinogen by a fibrinogen polymerization agent, advantageously thrombin, which can be added during and / or after encapsulation. Also, the polymerization of the fibrinogen solution by the thrombin solution occurs during and / or after encapsulation. If it occurs after encapsulation, the polymerization occurs within the newly formed droplets or capsules, i.e., after the outer layer has hardened.

[0100] In this case, the microcompartments are three-dimensional microcompartments bounded by an outer layer of hydrogel, inside which an inner part contains a layer or mesh of cells and hydrogel. Advantageously, this is of various spherical shapes. Advantageously, the three-dimensional microcompartments are hollow, and more preferentially, the hollow microcompartments are ovoid, cylindrical, spheroidal, spherical or teardrop-shaped.

[0101] For purposes of the present invention, a "hollow microcompartment" refers to a three-dimensional microcompartment having a cavity bounded by an outer layer of hydrogel, the cavity optionally containing a layer or mesh of hydrogel, culture medium, and a plurality of cells, the cavity forming an interior portion. By way of example, Figure 8 depicts a microcompartment comprising an outer layer of hydrogel, an interior portion formed by a hollow cavity, the interior portion comprising a heterogeneous mesh of hydrogel.

[0102] On the one hand, the outer layer of the hydrogel protects the cells from the external environment and limits differentiation in the event of uncontrolled cell proliferation and differentiation. On the other hand, the layer or mesh in the inner part of the hydrogel provides a suitable environment for cell growth and proliferation. Cell proliferation can optionally be controlled based on the concentration of the hydrogel as described above.

[0103] In the context of the present invention, the cells present in the microcompartments can be of any type, in particular they are eukaryotic cells, more preferentially they are human cells or plant or animal cells.

[0104] In certain embodiments, the microcompartments comprise pluripotent stem cells. Pluripotent stem cells or pluripotent cells refer to cells that have the ability to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Pluripotent stem cells may in particular be induced pluripotent stem cells (iPS), MUSE cells ("Multilineage-differentiating Stress Enduring") cells found in the skin and bone marrow of adult mammals, or embryonic stem cells (ES). According to one embodiment, the microcompartments according to the invention do not comprise embryonic stem cells (ES).

[0105] According to a particularly preferred variant of the invention, the microcompartments according to the invention contain human or animal induced pluripotent stem cells.

[0106] In another particular embodiment, the microcompartments according to the invention comprise human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells and / or cells undergoing differentiation. The multipotent and / or progenitor cells are preferentially obtained from pluripotent stem cells, in particular human pluripotent stem cells, or optionally from non-pluripotent human cells that have been artificially modified so that their transcriptional profile matches that of the particular multipotent and / or progenitor cells, typically by expressing transcription factors specific for the target cell phenotype. Preferentially, the multipotent and / or progenitor cells are obtained from pluripotent stem cells after contact with a solution capable of initiating differentiation of said stem cells.

[0107] According to another variant, the microcompartments according to the invention contain differentiated cells of a human or animal origin. The differentiated cells are preferentially obtained from pluripotent stem or progenitor cells, in particular human pluripotent stem or progenitor cells, or optionally from non-pluripotent human cells whose transcriptional profile has been artificially modified to connect to that of a particular differentiated cell, typically by expressing transcription factors specific for the target cell phenotype. Preferentially, the differentiated cells are obtained from pluripotent or multipotent, or progenitor, stem cells after contact with a solution capable of initiating differentiation of said stem cells. According to one variant, the cellular content of the microcompartments comprises homogeneous or mixed cellular identity.

[0108] The differentiated cells may in particular be in the form of at least one layer of cells, or in the form of a three-dimensional tissue, a cell aggregate, a microtissue, or a plurality of tissues or microtissues within a microcompartment. The differentiated cells may be in the form of a compacted or non-compacted tissue or microtissue, with or without a lumen.

[0109] Thus, the microcompartments according to the invention may contain several types of cells, in particular they may contain, for example, stem cells induced to pluripotency and / or multipotent cells and / or progenitor cells and / or cells in the process of differentiation and / or differentiated cells.

[0110] According to a particular inventive object, the microcompartments according to the invention are obtained after several cell division cycles. Indeed, the cells contained in the microcompartments according to the invention are cells obtained by amplification from at least one cell.

[0111] Also, the cells present in the microcompartments according to the invention were obtained after at least two cell division cycles following encapsulation of at least one cell in the outer layer of the hydrogel.

[0112] Preferably, the cells present in the microcompartments according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles after encapsulation in the outer layer of a hydrogel of at least one cell, preferentially 1-5, 1-10, 1-15, 1-20, 1-30, 1-40, 1-50, 1-60, 1-100 cells. For example, the cells present in the microcompartments have been obtained after at least 6 cell division cycles after encapsulation in the outer layer of a hydrogel of at least one cell, preferentially 1-50 cells.

[0113] Preferentially, the microcompartments are obtained after at least 2, more preferentially at least 3, 4, 5, 6, 7, 8, 9 or 10 passages after encapsulation, each passage lasting for example at least 1 day, or between 2 and 50 days, in particular between 3 and 10 days.

[0114] Preferentially, all of the cells initially encapsulated within the microcompartment prior to the first cell division cycle occupy less than 50% of the volume of the microcompartment in which they are encapsulated, more preferentially less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0115] Thus, according to one embodiment, the cells present in the microcompartments according to the invention are obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles after encapsulation in the outer layer of the hydrogel of cells occupying a volume of less than 50% of the volume of the microcompartment in which they are encapsulated, more preferentially less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0116] Preferentially, in the microcompartments according to the invention, the cells occupy more than 50% by volume of the volume of the microcompartment, and even more preferentially more than 60%, 70%, 75%, 80%, 85%, 90% by volume of the volume of the microcompartment.

[0117] The microcompartments according to the invention comprise several cells, preferentially at least 20 cells, even more preferentially at least 100, at least 500, at least 1000, at least 10,000 cells.

[0118] According to another preferred object of the invention, the capsules are obtained by encapsulation by co-injection carried out concentrically through a microfluidic injector which forms at the injector outlet a jet consisting of a mixture of the various useful solutions, said jet being divided into droplets which are then collected in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0119] According to a variant allowing the formation of tubes, the co-injection is also carried out concentrically via a microfluidic injector, the injector having a tip that comes into contact with the calcium solution and forms a jet at the injector outlet consisting of a mixture of the solutions, the jet forming a tube in the calcium solution.

[0120] The microcompartments according to the invention may also contain other elements, in particular a culture medium, which is a medium suitable for the cells present in the microcompartment according to the knowledge of the person skilled in the art.

[0121] According to another preferred object of the present invention, the microcompartments comprise at least one lumen, which may contain a liquid, in particular a culture medium and / or a liquid secreted by the cells. Advantageously, the presence of this hollow space allows the cells to have a small diffusion volume whose composition can be controlled, facilitating cell communication. The three-dimensional arrangement of a monolayer or spherical cell base surrounding a lumen or central lumen may also be called a cyst.

[0122] The lumen is preferentially generated during cyst formation by cells that grow and develop within the hydrogel that constitutes the layer or mesh of the inner portion of the cellular microcompartment.

[0123] According to another preferred object, the layer of cells, the layer or mesh of the inner part of the hydrogel, and the outer layer are organized around the lumen, more preferentially they are organized continuously around the lumen.

[0124] The cyst-shaped three-dimensional structure allows stem cells to be subjected to less pressure than in 2D cultures or aggregates. This configuration also reduces cell death rates and increases the culture expansion rate. As a result, this reduces the number of required passaging and dissociation steps, shortening the culture time required to reach the final required number of cells.

[0125] According to one embodiment, the microcompartment may comprise multiple cysts or tissues or microtissues.

[0126] The cell microcompartments according to the invention are preferentially closed or partially closed, i.e. the outer layer is closed or partially closed. More preferentially, the microcompartments are closed.

[0127] The microcompartments according to the invention can have any three-dimensional shape, i.e., the shape of any object in space. The microcompartments can have any shape suitable for cell encapsulation. Preferentially, the microcompartments according to the invention have a spherical or elongated shape. The microcompartments according to the invention can have an ovoid, cylindrical, spheroidal or spherical shape. The microcompartments according to the invention can in particular have the shape of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0128] The outer layer of the microcompartment, i.e. the hydrogel layer, gives the microcompartment according to the invention its size and shape. Preferentially, the minimum dimension of the microcompartment according to the invention is between 10 μm and 1 mm, preferentially between 100 μm and 700 μm. The minimum dimension of the microcompartment according to the invention may be between 200 μm and 600 μm, in particular between 300 μm and 500 μm.

[0129] Its largest dimension is preferentially greater than 10 μm, more preferentially between 10 μm and 1 m, even more preferentially between 10 μm and 50 cm.

[0130] Microcompartments according to the present invention may optionally be frozen for storage, after which they must be thawed before use.

[0131] The present invention also relates to a plurality of microcompartments taken together. Thus, the present invention also relates to a collection of said cellular microcompartments or a series of cellular microcompartments comprising at least one cellular microcompartment according to the present invention.

[0132] The present invention also relates to a collection or series of at least two three-dimensional cellular microcompartments, each microcompartment comprising at least one outer layer of hydrogel and, inside the outer layer, at least one layer or mesh of hydrogel and at least one cell, wherein at least one microcompartment is a microcompartment according to the present invention.

[0133] The aggregate of microcompartments according to the invention is preferably between 2 and 10 16 It contains microcompartments.

[0134] Preferably, the series of microcompartments according to the invention are in a culture medium, in particular at least partially in a convective culture medium.

[0135] According to a particularly preferred embodiment, the object of the present invention is the aggregation of cell microcompartments in a closed chamber, e.g. a bioreactor, preferentially in a culture medium in a closed chamber, e.g. a bioreactor.

[0136] The presence of an outer layer of hydrogel and, optionally, an intermediate layer of isotonic aqueous solution allows for a uniform distribution of cells among the microcompartments. Furthermore, this hydrogel layer makes it possible to prevent the microcompartments from fusing, which is a major source of undesirable variability in the phenotypic homogeneity of the cells.

[0137] Therefore, the microcompartment according to any one of the above-mentioned embodiments or the assembly of microcompartments according to any one of the above-mentioned embodiments is particularly suitable for use in cell culture, particularly in three-dimensional cell culture.This allows the production of a large amount of target cells or even target organoids, which are suitable for use in, for example, cell therapy.In addition, the present invention also relates to the microcompartment according to the present invention or the assembly of microcompartments according to the present invention for use as medicine.

[0138] Therefore, microcompartments are particularly suitable for use in clinical settings.

[0139] According to another object, the present invention relates to the use of microcompartments according to any one of the aforementioned objects for the production of cells, microtissues, tissues, preferentially for the large-scale production of such cells and / or tissues.

[0140] The microcompartments according to the invention can also be used for the production of animal or plant cells for human or animal consumption, this use being particularly useful for creating substitutes for meat products such as meat to limit the intake of meat products.

[0141] Preparation method The microcompartments can be obtained by various means known to those skilled in the art for preparing microcompartments or capsules.

[0142] According to another aspect of the present invention, a method for preparing the microcompartments according to the present invention has also been developed by the inventors.

[0143] Therefore, a particularly preferred method for preparing microcompartments according to the invention comprises the following steps: a. mixing cells, optionally pre-incubated in culture medium; b. encapsulating the mixture from step (a) in an outer layer of a hydrogel; c. Cultivating the capsules obtained in step (b) in a culture medium, preferentially in a bioreactor, preferentially for at least 1 day, preferentially for 3 to 50 days; d. Optionally, a step of recovering the obtained cell microcompartments, wherein during step (a) and / or step (b) a hydrogel solution of plant or synthetic origin is added, characterized in that the Young's modulus of the hydrogel solution of plant or synthetic origin is strictly smaller than the Young's modulus of the hydrogel used to form the outer hydrogel layer.

[0144] Advantageously, the method according to the invention may comprise an additional step: thus, preferentially, the cells are incubated before the step of mixing the cells with a hydrogel solution having a Young's modulus lower than that of the hydrogel of the outer layer (ii) in a suitable culture medium, preferentially comprising at least one cytoprotective factor, more preferentially at least one inhibitor of apoptosis.

[0145] The inhibitor of apoptosis can be, for example, one or more inhibitors of the RHO / ROCK (RHO-associated protein kinase) pathway, or any other inhibitor of apoptosis known to those skilled in the art, which should be able to promote cell survival and adhesion of cells to fibrin during the formation of the outer layer of the hydrogel.

[0146] The method according to the invention may comprise a step of dissociation of the cells by chemical, enzymatic or mechanical dissociation, which itself takes place before the mixing step a), carried out before or simultaneously with the cell incubation step, this step being particularly important in the case of adherent cells.

[0147] The encapsulated cells are suspended in the form of single cells and / or cell clusters. Preferably, single cells account for less than 50% of the total number of encapsulated cells, and more preferentially, the single cells are hPSC cells. Indeed, encapsulating clusters of cells is preferred, as it reduces the occurrence of mutagenesis.

[0148] Preferentially, the encapsulation step b) comprises the following sub-steps: contacting the mixture from step (a) with a hydrogel solution intended to form the outer layer (i) to form at least one droplet; collecting the droplets in a calcium bath that allows the hydrogel solution to harden and form the outer layer of each microcompartment.

[0149] When the outer hydrogel layer hardens with the calcium bath, microcompartments are formed, which can then be rinsed away. Advantageously, if the Young's modulus of the hydrogel in the inner portion is smaller than that of the hydrogel in the outer layer, hardening of the hydrogel in the inner portion with the calcium bath is prevented / limited. In particular, considering that the Young's modulus of the hydrogel in the inner portion is strictly smaller than that of the hydrogel in the outer layer, the hardening process using the calcium bath cannot harden the hydrogel in the inner portion when the capsule is in use, and therefore cell growth is not promoted.

[0150] Preferentially, step b) is carried out by simultaneous co-injection of a hydrogel solution (i) intended to form the outer layer, a mixture (ii) from step a) optionally comprising a hydrogel solution having a Young's modulus strictly smaller than that of the hydrogel of the outer layer, and optionally an intermediate solution (iii) optionally comprising a hydrogel solution (ii) having a Young's modulus strictly smaller than that of the hydrogel of the outer layer, said co-injection being carried out concentrically via a microfluidic or millifluidic injector forming at the injector outlet a jet composed of the mixture of said solutions, said jet breaking up into droplets.

[0151] If the intermediate solution (iii) contains a hydrogel solution (ii) having a Young's modulus lower than that of the hydrogel of the outer layer, then the hydrogel solution (ii) is not present in the mixture of step a).

[0152] According to a preferred object of the present invention, the isotonic intermediate solution is a sorbitol solution.

[0153] According to one object of the present invention, the final opening diameter of the microfluidic injector is between 50 and 800 μm, preferentially between 80 and 240 μm, and the flow rate of each of the solutions is between 0.1 and 2000 mL / h, preferentially between 10 and 2000 mL / h, more preferentially between 11 and 100 mL / h.

[0154] According to one variant, step b) is carried out by simultaneous co-injection of the hydrogel solution (i) intended to form the outer layer, the mixture (ii) from step a) optionally comprising a hydrogel solution having a Young's modulus strictly smaller than that of the hydrogel of the outer layer, and optionally an intermediate solution (iii) optionally comprising a hydrogel solution (ii) having a Young's modulus strictly smaller than that of the hydrogel of the outer layer, said co-injection being carried out concentrically via a microfluidic or millifluidic injector comprising a tip that is in contact with the calcium solution and forms a jet of the mixture of the solutions at the injector outlet, said jet forming a tube.

[0155] When the tip of the injector is in contact with the calcium solution, the final opening diameter of the microfluidic injector is preferentially 50-1000 µm, more preferentially 80-300 µm, and the flow rate of each solution is 1-100 mL / h.

[0156] According to another variant of the invention, the inner part comprising a layer or mesh of hydrogel also comprises at least a second hydrogel different from the first hydrogel constituting the layer or mesh of the inner part, more preferentially fibrin, preferentially obtained from the polymerization of fibrinogen by a fibrinogen polymerization agent, advantageously thrombin, which can be added during and / or after encapsulation.

[0157] Alternatively, the thrombin solution may be co-injected with other solutions, preferentially mixed with an isotonic intermediate solution, and the fibrinogen solution mixed with the mixture from step a).

[0158] Preferentially, the fibrinogen concentration is between 10 and 25 mg / mL, preferentially between 14 and 20 mg / mL, more preferentially 20 mg / mL.

[0159] According to another object of the present invention, the concentration of thrombin is preferentially 0.001 U / mL to 2 U / mL, more preferentially 0.01 U / mL to 1 U / mL, 0.01 U / mL to 0.05 U / mL, and even more preferentially 0.04 U / mL. "U" refers to the unit of enzyme activity (i.e., the enzyme concentration) that represents the amount of enzyme required to process 1 micromole of substrate per minute. It is understood that the indicated concentration is the concentration in the mixture. In practice, advantageously, thrombin is mixed with the other components in a 1:1 ratio. Also, if the concentration of thrombin in the capsule before mixing is 0.01 U / mL, the concentration in the capsule will be approximately 0.01 U / mL.

[0160] The steps following encapsulation can be carried out with or without stirring. Preferentially, the steps following encapsulation are carried out under permanent or continuous stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusion gradients. For example, this stirring allows for homogeneous control of the cellular oxygenation level, thus avoiding the phenomenon of necrosis associated with hypoxia or oxidative stress associated with hyperoxia. Therefore, this stirring avoids an increase in cell mortality and / or oxidative stress.

[0161] Preferentially, after the step of culturing the capsules obtained, the method comprises a step consisting of rinsing the capsules obtained from step (d) so as to advantageously eliminate cytoprotective factors such as inhibitors of apoptosis.

[0162] If the method according to the invention comprises a step of rinsing the capsules obtained, the solution constituting the calcium bath is removed and replaced by a suitable medium for culturing the microcompartments according to the invention, preferentially an isotonic solution, more preferentially a culture medium containing an inhibitor of apoptosis. The rinsing step of the capsules obtained is carried out after step d).

[0163] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the cells after step (d), preferentially after the rinsing step, if present after step (d). "At least one re-encapsulation of the cells" means at least two encapsulation cycles. Preferentially, each encapsulation cycle corresponds to a passage. In this variant of the method (at least one re-encapsulation of the cells after step (d)), the number of cell divisions in the entire method (for all passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0164] In the method according to the invention, there may be several reencapsulations, preferentially 1 to 100, in particular 1 to 10 reencapsulations.

[0165] Each re-encapsulation: a) dissociating the microcompartment or a series of microcompartments to obtain a suspension of cells or a suspension of cell clusters, wherein the outer layer of the hydrogel can be eliminated, in particular by hydrolysis, dissolution, perforation, and / or destruction by any biocompatible means, i.e., means that are not toxic to cells. For example, elimination can be achieved using phosphate-buffered saline, divalent ion chelators, enzymes, such as alginate-degrading enzymes if the hydrogel contains alginate, and / or laser microdissection; b. Re-encapsulating all or part of the cells or cell clusters into hydrogel capsules.

[0166] Reencapsulation is a preferred means to increase cell expansion resulting from the pluripotency step and reduce the risk of mutations.

[0167] According to a particular embodiment, the reencapsulation comprises: a. removing the outer layer of the hydrogel; b. resuspending the cells contained in the microcompartments so as to obtain single cells and / or at least one aggregate or cluster of cells in a culture medium containing an isotonic medium, preferentially containing an inhibitor of apoptosis; c. encapsulating the cell suspension in a hydrogel layer; d. Preferentially, culturing the obtained microcompartments in an isotonic solution containing an inhibitor of apoptosis, preferentially in a culture medium containing an inhibitor of apoptosis; e. Preferentially, rinsing the microcompartments so as to advantageously remove inhibitors of apoptosis; f. Cultivating the microcompartments in an isotonic solution, preferentially a culture medium, for at least one cell division cycle; g. Optionally, recovering the resulting cell micro-compartments.

[0168] Kits according to the present invention The present invention also relates to a kit comprising a hydrogel solution (i) having a Young's modulus strictly smaller than that of the hydrogel of the outer layer, and a hydrogel solution (ii) having a Young's modulus greater than that of the hydrogel of the inner part of the layer or mesh intended to form the outer layer, and optionally an intermediate solution (iii).

[0169] In another aspect, the present invention relates to the use of said kit for preparing microcompartments according to the invention, said kit comprising at least one hydrogel solution (ii) intended to form the outer layer of the microcompartments, a hydrogel solution (i) of plant or synthetic origin having a Young's modulus strictly smaller than that used to form the outer layer, and optionally an intermediate solution (iii).

[0170] Preferentially, the kit comprises a low molecular weight hydrogel solution (i) and a high molecular weight hydrogel solution (ii), and optionally an intermediate solution (iii), in particular the low molecular weight hydrogel being less than the high molecular weight hydrogel.

[0171] According to another object, the present invention also relates to a kit comprising at least one alginate solution of up to 75 kDa, an alginate solution of 150 to 250 kDa, an isotonic solution, preferentially a sorbitol solution, a calcium solution and a suitable culture medium. According to one variant, the kit is a kit of parts.

[0172] The invention will now be illustrated by non-limiting examples and results of compositions according to the invention. [Example]

[0173] Example 1 - Capsules according to one embodiment of the present invention. This example describes a specific embodiment of the invention, also shown in Figure 2, in which a 0.5% alginate solution intended to form an inner alginate layer as a matrix substitute is added to a sorbitol solution and co-injected via a microfluidic injector with a mixture of cells in culture medium and a 2% alginate solution intended to form an outer alginate layer.

[0174] The microfluidic injector, which allows for the co-injection of various solutions, contains three lines upstream of the nozzle. A solution containing sorbitol and 0.5% alginate is injected into line "IS." A 2% alginate solution and a cell suspension in culture medium are injected into lines "A" and "CS," respectively, followed by encapsulation.

[0175] After encapsulation, the droplets are collected in a CaCl bath, which allows the 2% alginate to harden and form an alginate shell, forming microcompartments or capsules. The CaCl solution containing the capsules is then rinsed with serum-free cell culture medium. In this case, the capsules have the following characteristics: an outer alginate layer with a Young's modulus of approximately 120 kPa, and an inner alginate layer (matrix substitute) with a Young's modulus of approximately 1 kPa.

[0176] Example 2 - Capsules according to one embodiment of the present invention. This example describes a specific embodiment of the invention, also shown in Figure 3, in which a 0.5% alginate solution intended to form an inner alginate layer as a matrix substitute is added to a sorbitol solution and co-injected via a microfluidic injector with a mixture of cells in culture medium and a 2% alginate solution intended to form an outer alginate layer.

[0177] The microfluidic injector, which allows for the co-injection of various solutions, contains three lines upstream of the nozzle. A solution containing sorbitol and 0.5% alginate is injected into line "IS". A 2% alginate solution and a cell suspension in culture medium are injected into lines "A" and "CS", respectively. In this embodiment, the tip of the microfluidic injector is brought into contact with a calcium solution, allowing for encapsulation in the form of a tube.

[0178] After encapsulation, the tubes are collected in a CaCl bath, which allows the 2% alginate to harden and form an alginate wall that forms the tube. The CaCl solution containing the tubes is then rinsed with serum-free cell culture medium. In this case, the tubes have the following characteristics: an outer alginate wall with a Young's modulus of approximately 120 kPa, and an inner alginate layer (matrix substitute) with a Young's modulus of approximately 1 kPa.

[0179] Example 3 - Measurement of viscosity of capsules according to the invention and capsules outside the scope of the invention. In connection with this study, the inventors used iPS cell lines generated according to the usual standards for two-dimensional iPS culture, then detached the cells from the flask via the action of enzymes according to the knowledge of those skilled in the art and transferred them to a culture medium suitable for the culture of iPS.

[0180] The iPS cells were mixed in a suitable culture medium, resulting in a cell density of approximately 1.5 M / mL. A 0.5% alginate solution intended to form an inner alginate layer as a matrix substitute was mixed with a sorbitol solution. A 2% alginate solution intended to form an outer alginate layer was also prepared. The various solutions were then loaded via dedicated lines and co-injected simultaneously by a microfluidic injector. The amount of encapsulated cells was approximately 0.6 * It is 10^6.

[0181] The same protocol was used to obtain capsules without exogenous extracellular matrix and capsules with Matrigel®, with the exception that sorbitol solution was injected alone and 0.5% alginate solution was replaced with Matrigel® solution.

[0182] The capsules are visually checked from day 1 to day 5 after encapsulation. On day 5, the appearance of the cells, the amount of cells, their viability and pluripotency are also observed.

[0183] The viscosity of the components of each microcompartment was measured using a rolling ball viscometer, which measures the time it takes for a ball to roll through an opaque and clear liquid according to the Hoppler falling ball method. The results show the viscosity of each component present in each type of microcompartment, respectively, including: - Matrigel®-like extracellular matrix (outside the scope of the present invention) - without exogenous extracellular matrix or extracellular matrix substitute, i.e., culture medium only (outside the scope of the present invention) -Alginate-like extracellular matrix (present invention).

[0184] The results are presented in Table 1 below.

[0185] [Table 1]

[0186] Capsules containing only cell culture medium, i.e., no exogenous extracellular matrix, had the lowest viscosity, while 0.5% alginate-based capsules had a 2.3-fold higher viscosity. Matrigel®-based capsules had the highest viscosity. However, Matrigel® is a very complex medium with large variations in viscosity within the matrix. Locally, the viscosity can be very high or very low, which means that the viscosity within Matrigel® is very heterogeneous and therefore there is a high degree of heterogeneity in the formation of cell aggregates within Matrigel®. Conversely, alginate has a viscosity very close to that of culture medium, which promotes cell growth. Furthermore, the viscosity is very uniform, resulting in good cell distribution within the alginate-based capsules.

[0187] Example 4 - Determination of the percentage of empty capsules. This study compares the seeding rates of capsules containing culture medium alone, Matrigel®, or 0.5% alginate (invention). Capsules are prepared according to the method described in Example 3, and the percentage of empty capsules is measured at DO using the following method: Ten photographs are taken for each condition. Using software such as ImageJ, each photograph is analyzed to count empty and filled capsules (at least one cell in the capsule); the software then determines the ratio between the two and multiplies this by 100 to obtain a percentage.

[0188] These results are shown in Figure 4. We observed better cell seeding in capsules containing 0.5% alginate compared to Matrigel® capsules and capsules containing only cell culture medium. This alginate-based matrix is ​​more viscous and has a more uniform viscosity, allowing cells to remain in suspension and thus be uniformly distributed inside the capsule during the process. This contrasts sharply with capsules containing Matrigel®, a matrix with a very non-uniform viscosity. Thus, capsules according to the present invention are largely devoid of cells, demonstrating a better capacity for cell seeding in capsules according to the present invention, and therefore resulting in a greater proportion of capsules containing cells, and therefore cysts, which can then be used, particularly in cell therapy.

[0189] Example 5 - Determination of capsule size and number of cysts per capsule. This study compares capsules containing culture medium alone, Matrigel®, or 0.5% alginate (invention). Capsules are prepared according to the method described in Example 3. Capsule size is measured at DO using, for example, ImageJ software with the area selection tool, and the amount of cysts per capsule is measured at DO according to the method described in Example 4.

[0190] The results are shown in Table 2 below and in Figures 5a, 5b, 5c, 6a, 6b and 6c.

[0191] [Table 2]

[0192] After 5 days of culture, dead cells could be observed inside some capsules when cultured without matrix, resulting in somewhat irregular outer layers of the cysts. In Matrigel®-based capsules, cells grew rapidly, and each capsule was filled with more or less aggregates. Several aggregates could also be observed within the same capsule, which tended to fuse to form larger aggregates. The outer layers of the cysts were smooth and free of dead cells. In 0.5% alginate-based capsules, we also observed several aggregates within the same capsule, fewer dead cells than in the matrix-free condition, but smaller structures than in Matrigel®. The lumen was still visible after 5 days of culture. The average size of Matrigel®-based capsules and 0.5% alginate-based capsules was similar, allowing cells to grow appropriately. Finally, we observed more cysts in alginate-based capsules than in cell culture medium-based capsules.

[0193] Example 6 - Measurement of expansion and pluripotency after 5 days in culture. This test compares capsules containing culture medium only, Matrigel®-based capsules, or 0.5% alginate-based capsules (invention). The capsules are prepared according to the method described in Example 3. The expansion and pluripotency of the cells present in each type of capsule is measured at D5 according to the following method: The ratio of the number of cells encapsulated at D0 to the number of cells obtained (and surviving) at D5 is determined. At D5, the capsules are dissolved, the aggregates are dissociated, and the cells are then counted.

[0194] The results are shown in Figure 7.

[0195] At D7, we observed better expansion of cells cultured in 0.5% alginate-based capsules compared to cells cultured in matrix-free capsules. Although the expansion factor was higher in Matrigel®-based capsules than in the other two conditions, the variability between experiments was also much higher, which is not compatible with the clinical use of this technology.

[0196] Regarding pluripotency, at D7 we also observed a lower mean percentage of Oct4 / Nanog-positive cells in 0.5% alginate versus Matrigel® conditions, but higher versus matrix-free conditions. Experimental variability is also reduced with 0.5% alginate.

[0197] As a result, the capsules according to the invention protect the cells and aggregates from the mechanical stresses of culture in a bioreactor, not only by virtue of the protective shell on the one hand, but also by the hydrogel as a matrix substitute in the capsule, which provides an additional protective mesh for the cells and aggregates, this mesh being loose enough to allow migration and expansion of the cells, while maintaining their pluripotent characteristics.

[0198] Example 7 - Measurement of expansion and pluripotency after 5 days of culture in cell microcompartments containing alginate functionalized with YIGSR motifs. The purpose of this study is to compare 0.5% alginate-based capsules functionalized with a YIGSR motif and a spacer containing 6, 9, and 12 glycine residues, respectively (SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3). The capsules are prepared according to the method described in Example 3. The expansion and pluripotency of the cells present in each type of capsule is measured at D5 according to the following method: The ratio of the number of cells encapsulated at D0 to the number of cells obtained (and surviving) at D5 is determined (Factor-X). At D5, the capsules are dissolved, the aggregates are dissociated, and the cells are then counted.

[0199] The results of amplification and pluripotency are shown in Figures 9 and 10, respectively.

[0200] The inventors then found that functionalizing alginate (a matrix substitute) with the YIGSR motif improved cell adhesion to the alginate-based matrix substitute, thereby enhancing cell survival and thereby cell proliferation. This effect was further enhanced when the YIGSR motif also contained a spacer of 6, 9, and 12 glycine residues, respectively (SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3).

[0201] Finally, we also found that functionalization with the YIGSR motif, optionally including a spacer, maintains pluripotency under all conditions.

[0202] Example 8 - Measurement of proliferation after 5 days of culture in microcompartments according to the invention containing alginate as matrix substitute and endoderm and mesoderm differentiated cells. The purpose of this study is to demonstrate the effectiveness of 0.5% alginate-based microcompartments containing differentiated cells, i.e., endoderm and mesoderm cells. Capsules are prepared according to the method described in Example 3. The capsules containing iPSCs are cultured for 4 days. The cells are then differentiated for 5 days by adding a medium that differentiates the iPSC cells into endoderm and mesoderm sheets.

[0203] The amplification of the cells present in each type of capsule is measured at D5 according to the following method: the ratio of the number of cells encapsulated at D0 to the number of cells obtained (and surviving) at D5 is determined (amplification factor). At D5, the capsules are dissolved, the aggregates are dissociated, and the cells are then counted.

[0204] The results are shown in Figure 11.

[0205] The inventors then observed better expansion of cells cultured in 0.5% alginate-based capsules compared to cells cultured in two dimensions, thereby demonstrating the ability of the microcompartments according to the invention to expand and culture differentiated cells, in particular endodermal and mesodermal cells.

Claims

1. A three-dimensional cellular microcompartment, - The outer layer of the hydrogel, - comprising an inner portion including at least one cell and at least one layer or mesh of a hydrogel of plant or synthetic origin, A cellular microcompartment in which the Young's modulus of the hydrogel in the inner portion is strictly less than the Young's modulus of the hydrogel in the outer layer.

2. The cell microcompartment according to claim 1, characterized in that the layer or mesh of the inner portion of the hydrogel is at least partially juxtaposed on the inner surface of the outer layer.

3. The cellular microcompartment according to claim 1, characterized in that the Young's modulus of the hydrogel in the inner portion is 0.01 to 200 kPa, preferably 0.1 to 60 kPa.

4. The cellular microcompartment according to claim 1, characterized in that the Young's modulus of the hydrogel in the inner portion is 0.1 to 5 kPa.

5. The cellular microcompartment according to claim 1, characterized in that the Young's modulus of the outer hydrogel layer is greater than 10 kPa, preferably greater than 60 kPa.

6. The cellular microcompartment according to claim 1, characterized in that the hydrogel in the outer layer and / or the inner portion is alginate or contains at least alginate.

7. The cell microcompartment according to claim 1, characterized in that the inner portion further comprises at least one culture medium.

8. The cellular microcompartment according to claim 1, characterized in that the inner portion includes a layer of at least one cell.

9. The cell microcompartment according to claim 1, characterized in that the inner portion of the hydrogel layer or the mesh is disposed between the outer layer of the hydrogel and the layer of at least one cell.

10. The cell microcompartment according to claim 1, characterized in that the layer or mesh of the inner portion of the hydrogel contains at least one peptide sequence.

11. The cell microcompartment according to claim 1, characterized in that the layer or mesh of the inner portion of the hydrogel comprises, in addition to the plant or synthetic hydrogel, at least one other hydrogel selected from fibrin, collagen, fibronectin, entactin, hyaluronic acid, and laminin.

12. A cellular microcompartment according to claim 1, characterized in that it is closed.

13. The microsectional area according to claim 1, characterized by having an oval, cylindrical, ellipsoidal, spherical, or teardrop shape.

14. The cellular microcompartment according to claim 1, characterized in that the cells present in the inner portion are selected from human, animal, and plant eukaryotic cells.

15. The cellular microcompartment according to claim 1, characterized in that the cells present in the inner portion are pluripotent cells and / or progenitor cells and / or differentiated cells and / or differentiated cells.

16. The cellular microcompartment according to claim 1, characterized in that the inner portion of the microcompartment comprises at least one layer of cells and at least one lumen, and the layer of cells, the hydrogel layer or mesh of the inner portion, and the outer layer are continuously organized around the lumen.

17. The cell microcompartment according to claim 1, characterized in that the inner portion of the microcompartment includes at least one three-dimensional cell aggregate and / or cell microtissue.

18. A three-dimensional cellular microcompartment aggregate characterized in that the at least one microcompartment is the microcompartment described in claim 1.

19. A cell microcompartment according to claim 1 or a cell microcompartment aggregate according to claim 18, for use as a pharmaceutical.

20. Use of the microcompartments described in claim 1 or the aggregate of cellular microcompartments described in claim 18 for the production of microtissue.

21. A method for preparing a cellular microcompartment according to claim 1, a. A step of mixing cells that have been pre-incubated in culture medium, at the discretion of the user, b. The step of encapsulating the mixture of step (a) in a hydrogel intended to form the outer layer of the hydrogel, c. The capsules obtained in step (b) are cultured in a culture medium, preferably in a bioreactor, preferably for at least 1 day, and more preferably for 3 to 50 days. d. A method comprising the optional step of collecting the obtained cellular microcompartments, characterized in that a hydrogel solution of plant or synthetic origin is added during step (a) and / or during step (b), wherein the Young's modulus of the plant or synthetic origin hydrogel solution is strictly less than the Young's modulus of the hydrogel used to form the outer layer of the hydrogel.

22. The method according to claim 21, wherein step b) is performed by simultaneously co-injecting a hydrogel solution (i) intended to form the outer layer, a mixture (ii) from step a) which optionally comprises a hydrogel solution of plant or synthetic origin with a Young's modulus strictly less than that of the hydrogel used to form the outer layer, and an intermediate solution (iii) which optionally comprises a hydrogel solution of plant or synthetic origin with a Young's modulus strictly less than that of the hydrogel used to form the outer layer, wherein the co-injection is performed concentrically through a microfluidic injector or millifluidic injector which forms a jet of the mixture of the solutions at the injector outlet, and the jet is divided into droplets.

23. The method according to claim 22, characterized in that the final opening diameter of the microfluidic injector is 50 to 800 μm, preferably 80 to 240 μm, and the flow rate of each of the solutions is 10 to 2000 mL / h, preferably 11 to 100 mL / h.

24. The method according to claim 21, wherein step b) is carried out by simultaneous co-injection of a hydrogel solution (i) intended to form the outer layer, a mixture (ii) from step a) which optionally comprises the hydrogel solution of plant or synthetic origin having a Young's modulus strictly less than that of the hydrogel used to form the outer layer, and an intermediate solution (iii) which optionally comprises the hydrogel solution of plant or synthetic origin having a Young's modulus strictly less than that of the hydrogel used to form the outer layer, wherein the co-injection is carried out concentrically via a microfluidic injector or millifluidic injector, the injector having a tip which is in contact with the calcium solution and forms a jet at the injector outlet which comprises the mixture of the solutions and which forms a tube.

25. The method according to claim 22, characterized in that the final opening diameter of the microfluidic injector is 50 to 1000 μm, preferably 80 to 300 μm, and the flow rate of each of the solutions is 0.1 to 100 mL / h.

26. Use of a kit for preparing a microcompartment according to claim 1, wherein the kit comprises at least one hydrogel solution (i) intended to form the outer layer of the microcompartment, and a hydrogel solution (ii) of plant or synthetic origin whose Young's modulus is strictly less than the Young's modulus of the hydrogel used to form the outer layer.

27. A peptide sequence, a. A sequence containing n glycine residues, and b. A peptide sequence comprising at least one YIGSR motif or at least one RGD motif.

28. The peptide sequence according to claim 27, characterized in that it is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

29. The cellular microcompartment according to claim 1, characterized in that the hydrogel mesh of plant or synthetic origin comprises at least one peptide sequence as described in any one of claims 27 or 28.

Images