Extracellular matrix substitutes in cell microcompartments

Abstract

The present invention relates to the field of three-dimensional cell culture and 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 and, in particular, to cell microcompartments that do not contain extracellular matrix of animal origin and / or derived from cancer cell lines, for the production of GMP-grade cells and tissues. Background Art

[0002] Professor Yamanaka discovered induced pluripotent stem cells (iPS or iPSC), which has injected new impetus into the field of cell culture. Clinical trials soon began with the aim of treating diseases that are usually rare and incurable with traditional drugs.

[0003] Historically, cells (including induced pluripotent stem cells) were cultured two-dimensionally (2D). Due to the limitations of 2D cell culture, three-dimensional (3D) culture systems have been developed in recent years, making it possible to partially overcome the disadvantages of 2D culture.

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

[0005] A particularly suitable technique is the one described in patent application WO 2018 / 096277, which describes three-dimensional microcompartments for stem cell culture, especially containing cells, a cell-like extracellular matrix layer, and a hydrogel outer layer.

[0006] Although three-dimensional culture is a very promising technique, in order to be able to be used under clinical conditions, it still has certain disadvantages. For this purpose, both the cells and the three-dimensional culture system from which the cells are derived must comply with the regulations regarding good manufacturing practice (GMP). However, most three-dimensional culture systems (such as cell microcompartments) include extracellular matrix of animal origin and / or from cancer cell lines (such as also known as Engelbreth-Holm-Swarm (EHS) matrix), which do not comply with such regulations.

[0007] Therefore, its widespread application in cell culture, especially in iPS cell culture, is questioned because it is derived from mouse sarcoma, which may introduce xenocontaminants into the cells obtained from the culture containing In addition, the complexity of its mixture and the variability of the biochemical and mechanical properties between and within batches mean that cell culture experiments lack reproducibility.

[0008] In view of the emergence of cell therapies based on this technology or the production of animal or plant cells for human or animal food consumption, there is a need to develop an alternative, a substitute that makes it possible to eliminate the presence of such extracellular matrices while maintaining the possibility of satisfactory cell adhesion and growth.

[0009] During their work, the inventors surprisingly discovered the use of a hydrogel with a high Young's modulus, which is intended to form the outer membrane or outer layer of a cell microcompartment, in combination with a second hydrogel with a Young's modulus less than that of the outer layer, which is intended to form a layer or network in the inner part of the microcompartment, on which at least one cell can be accommodated and grow. Advantageously, the present invention makes it possible to obtain cells in large quantities as well as rapid growth and a low mutation rate. Such results thus make it possible to envisage its use in the production of three-dimensional cell microcompartments for human and veterinary applications.

[0010] Research has definitely described the use of synthetic matrices, including three-dimensional uses. However, there is no description of a cell microcompartment containing two hydrogels, each with a different Young's modulus, especially when the Young's modulus of the hydrogel of the outer layer is strictly greater than the Young's modulus of the hydrogel forming the layer or network of the inner part. However, such a structure is complex to implement and requires the outer layer to be rigid enough to form the outer membrane of the cell microcompartment in order to protect the cell contents; the inner part of the microcompartment, delimited by the outer membrane of the cell microcompartment and containing at least the hydrogel layer or network, has a specific rheology as well as mechanical strength, thus allowing it to provide an environment for the cells that helps their dispersion, proliferation and migration.

[0011] Furthermore, in order to meet this need for cell microcompartments free of non-GMP extracellular matrices (such as ), which have a rheology 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, especially where the Young's modulus of the outer hydrogel is strictly greater than the Young's modulus of the hydrogel contained in the inner part and forming the layer or network in the microcompartment. Summary of the Invention

[0012] Thus, the present invention relates to a new three-dimensional cell microcompartment that comprises:

[0013] - an outer hydrogel layer, and

[0014] - an inner part that contains at least one cell and at least one hydrogel layer or hydrogel network of plant or synthetic origin,

[0015] wherein the Young's modulus of the hydrogel in the inner part is strictly less than the Young's modulus of the hydrogel in the outer layer.

[0016] Accordingly, the present invention relates to the use of two specific hydrogels, one hydrogel being intended to form an outer layer and the second hydrogel being intended to form a layer or network in the internal part, it being understood that the internal part is delimited by the hydrogel outer layer and that the Young's modulus of the hydrogel of the internal part is strictly less than the Young's modulus of the hydrogel of the outer layer.

[0017] On the one hand, the hydrogel outer layer forms a protective outer membrane and thus constitutes a capsule, and on the other hand, given the particularly suitable rheology of the hydrogel of the layer or network of the internal part, the less rigid and looser layer or network of the internal part allows cell growth.

[0018] Thus, the microcompartment according to the present invention consists of two different hydrogels. The Young's modulus of the hydrogel constituting the outer layer is strictly greater than the Young's modulus of the hydrogel of the layer or network constituting the internal part, which improves the protection of the cells and cell aggregates present in the cell microcompartment.

[0019] In fact, advantageously, the outer layer provides a first protective envelope. This effect is enhanced by at least one hydrogel layer or network in the internal part, which provides an additional protective network for the cells and aggregates present in the microcompartment while being loose enough to allow cell migration and expansion and to maintain their pluripotency. In contrast, the known prior art consisting of a single layer of hydrogel is either too rigid, preventing cell migration and expansion and inducing apoptosis, or on the contrary too loose and thus not suitable for culturing in a bioreactor. In fact, the mechanical constraints of culturing in a bioreactor, especially due to the high shear forces generated during culturing, require a suitable cell microcompartment.

[0020] Thus, the hydrogel outer layer with a high Young's modulus forms the outer membrane of the microcompartment or capsule according to the present invention, thus allowing the protection of the content of the capsule from the external environment, especially when the capsule is cultured in a bioreactor. This is further enhanced by the presence of a hydrogel layer or network on the internal part of the microcompartment. Advantageously, the hydrogel layer or network of the internal part is at least partially juxtaposed with the inner face of the outer layer.

[0021] According to a preferred object of the present invention, the Young's modulus of the hydrogel of the network or layer in the internal part is from 0.01 kPa to 200 kPa, more preferably from 0.1 kPa to 60 kPa, even more preferably from 0.1 kPa to 5 kPa.

[0022] Preferably, the Young's modulus of the hydrogel of the outer layer is greater than 10 kPa, more preferably greater than 60 kPa, even more preferably greater than 100 kPa.

[0023] For example, when the Young's modulus of the hydrogel present in the inner part is 10 kPa, preferably 60 kPa, more preferably 100 kPa, the Young's modulus of the hydrogel in the outer layer must be strictly greater than 10 kPa, preferably greater than 60 kPa, more preferably greater than 100 kPa.

[0024] According to a particularly preferred embodiment of the present invention, the hydrogel constituting the outer layer and / or the inner part of the microcompartment consists of or consists only of alginate.

[0025] Alginate is a monomer of β-D-mannuronic acid (M unit) and α-L-guluronic acid (G unit) with (1-4) bonds, and the amounts and sequential distributions along the polymer chain vary. Divalent cations such as Ca2+ bind cooperatively between the G blocks of adjacent alginate chains, generating interchain ionic bridges and causing the gelation of the alginate aqueous solution.

[0026] When the hydrogel contained in the inner part is alginate, the concentration of the alginate solution for forming the layer or network of alginate in the inner part of the microcompartment is preferably 0.25% to 2%, more preferably 0.25% to 1%, even more preferably 0.5% (±0.1%). When the concentration of the alginate solution for forming the layer or network of alginate in the inner part of the microcompartment is 0.5%, the viscosity of the alginate is preferably 3 mPa / s.

[0027] Advantageously, the inner part further comprises at least one culture medium.

[0028] According to a preferred object of the present invention, the hydrogel layer or network of the inner part is arranged between the hydrogel outer layer and the at least one cell layer. More preferably, the inner part contains at least one cell layer.

[0029] According to another preferred object, the molecular weight of the hydrogel in the inner part is strictly less than the molecular weight of the hydrogel in the outer layer. More preferably, the alginate in the inner part has a molecular weight less than or equal to 75 kDa and / or the alginate in the outer layer has a molecular weight of 150 kDa to 250 kDa, which in particular enables it to have a sufficiently hard envelope to protect the contents of the capsule and a looser layer or network in the inner part, which can act as an extracellular matrix substitute for the cells present in the capsule and enhance the protection effect. The lower molecular weight alginate with shorter chains results in a lower degree of polymerization and viscosity of the gel. As a result, the looser hydrogel allows cell migration, growth and the formation of cell aggregates, or even one or more cysts.

[0030] According to another object, the hydrogel layer or network of the internal part may contain other components. Thus, the hydrogel layer or network preferably contains at least one peptide sequence, more preferably a peptide sequence of interest capable of interacting with cells, which cells particularly constitute the cell layer present in the microcompartments according to the invention, which makes it possible to improve the adhesion and survival of cells within the microcompartments. By way of example, the peptide sequence may be a peptide or a protein, preferably, the peptide sequence is derived from extracellular matrix proteins selected from collagen, fibronectin, laminin, etc. More preferably, the peptide sequence is an RGD motif or a YIGSR motif.

[0031] The RGD motif enables cell adhesion through the interaction of the RGD peptide sequence with integrins on the cell. The RGD motif means a peptide having an RGD sequence, which RGD sequence may contain one or two other residues before and after the RGD motif to enhance the cell detection of the RGD motif.

[0032] The YIGSR motif enables cell adhesion through the interaction of the YIGSR peptide sequence with the laminin receptor on the cell. The YIGSR motif means a peptide having a YIGSR sequence, which YIGSR sequence may contain one or two other residues before and after the YIGSR motif to enhance the cell detection of the YIGSR motif.

[0033] Advantageously, the hydrogen layer or hydrogen network of the internal part further contains at least a second hydrogel different from the first hydrogel of the internal part, more preferably, the second hydrogel is selected from fibrin, laminin, collagen, fibronectin, nestin and hyaluronic acid.

[0034] According to another object of the invention, the microcompartment is preferably closed. The microcompartment according to the invention may also have different shapes, preferably the shape of an ovoid, a cylinder, a spheroid, a sphere or a teardrop.

[0035] Furthermore, the microcompartment according to the invention consists of three main components: a hydrogel outer layer forming the envelope of the microcompartment having a high Young's modulus, a hydrogel layer or network having a low Young's modulus in the internal part of the cell microcompartment, the Young's modulus of said layer or network being strictly less than the Young's modulus of the hydrogel intended to form the outer layer, said layer or network being intended to serve as a substitute for the extracellular matrix, in particular to replace Finally, the internal part contains at least one cell.

[0036] The cells present in the internal part advantageously constitute a cell layer, these cells can be of all cell types, preferably said cells are not derived from human embryos or cells that require the destruction of human embryos, more preferably, these cells are selected from human, animal and plant eukaryotic cells, even more preferably pluripotent cells, progenitor cells, cells in the process of differentiation and differentiated cells.

[0037] When the microcompartment comprises the aforementioned three components, the microcompartment preferably comprises at least one cell layer and at least one lumen. When the microcompartment comprises at least one lumen, the cell layer, the hydrogel layer or net of the inner part and the outer layer are preferably continuously organized around the lumen.

[0038] Preferably, the interior portion of the microcompartment comprises at least one three-dimensional cell aggregate and / or cell microtissue.

[0039] According to another aspect, the present invention also relates to a microcompartment assembly, wherein the assembly comprises at least one microcompartment according to any one of the preceding embodiments.

[0040] The microcompartment according to the present invention or the microcompartment assembly according to the present invention is also particularly suitable for cell culture, in particular three-dimensional cell culture, which allows the production of cells, microtissues or even organoids of interest in large quantities, suitable for example for cell therapy. In addition, the present invention also relates to a microcompartment according to the present invention or a microcompartment assembly according to the present invention used as a medicine.

[0041] According to a variant, the present invention also relates to the use of a microcompartment according to the present invention or a microcompartment assembly according to the present invention for the manufacture of microtissues. It should be understood that in this particular embodiment, the microtissues are not implanted in humans or animals. For example, they can be used as ex vivo models.

[0042] On the other hand, the microcompartment according to the invention can be produced in different ways, however, according to a specific aspect, the invention relates to a method for preparing a cell microcompartment according to the invention, the method comprising the following steps:

[0043] a. mixing cells optionally previously incubated in culture medium,

[0044] b. encapsulating the mixture from step (a) in a hydrogel intended to form an outer layer;

[0045] c. culturing the capsules obtained in step (b) in a culture medium, preferably in a bioreactor, preferably for at least 1 day, even more preferably for 3 to 50 days, and

[0046] d. optionally recovering the obtained cell microcompartments,

[0047] Characterized in that, in step (a) and / or step (b), a hydrogel solution of plant or synthetic origin is added, whose Young's modulus is strictly smaller than the Young's modulus of the hydrogel used to form the hydrogel outer layer.

[0048] According to a specific object of the present invention, step (b) comprises the following sub-steps:

[0049] - contacting the mixture from step (a) with a hydrogel solution intended to form the outer layer (i) to form at least one droplet, and

[0050] - collecting the droplets in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0051] In a particularly preferred manner, step b) is carried out by co-injecting simultaneously the hydrogel solution intended to form the outer layer (i), the mixture from step a) and optionally an intermediate solution (iii), the mixture optionally comprising a 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 (ii), the intermediate solution optionally comprising a 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; said co-injection is carried out concentrically via a microfluidic or millifluidic syringe, forming a jet consisting of a mixture of said solutions at the syringe outlet, and said jet breaks up into droplets.

[0052] Advantageously, the final opening diameter of the microfluidic syringe is from 50 μm to 800 μm, preferably from 80 μm to 240 μm, and the flow rate of each solution is from 0.1 mL / h to 2000 mL / h, preferably from 10 mL / h to 2000 mL / h, more preferably from 10 mL to 150 mL, even more preferably from 11 mL / h to 100 mL / h.

[0053] According to a variant of the method of the invention, step b) is carried out by co-injecting simultaneously the hydrogel solution intended to form the outer layer (i), the mixture from step a) and optionally an intermediate solution (iii), the mixture optionally comprising a 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 (ii), the intermediate solution optionally comprising a 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; said co-injection is carried out concentrically via a microfluidic or millifluidic syringe, said syringe comprising a tip which is in contact with the calcium solution, forming a jet consisting of a mixture of said solutions at the syringe outlet, and said jet forms a tube.

[0054] When the syringe comprises a tip which is in contact with the calcium solution, the final opening diameter of the microfluidic syringe is preferably from 50 μm to 1000 μm, more preferably from 80 μm to 300 μm, and the flow rate of each solution is from 1 mL / h to 100 mL / h.

[0055] According to a last aspect, the invention relates to a kit, said kit comprising a hydrogel solution intended to form the outer layer (i) of the microcompartment, a hydrogel solution of vegetable or synthetic origin, preferably alginate, whose Young's modulus is strictly less than that of the hydrogel used to form the outer layer (ii), and an optional intermediate solution (iii), preferably a sorbitol solution.

[0056] Finally, the invention also relates to the use of a kit intended for the preparation of a microcompartment according to the invention.

[0057] Other features and advantages will become apparent from the detailed description, examples and the following drawings of the invention. Description of the Drawings

[0058] Figure 1 shows a cell microcompartment 1 according to a specific embodiment of the invention, which comprises a hydrogel outer layer 2 and an internal part 3 delimited by said outer layer 2. Said internal part 3 comprises a hydrogel layer or network 30, a cell layer 31 and a lumen 32, the Young's modulus of which hydrogel layer or network is strictly less than that of the hydrogel of the outer layer 2. Said layer or network 30 is arranged between the inner face 20 of the hydrogel outer layer 2 and the cell layer 31.

[0059] Figure 2 is an embodiment of the invention in which a 0.5% alginate solution is mixed with sorbitol and introduced into a first injection line "IS". Two other solutions are present in two other injection lines, a 2% alginate "A" to form the outer layer, or cells suspended in a culture medium "CS". The three lines are then co-injected using a microfluidic syringe with the various solutions forming a jet, which is split into droplets in a CaCl2 bath, said bath hardening the outer layer of the 2% alginate solution, thus forming capsules.

[0060] Figure 3 shows a second embodiment of the invention in subfigure A, in which a 0.5% alginate solution is mixed with sorbitol and introduced into a first injection line "IS". Two other solutions are present in two other injection lines, a 2% alginate "A", or cells suspended in a culture medium "CS". The three lines are then co-injected using a microfluidic syringe, with the tip of the syringe in contact with a CaCl2 bath, forming a jet which takes the form of a tube in the CaCl2 bath, said bath hardening the outer layer from the 2% alginate solution, thus forming a tube. Subfigure B is a 4x magnified image of the cells in the tube containing 0.5% alginate at D4 and D5 taken using an EVOS microscope.

[0061] Figure 4 shows capsules without exogenous extracellular matrix (outside the scope of the invention), based on ​​​​The percentage of empty capsules without cells or encapsulations in capsules (outside the scope of the present invention) and 0.5% alginate-based capsules (present invention) as a matrix substitute.

[0062] [Figure 5a] is a 4x magnified image of cells in a capsule without exogenous extracellular matrix (outside the scope of the present invention) taken using a transmission light microscope at D0.

[0063] [Figure 5b] is a 4x magnified image of cells in a capsule based on (outside the scope of the present invention) taken using a transmission light microscope at D0.

[0064] [Figure 5c] is a 4x magnified image of cells in a 0.5% alginate-based capsule (present invention) taken using a transmission light microscope at D0.

[0065] [Figure 6a] is a 4x magnified image of cells in a capsule without exogenous extracellular matrix (outside the scope of the present invention) taken using a transmission light microscope at D5.

[0066] [Figure 6b] is a 4x magnified image of cells in a capsule based on (outside the scope of the present invention) taken using a transmission light microscope at D5.

[0067] [Figure 6c] is a 4x magnified image of cells in a 0.5% alginate-based capsule (present invention) taken using a transmission light microscope at D5.

[0068] Figure 7 shows the expansion and pluripotency of cells present in a capsule without exogenous extracellular matrix (outside the scope of the present invention), a capsule based on (outside the scope of the present invention), and a 0.5% alginate-based capsule (present invention) at D5.

[0069] Figure 8 shows a three-dimensional cell microcompartment 1 according to an embodiment of the present invention, which comprises a hydrogel outer layer 2 forming a hollow cavity, i.e., an inner part 3 of the microcompartment 1 according to the present invention. The hollow cavity 3 includes a hydrogel network 4 in contact with the outer layer 2 and a plurality of cells forming a capsule 5 in 3D tissue, and the capsule 5 includes an inner cavity 6 at its center.

[0070] Figure 9 shows the expansion of cells present in a 0.5% alginate-based capsule at D5, and the 0.5% alginate-based capsule is functionalized with at least one YIGSR motif, which also comprises a spacer consisting of 6, 9, 12 glycine residues.

[0071] Figure 10 ​​​​Shows the pluripotency of cells present in 0.5% alginate capsules at D5. The 0.5% alginate capsules are functionalized with at least one YIGSR motif, which also contains a spacer consisting of 6, 9, or 12 glycine residues.

[0072] Figure 11 Shows the amplification results of differentiated cells (i.e., endoderm cells (subfigure A) and mesoderm cells (subfigure B) present in 0.5% alginate capsules) at D5. Detailed Description

[0073] Definition

[0074] "Hydrogels of plant or synthetic origin" means hydrogels that are not of animal origin and / or not derived from cancer cell lines, such as

[0075] For the purposes of the present invention, a "hydrogel with a high Young's modulus" is a hydrogel whose Young's modulus is strictly greater than that of the hydrogel constituting the layer or network in the inner part of the cell microcompartment according to the present invention. Additionally, the Young's modulus of the outer layer hydrogel is strictly greater than that of the hydrogel present in the inner part of the layer or network intended to replace the extracellular matrix of animal origin, such as )

[0076] Conversely, "low Young's modulus" means that the Young's modulus of the hydrogel is strictly less than that of the hydrogel intended to form the outer layer of the microcompartment according to the present invention.

[0077] For the purposes of the present invention, a "high molecular weight hydrogel" means a higher molecular weight hydrogel constituting the outer layer of the cell microcompartment according to the present invention, as compared to the hydrogel constituting the layer or network in the inner part of the cell microcompartment with a lower molecular weight. Additionally, the molecular weight of the outer layer hydrogel is greater than the molecular weight of the hydrogel present in the inner layer.

[0078] Conversely, for the purposes of the present invention, a "low molecular weight hydrogel" means a hydrogel with a lower molecular weight, i.e., a lower molecular weight than the hydrogel constituting the outer layer of the cell microcompartment, and the low molecular weight hydrogel constitutes the layer or network in the inner part of the cell microcompartment according to the present invention. Moreover, the molecular weight of the hydrogel present in the inner part is less than the molecular weight of the outer layer hydrogel.

[0079] ​For the purposes of the present invention, a "microcompartment" or "capsule" also means a partially or completely enclosed three-dimensional structure containing a plurality of cells. This is formed by a matrix of polymer chains (e.g., alginate), which swells by a liquid and preferably by water. Thus, the structure consists of a hardened outer layer of hydrogel forming a hollow cavity or an inner part and a hydrogel layer or network suitable for cell culture and the growth of said cells, the hollow cavity or inner part containing at least one cell, preferably a plurality of cells.

[0080] For the purposes of the present invention, a "droplet" also means a three-dimensional structure formed from at least one liquid solution of hydrogel precursor elements, the at least one liquid solution containing components of a non-hardened hydrogel (polymerization precursors, non-crosslinked or partially crosslinked polymer chains, etc.). Additionally, the droplet constitutes a transition state between the co-injection of various components and the microcompartments according to the present invention.

[0081] For the purposes of the present invention, a "differentiated" cell means a cell with a specific phenotype, as opposed to an undifferentiated pluripotent stem cell or a progenitor cell undergoing differentiation.

[0082] For the purposes of the present invention, a "human cell" means a human cell or an immunohumanized non-human mammalian cell. Even when this is not stated, the cells, stem cells, progenitor cells, and tissues according to the present invention consist of or are obtained from human cells or immunohumanized non-human mammalian cells.

[0083] For the purposes of the present invention, the term "mutant cell" refers to a cell carrying at least one mutation.

[0084] For the purposes of the present invention, a "progenitor cell" means a stem cell that has participated in cell differentiation but has not yet differentiated.

[0085] For the purposes of the present invention, an "embryonic stem cell" means a pluripotent stem cell of cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be evaluated by the presence of markers such as the transcription factors OCT4, NANOG, and SOX2 and surface markers such as SSEA4 / 5, Tra-1-60, and Tra-1-81. The embryonic stem cells used in the context of the present invention are obtained without destroying the embryo from which they originate, for example, using the technique described by Chang et al. (Cell Stem Cell, 2008, 2(2): 113-117). Optionally, human embryonic stem cells can be excluded.

[0086] For the purposes of the present invention, a "pluripotent stem cell" or "pluripotent cell" means a cell that has the ability to form all of the tissues present in the originating organism as a whole, yet is not capable of forming the whole organism itself. In the context of the present invention, human pluripotent stem cells may be referred to as hPSCs. These cells may in particular be induced pluripotent stem cells (iPSCs or human induced pluripotent stem cells hiPSCs), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0087] For the purposes of the present invention, an "induced pluripotent stem cell" means a pluripotent stem cell that has been induced to become pluripotent by genetic reprogramming of a differentiated somatic cell. These cells are in particular positive for pluripotency markers such as staining with alkaline phosphatase and the expression of the proteins NANOG, SOX2, OCT4 and SSEA4 / 5. 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).

[0088] For the purposes of the present invention, a "cell layer" or "cell basement layer" is understood to mean a plurality of cells that form a layer or a basement that can surround a lumen structure, which may for example be a cell tissue or a microtissue or a three-dimensional grouped culture. The thickness of the cell layer may be variable. The cell layer is organized in three dimensions in a microcompartment.

[0089] For the purposes of the present invention, a "tissue" or "biological tissue" has the usual meaning of a tissue in biology, that is to say, the intermediate tissue level between cells and organs. A tissue is a group of similar cells and cells of the same origin (most usually derived from a common cell line, although they may originate from the union of different cell lines), grouped into clusters, networks or bundles (fibers). Tissues form functional components, that is to say, their cells contribute to the same function. Biological tissues are regularly regenerated and assembled together to form organs.

[0090] For the purposes of the present invention, a "lumen" means a defined volume of aqueous solution that is topologically surrounded by cells. Preferably, the content of the lumen is not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0091] Microcompartment according to the present invention

[0092] The object of the present invention is a three-dimensional cell microcompartment, which comprises:

[0093] - a hydrogel outer layer, and

[0094] - An internal part that contains at least one cell and at least one hydrogel layer or hydrogel network of plant or synthetic origin,

[0095] wherein the Young's modulus of the hydrogel of the layer or network of the internal part is strictly less than the Young's modulus of the hydrogel of the outer layer.

[0096] In the context of the present invention, the internal part is delimited by an outer hydrogel layer, the Young's modulus of which is strictly greater than the Young's modulus of the hydrogel of the layer or network forming the internal part, thereby replacing an extracellular matrix of animal origin such as Thus, the present invention relates to a three-dimensional cell microcompartment comprising two different hydrogels, one forming the outer layer and thus forming a hollow cavity, and the second being a layer or network present in the internal part (i.e., the hollow cavity) of the microcompartment.

[0097] On the one hand, the outer hydrogel layer forms a protective outer membrane and thus constitutes a capsule. On the other hand, given that the rheology of the internal part is particularly suitable for cell growth and survival, the less rigid and looser layer or network of the internal part allows cell growth. To achieve this, in the context of the present invention, the Young's modulus of the hydrogel of the internal part is strictly less than the Young's modulus of the hydrogel of the outer layer.

[0098] Such a microcompartment according to the present invention consists of two different hydrogels, wherein the Young's modulus of the hydrogel constituting the outer layer is strictly greater than the Young's modulus of the hydrogel of the layer or network constituting the internal part, which improves the protection of the cells and / or cell aggregates present in the microcompartment, especially in the case of culturing in a bioreactor, which can lead to high shear forces.

[0099] Advantageously, the outer layer provides a first protective envelope, which is strengthened by adding at least one hydrogel layer or hydrogel network, which provides an additional protective network for the cells and aggregates present in the microcompartment, while being loose enough to allow cell migration and expansion and to maintain their pluripotency. In contrast, the prior art known to consist of a single hydrogel layer is either too rigid, preventing cell migration and expansion and inducing apoptosis, or on the contrary not rigid enough and thus not suitable for culturing in a bioreactor.

[0100] Thus, the outer hydrogel layer forms the outer membrane of the microcompartment or capsule according to the present invention, thereby protecting its contents from the external environment, especially when culturing the capsule in a bioreactor. This is further enhanced by adding a hydrogel layer or hydrogel network to the internal part of the microcompartment.

[0101] Advantageously, the hydrogel layer or hydrogel network of the internal part is at least partially juxtaposed with the inner surface of the outer layer.

[0102] According to a preferred object of the present invention, when the Young's modulus of the hydrogel of the inner part is from 0.01 kPa to 200 kPa, more preferably from 0.1 kPa to 60 kPa, and even more preferably from 0.1 kPa to 5 kPa, the above advantages are further enhanced. Preferably, the Young's modulus of the hydrogel of the outer layer is greater than 10 kPa, more preferably greater than 60 kPa, and even more preferably greater than 100 kPa.

[0103] Preferably, the hydrogel of the layer or network of the inner part is entangled with the hydrogel of the outer layer, especially on the inner surface of the outer layer. Additionally, although the Young's moduli of the two hydrogels are different, the boundary between them may not be very distinct. As a result, at least a part of the hydrogel of the layer or network of the inner part can be entangled with the inner surface of the outer layer.

[0104] Since the Young's modulus of the hydrogel of the inner part is strictly less than that of the hydrogel of the outer layer, the outer layer will be harder than the layer or network of the inner part (its hydrogel is looser and more relaxed, especially due to the presence of shorter chains), thus promoting cell growth.

[0105] Preferably, the hydrogel used is biocompatible, that is, it is non-toxic to cells. The hydrogel must allow the diffusion of oxygen and nutrients in order to supply the cells contained in the microcompartment and enable them to survive. According to a particularly preferred embodiment, the hydrogel outer layer contains at least alginate. It may consist only of alginate.

[0106] The alginate can in particular be sodium alginate consisting of 80% α-L-guluronate and 20% β-D-mannuronate, which has a Young's modulus greater than 10 kPa, preferably greater than 60 kPa, and more preferably greater than 100 kPa.

[0107] According to another object, the alginate advantageously has an average molecular weight of 100 kDa to 400 kDa, and more preferably, the outer layer has a molecular weight of 150 kDa to 250 kDa. When the hydrogel of the outer layer is alginate, the concentration of the alginate solution for forming the outer layer of the microcompartment is preferably from 0.5% by mass to 5% by mass, and more preferably, the concentration is equal to 2% by mass (±0.5% by mass).

[0108] When the concentration of the alginate solution for forming the outer layer of the microcompartment is equal to 2%, the viscosity of the alginate is preferably equal to 144 mPa / s.

[0109] Advantageously, the hydrogel outer layer is free of cells.

[0110] Therefore, the hydrogel outer layer makes it possible to protect the cells from the external environment, thereby limiting the uncontrolled proliferation of the cells and, in the case of differentiation, limiting their differentiation.

[0111] According to a particularly preferred embodiment, the layer or net of the inner part of the hydrogel also contains at least alginate. It can consist only of alginate. The alginate can especially be sodium alginate consisting of 80% α-L-guluronate and 20% β-D-mannuronate, having a Young's modulus of 0.01 kPa to 200 kPa, preferably 0.1 kPa to 60 kPa, more preferably 0.1 kPa to 5 kPa.

[0112] According to another object, the average molecular weight of the alginate is 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 microcompartment is preferably 0.25% to 2%, more preferably 0.25% to 1%, even more preferably 0.5%. When the concentration of the alginate solution intended to form the outer layer of the microcompartment is 0.5%, the viscosity of the alginate is preferably 3 mPa / s.

[0113] The layer or mesh of the inner part with low Young's modulus then has viscoelastic and viscoplastic properties, which are particularly advantageous for obtaining an extracellular matrix substitute in which cells can be accommodated and grow satisfactorily. In addition, alginate hydrogels have rheological properties, i.e., their viscosity decreases with increasing shear rate, which is particularly advantageous when passing through a microfluidic syringe. Finally, alginates with low Young's modulus will also exhibit faster relaxation properties, also known as "fast relaxation", which allows cells to move, change shape, swell, proliferate and mechanically reshape alginate-based matrices.

[0114] Furthermore, the hydrogel of the outer layer and / or the layer or web of the inner part is very preferably alginate.

[0115] Although mammalian cells cannot interact with alginate, especially because it allows minimal protein adsorption, alginate can be used as an extracellular matrix substitute, such as Alginate has the following advantages: Alginate has good properties, is easy to sterilize and store, it can optionally be chemically modified, and provides interesting mechanical properties. Furthermore, alginate is particularly suitable in the context of the present invention and provides good growth, low cell death and good expansion.

[0116] According to another preferred object of the present invention, the cell microcompartment comprises cells, an outer layer of hydrogel and a layer or network in the inner part of the hydrogel, the molecular weight of the layer or network being lower than that of the outer layer of hydrogel.

[0117] Advantageously, the microcompartment according to any one of the foregoing embodiments further comprises at least one cell layer and / or at least one culture medium. When the microcompartment according to the present invention comprises a layer or a net in the inner part of the hydrogel, the layer or the net is arranged between the outer layer of the hydrogel and the cell layer. It should be understood that the microcompartment may also include cells suspended in the culture medium or cells optionally contained in the hydrogel.

[0118] According to a variant, the present invention also relates to a cell microcompartment comprising:

[0119] - at least one cell layer,

[0120] - a hydrogel outer layer, and

[0121] - a layer or mesh in the inner part of the hydrogel, the layer or mesh being arranged between the outer layer of the hydrogel and said layer of cells,

[0122] Characterized in that the inner part comprises a hydrogel, the molecular weight of which is strictly smaller than the molecular weight of the hydrogel of the outer layer.

[0123] Advantageously, the inventors observed better expansion and the presence of cysts (mostly rounded) at a concentration of 0.5%. At 2%, the cysts were essentially elongated in shape and the microcompartments had fewer cysts relative to lower alginate concentrations. The higher the concentration of alginate, the greater the physical constraints in the capsule, making the gel tighter, thus giving the cells less space to reproduce and move. The cysts then took on a substantially elongated shape.

[0124] Thus, depending on the purpose or cell type sought, the alginate constituting the layer or network of the inner part of the microcompartment may be present in a total concentration of 0.25% to 2% by mass, which makes it possible to obtain good expansion and good pluripotency, thus enabling its use in three-dimensional cell culture.

[0125] The layer or net of the inner part of the hydrogel (preferably alginate) may contain other ingredients, so the layer or net of the inner part of the hydrogel preferably comprises at least one peptide sequence, more preferably a peptide sequence of interest capable of interacting with cells, which cells particularly constitute the cell layer present in the microcompartment according to the present invention. For example, the peptide sequence can be a peptide or a protein.

[0126] 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, having a tyrosine-isoleucine-glycine-serine-arginine sequence, which promotes cell adhesion to the matrix. The RGD motif is a peptide having an arginine-glycine-asparagine sequence, which also promotes cell adhesion to the matrix.

[0127] Thus, in order to further improve the properties of such a hydrogel-based extracellular matrix substitute, the layer or the network of the inner part of the hydrogel preferably comprises at least one peptide sequence, namely the YIGSR motif or the RGD motif. The efficiency is further enhanced when the YIGSR or RGD motif comprises a spacer located between the motif of interest (YIGSR or RGD) and the hydrogel of the inner part. Preferably, the spacer is a peptide sequence which comprises n glycine residues, thereby improving the accessibility of the sequence of interest to cells while enhancing the polymerization of the hydrogel of the inner part. More preferably, the spacer comprises at least 3 glycine residues, preferably from 3 to 30 glycine residues, even more preferably from 6 to 15 glycine residues. The potency is particularly enhanced for cell expansion when the spacer comprises 12 glycine residues.

[0128] Furthermore, according to another object, 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. According to a preferred object, the peptide sequence is selected from 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.

[0129] Thus, the hydrogel network of plant or synthetic origin of the cell microcompartment according to the invention preferably comprises at least one peptide sequence comprising n glycine residues, and at least one YIGSR motif or at least one RGD motif. The hydrogel network of plant or synthetic origin of the cell microcompartment according to the invention is then functionalized with the peptide sequence comprising n glycine residues and at least the YIGSR motif or at least the RGD motif, such that the accessibility of the motif of interest to cells can be improved while improving the polymerization of the hydrogel of the inner part in order to improve cell survival and expansion.

[0130] According to a 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.

[0131] According to another preferred object of the invention, the layer or the network of the inner part of the hydrogel comprises at least a second hydrogel, which is different from the first hydrogel of the layer or the network of the inner part, and more preferably, the second hydrogel is selected from fibrin, laminin, fibronectin, nidogen, hyaluronic acid and collagen.

[0132] When a layer or network of the inner part of the hydrogel contains fibrin, the fibrin is preferably obtained by polymerization of fibrinogen by a fibrinogen polymerizing agent, advantageously, the reagent is thrombin, and the reagent can be added during and / or after encapsulation. Additionally, the polymerization of the fibrinogen solution by the thrombin solution occurs during and / or after encapsulation. When the polymerization occurs after encapsulation, the polymerization occurs within the newly formed droplets or capsules, i.e., once the outer layer has hardened.

[0133] The microcompartments are three-dimensional microcompartments delimited by an outer hydrogel layer, and within said outer layer, the inner part contains cells and a hydrogel layer or hydrogel network. Advantageously, it has a variety of spherical shapes. Advantageously, the three-dimensional microcompartment is hollow, and more preferably, the hollow microcompartment is in the shape of an ovoid, cylinder, spheroid, sphere or teardrop.

[0134] For the purposes of the present invention, a "hollow microcompartment" refers to a three-dimensional microcompartment having a cavity delimited by an outer hydrogel layer, said cavity optionally containing a hydrogel layer or hydrogel network, a culture medium and a plurality of cells, and said cavity forms the inner part. By way of example, Figure 8 A microcompartment is described that includes an outer hydrogel layer and an inner part formed by a hollow cavity, the latter containing a heterogeneous hydrogel network.

[0135] On the one hand, the outer hydrogel layer protects the cells from the external environment and limits uncontrolled cell proliferation and differentiation in the case of differentiation; on the other hand, the layer or network in the inner part of the hydrogel provides a suitable environment for cell growth and reproduction. Cell proliferation can optionally be controlled based on the concentration of the hydrogel as described above.

[0136] In the context of the present invention, the cells present in the microcompartments can be any type of cell, and in particular the cells are eukaryotic cells. More preferably, the cells are human or plant or animal cells.

[0137] In a specific embodiment, the microcompartment includes pluripotent stem cells. Pluripotent stem cells or pluripotent cells refer to cells that have the ability to form all tissues present in the entire originating organism, but cannot form a complete organism as such. Pluripotent stem cells can in particular be induced pluripotent stem cells (iPS), MUSE cells ("Multilineage-differentiating Stress Enduring") found in the skin and bone marrow of adult mammals, or embryonic stem cells (ES). According to one embodiment, the microcompartments according to the present invention do not include embryonic stem cells (ES).

[0138] According to a particularly suitable variant of the present invention, the microcompartments according to the present invention include human or animal induced pluripotent stem cells.

[0139] In another specific embodiment, the microcompartments according to the invention comprise human or animal committed cells and / or human or animal progenitor cells derived from these committed cells and / or cells in the process of differentiation. The pluripotent cells and / or progenitor cells are preferably obtained from pluripotent stem cells (in particular human pluripotent stem cells) or optionally from non-pluripotent human cells, the transcriptional profiles of these pluripotent stem cells being artificially modified to match the transcriptional profiles of specific committed cells and / or progenitor cells by forced expression of specific transcription factors characteristic of the target cell phenotype. Preferably, the committed cells and / or progenitor cells are obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells.

[0140] According to another variant, the microcompartments according to the invention comprise differentiated human or animal cells. The differentiated cells are preferably obtained from pluripotent stem cells or progenitor cells (in particular human pluripotent stem cells or human progenitor cells, or optionally non-pluripotent human cells), the transcriptional profiles of these pluripotent stem cells or progenitor cells being artificially modified to match the transcriptional profiles of specific differentiated cells by forced expression of specific transcription factors characteristic of the target cell phenotype. Preferably, the differentiated cells are obtained from pluripotent cells or committed cells or progenitor stem cells after contact with a solution capable of initiating the differentiation of said stem cells. According to one variant, the cellular content of the microcompartment comprises homogeneous or mixed cell labels.

[0141] The differentiated cells can in particular be in the form of at least one cell layer, or in the form of a three-dimensional tissue, cell aggregate or microtissue, or in the form of multiple tissues or microtissues in the microcompartment. It can be a dense or non-dense tissue or microtissue, with or without a lumen.

[0142] Thus, the microcompartments according to the invention can comprise several types of cells. In particular, the microcompartments according to the invention can comprise, for example, stem cells induced to pluripotency and / or committed cells and / or progenitor cells and / or cells in the process of differentiation and / or differentiated cells.

[0143] According to a specific object of the invention, the microcompartments according to the invention are obtained after several cell division cycles. In fact, the cells comprised in the microcompartments according to the invention are cells obtained by amplification from at least one cell.

[0144] In addition, the cells present in the microcompartments according to the invention are obtained after at least two cell division cycles after being encapsulated in a hydrogel outer layer of at least one cell.

[0145] Preferably, the cells present in the microcompartments according to the invention are obtained after encapsulating at least 1 cell, preferably 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 100 cells in the hydrogel outer layer and after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles. For example, the cells present in the microcompartments are obtained after at least six cell division cycles after encapsulating at least 1 cell, preferably 1 to 50 cells in the hydrogel outer layer.

[0146] Preferably, the microcompartments are obtained after at least 2 passages, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passages after encapsulation. Each passage can last, for example, at least 1 day, or 2 to 50 days, especially 3 to 10 days.

[0147] Preferably, all of the cells in the cells initially encapsulated in the microcompartments before the first cell division cycle occupy less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0148] 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 encapsulating cells in the hydrogel outer layer and occupy less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0149] Preferably, in the microcompartments according to the invention, the cells occupy more than 50% by volume relative to the volume of the microcompartment, even more preferably more than 60% by volume, 70% by volume, 75% by volume, 80% by volume, 85% by volume, 90% by volume relative to the volume of the microcompartment.

[0150] The microcompartments according to the invention comprise a plurality of cells, preferably at least 20 cells, even more preferably at least 100, at least 500, at least 1000, at least 10,000 cells.

[0151] According to another preferred object of the present invention, the capsules are obtained by co-injection carried out concentrically via a microfluidic syringe, which forms a jet composed of a mixture of various useful solutions at the syringe outlet, and the jet breaks up into droplets. Then, the droplets are collected in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0152] According to a variant allowing the formation of tubes, the co-injection is also carried out concentrically via a microfluidic syringe, the syringe comprising a tip which is in contact with the calcium solution and forms a jet composed of a mixture of said solutions at the syringe outlet, and the jet forms tubes in the calcium solution.

[0153] The microcompartments according to the present invention may also contain other elements, in particular culture media. According to the knowledge of those skilled in the art, the culture medium is a culture medium suitable for the cells present in the microcompartments.

[0154] According to another preferred object of the present invention, the microcompartment comprises at least one inner cavity. The inner cavity may contain a liquid, in particular a culture medium and / or a liquid secreted by the cells. Advantageously, the presence of this hollow part enables the cells to have a small diffusion volume, and these cells can control the composition of this volume, thus promoting cell communication. The three-dimensional arrangement of a monolayer or spherical cell substrate around the inner cavity or central inner cavity may also be referred to as encapsulation.

[0155] The inner cavity is preferably generated by cells that multiply and develop within the hydrogel of the layer or network constituting the inner part of the cell microcompartment during encapsulation formation.

[0156] According to another preferred object, the cell layer, the layer or network of the inner part of the hydrogel, and the outer layer are organized around the inner cavity, and more preferably they are continuously organized around the inner cavity.

[0157] Compared with 2D cultures or aggregates, the conformation of the encapsulation shape makes it possible to reduce the stress experienced by the stem cells. This configuration also makes it possible to reduce cell mortality and increase the culture expansion factor. As a result, this makes it possible to reduce the number of passages and dissociations required, thus reducing the culture time required to reach the final desired number of cells.

[0158] According to one embodiment, the microcompartment may comprise a plurality of encapsulations or tissues or microtissues.

[0159] The cell microcompartments according to the present invention are preferably closed or partially closed, that is to say, the outer layer is closed or partially closed. Preferably, the microcompartment is closed.

[0160] The microcompartments according to the invention can be of any three-dimensional form, i.e., it can have the shape of any object in space. The microcompartments can have any form compatible with cell encapsulation. Preferably, the microcompartments according to the invention are spherical or elongated in form. It can have the shape of an ovoid, a cylinder, a spheroid or a sphere. In particular, it can be in the form of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0161] It is the outer layer of the microcompartment (i.e., the hydrogel layer), which gives the microcompartment according to the invention its size and shape. Preferably, the minimum size of the microcompartments according to the invention is from 10 μm to 1 mm, preferably from 100 μm to 700 μm. It can be from 200 μm to 600 μm, especially from 300 μm to 500 μm.

[0162] Its maximum size is preferably greater than 10 μm, more preferably from 10 μm to 1 m, even more preferably from 10 μm to 50 cm.

[0163] The microcompartments according to the invention can optionally be stored frozen. Then it must be thawed before use.

[0164] The invention also relates to a plurality of microcompartments together. In addition, the invention also relates to a cell microcompartment assembly or a series of cell microcompartments as described above, comprising at least one cell microcompartment according to the invention.

[0165] The invention also relates to a microcompartment assembly or a series of microcompartments of at least two three-dimensional cell microcompartments, each microcompartment comprising at least one hydrogel outer layer and at least one hydrogel layer or hydrogel network within said outer layer and at least one cell, wherein at least one microcompartment is a microcompartment according to the invention.

[0166] The microcompartment assembly according to the invention preferably comprises from 2 to 10 16 microcompartments.

[0167] Preferably, the series of microcompartments according to the invention is in a culture medium, especially in a culture medium with at least partial convection.

[0168] According to a particularly suitable embodiment, the object of the invention is a cell microcompartment assembly in a closed chamber (such as a bioreactor), preferably in a culture medium in a closed chamber (such as a bioreactor).

[0169] The presence of the hydrogel outer layer and an optional intermediate layer of isosmotic aqueous solution enables the cells to be evenly distributed between the microcompartments. In addition, this hydrogel layer makes it possible to prevent the microcompartments from merging, which are the main source of variability that is not conducive to the uniformity of cell phenotype.

[0170] Thus, the microcompartments according to any of the foregoing embodiments or the microcompartment assemblies according to any of the foregoing embodiments are also particularly suitable for cell culture, especially three-dimensional cell culture. These are capable of producing large amounts of cells of interest or even organoids of interest and are suitable for, for example, cell therapy. Additionally, the present invention also relates to the microcompartments according to the present invention or the microcompartment assemblies according to the present invention for use as a medicament.

[0171] Thus, the microcompartments are particularly suitable for the clinical setting.

[0172] According to another object, the present invention relates to the use of a microcompartment according to any of the foregoing objects for the production of cells, microtissues, tissues, preferably for the large-scale production of such cells and / or tissues.

[0173] The microcompartments according to the present invention can also be used for the production of animal or plant cells for human or animal food consumption. Such use is particularly useful for producing alternatives to meat products (such as meat) to limit the consumption of meat products.

[0174] Preparation method

[0175] The microcompartments can be obtained by different ways known to those skilled in the art for preparing microcompartments or capsules.

[0176] According to another aspect of the present invention, the inventors have also developed a method for preparing the microcompartments according to the present invention.

[0177] Thus, a particularly suitable method for preparing the microcompartments according to the present invention comprises the following steps:

[0178] a. Mixing cells, optionally previously incubated in a culture medium,

[0179] b. Encapsulating the mixture from step (a) in a hydrogel outer layer;

[0180] c. Culturing the capsules obtained in step (b) in a culture medium, preferably in a bioreactor, preferably for at least 1 day, more preferably for 3 days to 50 days, and

[0181] d. Optionally recovering the obtained cell microcompartments, characterized in that, during step (a) and / or during step (b), a hydrogel solution of plant or synthetic origin is added, the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the hydrogel outer layer.

[0182] Advantageously, the method according to the invention may include additional steps. Thus, preferably, the cells are incubated before the step of mixing the cells with a hydrogel solution having a Young's modulus less than that of the hydrogel of the outer layer (ii) in a suitable culture medium. The culture medium preferably contains at least one cell protection factor, more preferably at least one apoptosis inhibitor.

[0183] The apoptosis inhibitor may be, for example, one or more inhibitors of the RHO / ROCK (RHO-associated protein kinase) pathway, or any other apoptosis inhibitor known to those skilled in the art. The apoptosis inhibitor must make it possible to promote cell survival and promote the adhesion of cells to fibrin during the formation of the hydrogel outer layer.

[0184] The method according to the invention may include a cell dissociation step by chemical, enzymatic or mechanical dissociation carried out before or simultaneously with the cell incubation step, which step itself is carried out before the mixing step a). This step is particularly important in the case of adherent cells.

[0185] The encapsulated cells are suspended in the form of single cells and / or cell clusters. Preferably, the single cells account for less than 50% of the total number of encapsulated cells, and more preferably, the single cells are hPSC cells. In fact, it is preferred to encapsulate cell clusters because this reduces the occurrence of mutagenesis phenomena.

[0186] Preferably, the encapsulation step b) includes the following sub-steps:

[0187] - bringing the mixture from step (a) into contact with a hydrogel solution intended to form the outer layer (i) to form at least one droplet, and

[0188] - collecting the droplets in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0189] Once the outer hydrogel layer has been hardened by the calcium bath, microcompartments are formed. Then it can be rinsed off. Advantageously, the Young's modulus of the hydrogel in the inner part is less than the Young's modulus of the hydrogel in the outer layer, preventing / limiting the hardening of the hydrogel in the inner part due to the calcium bath. In particular, given that the Young's modulus of the hydrogel in the inner part is strictly less than the Young's modulus of the hydrogel in the outer layer, the hardening process using the calcium bath does not allow the hydrogel in the inner part to harden during the use of the capsule, and thus does not promote cell proliferation.

[0190] Preferably, step b) is carried out by co-injecting simultaneously the hydrogel solution intended to form the outer layer (i), the mixture from step a) which optionally comprises a hydrogel solution having a Young's modulus strictly lower than that of the hydrogel of the outer layer (ii), and optionally an intermediate solution (iii) which optionally comprises a hydrogel solution having a Young's modulus strictly lower than that of the hydrogel of the outer layer (ii); said co-injection is carried out concentrically via a microfluidic or a milli-fluidic syringe, a jet consisting of the mixture of said solutions being formed at the outlet of the syringe, said jet breaking up into droplets.

[0191] When the intermediate solution (iii) comprises a hydrogel solution having a Young's modulus lower than that of the hydrogel of the outer layer (ii), the hydrogel solution (ii) is not present in the mixture of step a).

[0192] According to a preferred object of the invention, the isotonic intermediate solution is a sorbitol solution.

[0193] According to one object of the invention, the final orifice diameter of the microfluidic syringe is from 50 μm to 800 μm, preferably from 80 μm to 240 μm, and the flow rate of each solution is from 0.1 mL / h to 2000 mL / h, preferably from 10 mL / h to 2000 mL / h, more preferably from 11 mL / h to 100 mL / h.

[0194] According to a variant, step b) is carried out by co-injecting simultaneously the hydrogel solution intended to form the outer layer (i), the mixture from step a) which optionally comprises a hydrogel solution having a Young's modulus strictly lower than that of the hydrogel of the outer layer (ii), and optionally an intermediate solution (iii) which optionally comprises a hydrogel solution having a Young's modulus strictly lower than that of the hydrogel of the outer layer (ii); said co-injection is carried out concentrically via a microfluidic or a milli-fluidic syringe, said syringe comprising a tip which is in contact with a calcium solution, a jet consisting of the mixture of said solutions being formed at the outlet of the syringe, said jet forming a tube.

[0195] When the syringe tip is in contact with the calcium solution, the final orifice diameter of the microfluidic syringe is preferably from 50 μm to 1000 μm, more preferably from 80 μm to 300 μm, and the flow rate of each solution is from 1 mL / h to 100 mL / h.

[0196] According to another variant of the invention, the internal part comprising a hydrogel layer or a hydrogel network further comprises at least a second hydrogel, which is different from the first hydrogel constituting the layer or the network of the internal part, more preferably fibrin. The fibrin is preferably obtained by polymerization of fibrinogen by means of a fibrinogen polymerizing agent, advantageously, said agent is thrombin, said agent being able to be added during and / or after encapsulation.

[0197] In addition, the thrombin solution is co-injected with other solutions, preferably mixed with an isotonic intermediate solution, and the fibrinogen solution is mixed with the mixture from step a).

[0198] Preferably, the fibrinogen concentration is between 10 mg / mL and 25 mg / mL, preferably between 14 mg / mL and 20 mg / mL, more preferably between 20 mg / mL.

[0199] According to another object of the present invention, the concentration of thrombin is preferably from 0.001 U / mL to 2 U / mL, more preferably from 0.01 U / mL to 1 U / mL, from 0.01 U / mL to 0.05 U / mL, and even more preferably 0.04 U / mL. "U" refers to the enzyme activity unit (i.e., the concentration of the enzyme), which represents the amount of enzyme necessary to process one micromole of substrate in one minute. It should be understood that the indicated concentration is the concentration in the mixture. In fact, advantageously, thrombin is mixed with other components in a 1:1 ratio. Additionally, inside the capsule, when the concentration of thrombin before mixing is 0.01 U / mL, the concentration inside the capsule is approximately 0.01 U / mL.

[0200] The post-encapsulation step can be carried out with or without agitation. Preferably, the post-encapsulation step is carried out with permanent or continuous agitation. This agitation is important because it maintains the uniformity of the culture environment and avoids the formation of any diffusion gradients. For example, it allows for uniform control of the cell oxygenation level; thus avoiding phenomena such as hypoxia-related necrosis or hyperoxia-related oxidative stress. Therefore, it avoids an increase in cell mortality and / or oxidative stress.

[0201] Preferably, after the step of culturing the obtained capsules, the method includes a step consisting of rinsing the capsules obtained from step (d), advantageously in order to eliminate cell protection factors such as apoptosis inhibitors.

[0202] When the method according to the present invention includes a step of rinsing the obtained capsules, the solution constituting the calcium bath is removed and replaced with a suitable culture medium for culturing the microcompartments according to the present invention, preferably an isotonic solution, more preferably a culture medium containing an apoptosis inhibitor. The rinsing step of the obtained capsules is carried out after step d).

[0203] In a preferred variant, the method according to the invention comprises, after step (d), preferably after a rinsing step (if such a step exists after step d), at least one re-encapsulation of the cells. "At least one re-encapsulation of the cells" means at least two encapsulation cycles. Preferably, each encapsulation cycle corresponds to one passage. In this variant of the method (at least one re-encapsulation of the cells after step (d)), the number of cell divisions for 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.

[0204] In the method according to the invention, there may be several re-encapsulations, preferably from 1 to 100 times, in particular from 1 to 10 re-encapsulations.

[0205] Each re-encapsulation may comprise:

[0206] a. A step consisting of: dissociating the microcompartment or the series of microcompartments in order to obtain a suspension of cells or a suspension of cell clusters; which may in particular be carried out by hydrolysis, dissolution, piercing and / or

[0207] or by any biocompatible means (that is to say, means that are non-toxic to the cells) for decomposition to eliminate the hydrogel outer layer. For example, this elimination may be achieved using: phosphate buffered saline, divalent ion chelators, enzymes such as alginate lyase (if the hydrogel contains alginate) and / or laser microdissection, and

[0208] b. A step of re-encapsulating all or part of the cells or cell clusters in the cells or cell clusters in a hydrogel capsule.

[0209] Re-encapsulation is a means suitable for increasing cell expansion resulting from the pluripotent step and reducing the risk of mutations.

[0210] According to a specific embodiment, the re-encapsulation comprises the following steps:

[0211] a. Eliminating the hydrogel outer layer,

[0212] b. Resuspending the cells contained in the microcompartment in order to obtain single cells and / or at least one group or cluster of cells in an isotonic medium, preferably a medium containing a cell apoptosis inhibitor,

[0213] c. Encapsulating the suspension of cells in a hydrogel layer;

[0214] d. Preferably, culturing the obtained microcompartments in an isotonic solution containing a cell apoptosis inhibitor, preferably in a medium containing a cell apoptosis inhibitor;

[0215] e. Preferably, rinsing the microcompartments, advantageously in order to eliminate the cell apoptosis inhibitor;

[0216] f. Culturing the microcompartments in an isotonic solution, preferably a culture medium, for at least one cell division cycle, and

[0217] g. Optionally recovering the obtained cell microcompartments.

[0218] Kit according to the present invention

[0219] The present invention also relates to a kit, the kit comprising a hydrogel solution with a Young's modulus strictly less than that of the hydrogel of the outer layer (i), a hydrogel solution with a Young's modulus greater than that of the hydrogel of the layer or network of the internal part intended to form the outer layer (ii), and an optional intermediate solution (iii).

[0220] According to another aspect, the present invention relates to the use of the kit for preparing the microcompartments according to the present invention, the kit comprising at least one hydrogel solution intended to form the outer layer (ii) of the microcompartments, a hydrogel solution of plant or synthetic origin with a Young's modulus strictly less than that of the hydrogel solution used to form the outer layer (i), and an optional intermediate solution (iii).

[0221] Preferably, the kit comprises a low molecular weight hydrogel solution (i) and a high molecular weight hydrogel solution (ii), and an optional intermediate solution (iii), in particular the low molecular weight hydrogel is less than the high molecular weight hydrogel.

[0222] According to another object, the present invention also relates to a kit, which comprises at least one alginate solution up to 75 kDa, an alginate solution of 150 kDa to 250 kDa, an isotonic solution (preferably a sorbitol solution), a calcium solution, and a suitable culture medium. According to a variant, the kit is a multi-part kit.

[0223] The present invention is now illustrated by non-limiting examples and results of the compositions according to the present invention.

[0224] Example

[0225] Example 1 - Capsule according to an embodiment of the present invention .

[0226] This example describes a specific embodiment of the present invention, as also shown in Figure 2 , in which a 0.5% alginate solution intended to form the inner alginate layer as a matrix substitute is added to a sorbitol solution and co-injected via a microfluidic syringe with a cell mixture in a culture medium and a 2% alginate solution intended to form the outer alginate layer.

[0227] A microfluidic syringe capable of co-injecting various solutions includes three pipelines upstream of the nozzle. A solution containing sorbitol and 0.5% alginate is injected into pipeline "IS". A 2% alginate solution and a cell suspension in a culture medium are respectively injected into pipeline "A" and pipeline "CS", and then encapsulation is carried out.

[0228] Once encapsulation has been carried out, the droplets are collected in a CaCl2 bath, allowing the hardening of the 2% alginate and the formation of an alginate shell that forms microcompartments or capsules. Then, the CaCl2 solution containing the capsules is rinsed with a serum-free cell culture medium. The capsules then 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.

[0229] Example 2 - Capsule according to an embodiment of the present invention 。

[0230] This example describes a specific embodiment of the present invention, also as Figure 3 shown, 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 syringe with a cell mixture in a culture medium and a 2% alginate solution intended to form an outer alginate layer.

[0231] A microfluidic syringe capable of co-injecting various solutions includes three pipelines upstream of the nozzle. A solution containing sorbitol and 0.5% alginate is injected into pipeline "IS". A 2% alginate solution and a cell suspension in a culture medium are respectively injected into pipeline "A" and pipeline "CS". In this embodiment, the tip of the microfluidic syringe is brought into contact with a calcium solution, thus allowing encapsulation in the form of a tube.

[0232] Once encapsulation has been carried out, the tubes are collected in a CaCl2 bath, allowing the hardening of the 2% alginate and the formation of an alginate wall that forms the tubes. Then, the CaCl2 solution containing the tubes is rinsed with a serum-free cell culture medium. The tubes then 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.

[0233] Example 3 - Measuring the viscosity of capsules according to the present invention and outside the scope of the present invention 。

[0234] In the context of this test, the inventors used an iPS cell line generated according to the conventional standards of two-dimensional iPS culture, and then, according to the knowledge of those skilled in the art, separated the cells from the flask via the action of enzymes and transferred them to a culture medium suitable for iPS culture.

[0235] Mix iPS cells in a suitable culture medium to obtain a cell density of approximately 1.5 M / mL. Mix a 0.5% alginate solution, which is intended to form an inner alginate layer as a matrix substitute, with a sorbitol solution. Also prepare a 2% alginate solution, which is intended to form an outer alginate layer. Then load the various solutions via dedicated pipelines and co-inject them simultaneously through a microfluidic syringe. The amount of encapsulated cells is approximately 0.6 * 10^6.

[0236] Use the same protocol to obtain capsules without exogenous extracellular matrix and capsules with The difference is that only the sorbitol solution is injected, and The solution is used instead of the 0.5% alginate solution.

[0237] From D1 to D5 after encapsulation, visually inspect the capsules. At D5, also observe the appearance of the cells, the amount of cells, their viability, and pluripotency.

[0238] Measure the viscosity of the components of each microcompartment using a rolling ball viscometer, and measure the rolling time of the ball through the opaque liquid and the transparent liquid according to The falling ball principle. The results show the viscosity of each component present in each type of microcompartment, which respectively include:

[0239] - Cell-like extracellular matrix (outside the scope of the present invention),

[0240] - Absence of exogenous extracellular matrix or extracellular matrix substitute, i.e., only the culture medium (outside the scope of the present invention),

[0241] - Alginate-like extracellular matrix (the present invention).

[0242] The results are presented in Table 1 below.

[0243] [Table 1]

[0244]

[0245] Capsules containing only cell culture medium without exogenous extracellular matrix have the lowest viscosity, while capsules based on 0.5% alginate have a viscosity 2.3 times higher. Capsules based on Have the highest viscosity. However, Is a highly complex medium, and the viscosity within the matrix varies greatly. Locally, the viscosity can be very high or very low, which means Very uneven viscosity within Highly heterogeneous formation of cell aggregates within the capsules. In contrast, alginate has a viscosity quite close to that of the culture medium, promoting cell growth. Moreover, the viscosity is very uniform, leading to good cell distribution within the alginate-based capsules.

[0246] Example 4 - Measuring the percentage of empty capsules .

[0247] The test compared Or the inoculation rate of 0.5% alginate (the present invention) capsule.Prepare capsule according to the method described in Example 3, and use the following method to measure the empty capsule percentage at D0.Ten photos are taken for each condition.Use software such as ImageJ, analyze each photo to count empty capsules and full capsules (at least one cell in the capsule), and then the software determines the ratio between the two and multiplies it by 100 to obtain the percentage.

[0248] The results are shown in [ Figure 4 ]. The inventors observed that relative to capsules and capsules containing only cell culture medium, with better cell seeding rates in capsules with 0.5% alginate. Since this alginate-based matrix is more viscous and has a more uniform viscosity, it enables the cells to remain suspended, thereby distributing themselves evenly within the capsule during the process. This is in contrast to capsules containing The capsule is in sharp contrast to the The matrix has a highly heterogeneous viscosity. Thus, there are almost no capsules without cells in the capsules according to the invention, indicating that the capsules according to the invention have a better cell seeding capacity, so that a greater proportion of capsules contain cells and thus subsequently encapsulate, allowing their subsequent use, in particular in cell therapy.

[0249] Example 5 - Measuring the capsule size and the number of encapsulations per capsule .

[0250] The test compared or 0.5% alginate (invention). Capsules were prepared according to the method described in Example 3. The size of the capsules was measured at D0, for example using ImageJ software including a region selection tool, and the amount of encapsulation per capsule was measured at D0 according to the method described in Example 4.

[0251] The results are presented in the following Table 2 and [Figure 5a], [Figure 5b], [Figure 5c], [Figure 6a], [Figure 6b], [Figure 6c].

[0252] [Table 2]

[0253]

[0254] After 5 days of culture, when cultured without a substrate, dead cells were observable in some of the capsules, making the outer layer of the capsule somewhat irregular. In capsules based on , the cells grew rapidly and each capsule was more or less filled with aggregates. Several aggregates were also observable in the same capsules and they tended to merge to form larger aggregates. The outer layer of the capsule was smooth and there were no dead cells. In capsules based on 0.5% alginate, the inventors also observed several aggregates in the same capsules, with fewer dead cells compared to the substrate-free condition, but smaller in structure compared to . The lumen was still visible after 5 days of culture. The average sizes of capsules based on and on 0.5% alginate were similar, allowing the cells to reproduce properly. Finally, the inventors observed that the number of capsules in alginate-based capsules was greater than the number of capsules in cell culture medium-based capsules.

[0255] Example 6 - Measuring the expansion and pluripotency after 5 days of culture .

[0256] This test compared capsules containing only the culture medium, capsules based on or capsules based on 0.5% alginate (the present invention). The capsules were prepared according to the method described in Example 3. The expansion and pluripotency of the cells present in each type of capsule were 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 was determined. At D5, the capsules were dissolved and the aggregates were dissociated, and then the cells were counted.

[0257] The results are shown in Figure 7 .

[0258] At D7, the inventors observed better expansion of the cells cultured in capsules based on 0.5% alginate compared to the cells cultured in substrate-free capsules. In capsules based on , the expansion factor was higher than in the other two conditions, but the variability between experiments was also much higher, which is not compatible with the clinical use of this technology.

[0259] Regarding pluripotency, at D7, the inventors also observed that the average percentage of Oct4 / Nanog-positive cells in 0.5% alginate was lower compared to the condition, but higher compared to the substrate-free condition. 0.5% alginate also reduced the variability between experiments.

[0260] Thus, the capsules according to the present invention protect the cells and aggregates from the mechanical stress of culture in a bioreactor by the action of the protective shell on the one hand, but also have the hydrogel as a substrate substitute in the capsule, providing an additional protective network for the cells and aggregates. This network is loose enough to allow cell migration and expansion while maintaining their pluripotent characteristics.

[0261] Example 7 - Measuring the expansion after 5 days of culture in cell microcompartments containing alginate functionalized with the YIGSR motif and pluripotency 。

[0262] The purpose of this test was to compare 0.5% alginate-based capsules functionalized with the YIGSR motif and spacers containing 6, 9, and 12 glycine residues, respectively (SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3). The capsules were prepared according to the method described in Example 3. The expansion and pluripotency of the cells present in each type of capsule were measured at D5 according to the following method. The ratio (factor X) of the number of cells encapsulated at D0 to the number of cells obtained (and surviving) at D5 was determined. At D5, the capsules were dissolved and the aggregates were dissociated, and then the cells were counted.

[0263] The results of expansion and pluripotency are shown respectively in Figure 9 and Figure 10 .

[0264] Then, the inventors found that functionalization of alginate (matrix substitute) with the YIGSR motif improved cell adhesion to the alginate-based matrix substitute, thus enhancing the survival of the cells and thereby enhancing the expansion of the cells. This effect was further enhanced when the YIGSR motif also contained spacers of 6, 9, and 12 glycine residues, respectively (SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3).

[0265] Finally, the inventors also found that functionalization with the YIGSR motif (optionally containing a spacer) maintained pluripotency under each condition.

[0266] Example 8 - Measuring the expansion after 5 days of culture in microcompartments according to the present invention containing alginate as a matrix substitute and differentiated cells of the endoderm and mesoderm Figure 11 。

[0267] The purpose of this test was to demonstrate the potency of 0.5% alginate-based microcompartments containing differentiated cells (i.e., cells of the endoderm and mesoderm). The capsules were prepared according to the method described in Example 3. The capsules containing iPSCs were cultured for 4 days. Then, the cells were differentiated for 5 days by adding a medium that allowed the iPSC cells to differentiate into endoderm and mesoderm sheets.

[0268] The expansion of the cells present in each type of capsule was measured at D5 according to the following method. The ratio (expansion factor) of the number of cells encapsulated at D0 to the number of cells obtained (and surviving) at D5 was determined. At D5, the capsules were dissolved and the aggregates were dissociated, and then the cells were counted.

[0269] The expansion results are shown respectively in ​ .

[0270] Then, the inventors observed that the cells cultured in 0.5% alginate-based capsules had better proliferation compared to cells cultured two-dimensionally, thus demonstrating the proliferation and culturing ability of the microcompartments according to the invention for differentiated cells, in particular cells of the endoderm and mesoderm.

Claims

1. A three-dimensional cell microcompartment, the cell microcompartment comprising: - A hydrogel outer layer, and - An inner portion containing at least one cell and at least one hydrogel layer or hydrogel network of plant or synthetic origin, Characterized in that 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 the previous claim, characterized in that, The hydrogel layer or the hydrogel network of the inner portion is at least partially juxtaposed with the inner surface of the outer layer.

3. The cell microcompartment according to one of the preceding claims, characterized in that, The Young's modulus of the hydrogel in the inner portion is from 0.01 kPa to 200 kPa, preferably from 0.1 kPa to 60 kPa.

4. The cell microcompartment according to one of the preceding claims, characterized in that, The Young's modulus of the hydrogel in the inner portion is from 0.1 kPa to 5 kPa.

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

6. The cell microcompartment according to one of the preceding claims, 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 one of the preceding claims, characterized in that, The inner portion further contains at least one culture medium.

8. The cell microcompartment according to one of the preceding claims, characterized in that, The inner portion contains at least one cell layer.

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

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

11. The cell microcompartment according to one of the preceding claims, characterized in that, In addition to the hydrogel of plant or synthetic origin, the hydrogel layer or the hydrogel network of the inner portion further contains at least one other hydrogel selected from fibrin, collagen, fibronectin, nestin, hyaluronic acid and laminin.

12. The cell microcompartment according to one of the preceding claims, wherein, The microcompartment is closed.

13. The microcompartment according to one of the preceding claims, characterized in that, The microcompartment has the shape of an ovoid, cylinder, spheroid, sphere or teardrop.

14. The cell microcompartment according to one of the preceding claims, characterized in that, The one or more cells present in the inner portion are selected from human, animal and plant eukaryotic cells.

15. The cell microcompartment according to one of the preceding claims, characterized in that, The one or more cells present in the inner portion are pluripotent cells and / or progenitor cells and / or cells undergoing differentiation and / or differentiated cells.

16. The cell microcompartment according to one of the preceding claims, characterized in that, The inner portion of the microcompartment contains at least one cell layer and at least one lumen, and the cell layer, the hydrogel layer or the hydrogel network of the inner portion and the outer layer are continuously organized around the lumen.

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

18. A three-dimensional cell microcompartment assembly, characterized in that, At least one microcompartment is the microcompartment according to one of the preceding claims.

19. The cell microcompartment according to one of claims 1 to 17 or the microcompartment assembly according to claim 18, for use as a medicament.

20. Use of the microcompartment according to one of claims 1 to 17 or the cell microcompartment assembly according to claim 18 for the manufacture of microtissue.

21. A method for preparing the cell microcompartment according to one of claims 1 to 17, the method comprising the following steps: a. Mixing cells, optionally previously incubated in a culture medium, b. Encapsulating the mixture from step (a) in a hydrogel intended to form the hydrogel outer layer, c. Culturing the obtained capsules in a culture medium, preferably in a bioreactor, preferably for at least 1 day, even more preferably for 3 to 50 days, and d. Optionally recovering the obtained cell microcompartments, characterized in that during step (a) and / or during step (b), a hydrogel solution of plant or synthetic origin is added, the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the hydrogel outer layer.

22. The method according to the preceding claim, characterized in that, Step b) is carried out by co-injecting simultaneously the hydrogel solution intended to form the outer layer (i), the mixture from step a) and an optional intermediate solution (iii), the mixture optionally comprising a hydrogel solution of plant or synthetic origin the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the outer layer (ii), the intermediate solution optionally comprising a hydrogel solution of plant or synthetic origin the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the outer layer; the co-injection is carried out concentrically via a microfluidic or millifluidic syringe, a jet composed of the mixture of the solutions being formed at the outlet of the syringe, the jet splitting into droplets.

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

24. The method according to claim 21, wherein Step b) is carried out by co-injecting simultaneously the hydrogel solution intended to form the outer layer (i), the mixture from step a) and an optional intermediate solution (iii), the mixture optionally comprising a hydrogel solution of plant or synthetic origin the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the outer layer (ii), the intermediate solution optionally comprising a hydrogel solution of plant or synthetic origin the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the outer layer; The co-injection is carried out concentrically via a microfluidic or millifluidic syringe, the syringe comprising a tip which is in contact with the calcium solution, a jet composed of the mixture of the solutions being formed at the outlet of the syringe, the jet forming a tube.

25. The method according to the preceding claim, characterized in that, The final opening diameter of the microfluidic syringe is from 50 μm to 1000 μm, preferably from 80 μm to 300 μm, and the flow rate of each of the solutions in the solutions is from 1 mL / h to 100 mL / h.

26. Use of a kit for preparing a microcompartment according to one of claims 1 to 17, the kit comprising at least one hydrogel solution intended to form the outer layer (i) of the microcompartment and a hydrogel solution of plant or synthetic origin the Young's modulus of which is strictly less than the Young's modulus of the hydrogel used to form the outer layer (ii).

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

28. The peptide sequence according to the previous claim, characterized in that, The peptide sequence is selected from 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 cell microcompartment according to one of claims 1 to 17, characterized in that, The hydrogel network of plant or synthetic origin comprises at least one peptide sequence according to one of claims 27 or 28.

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