Extracellular matrix substitutes in cellular microcompartments

Abstract

The present invention relates to a clinically usable cellular microcompartment, referred to as "GMP," comprising at least a. a cell layer, b. an outer hydrogel layer, and c. a fibrin mesh interposed between the outer hydrogel layer and the cell layer. The present invention also relates to a method for preparing the microcompartment, and to the use of a kit comprising a fibrinogen solution and a thrombin solution for preparing the cellular microcompartment.

Description

[Technical Field]

[0001] The present invention relates to the field of three-dimensional cell culture, and in particular to cell microcompartments for the production of cells and tissues that can be used under GMP clinical conditions for human and veterinary use. [Background technology]

[0002] Cell culture has been an area of ​​growing interest since Professor Yamanaka's discovery of induced pluripotent stem cells (iPS or iPSC).

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

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

[0005] A particularly suitable technique is described in patent application WO 2018 / 096277, which consists of three-dimensional microcompartments for culturing stem cells.

[0006] Although this is a very promising technology, 3D culture still has certain drawbacks that make it unsuitable for clinical use. To do this, both the cells and the 3D culture system from which they are derived must comply with Good Manufacturing Practices (GMP) regulations. However, most 3D culture systems, such as cell microcompartments, contain extracellular matrices from animal origin and / or cancer cell lines, which do not comply with such regulations.

[0007] Therefore, to allow for the emergence of cell therapies based on this technology, but also for the production of animal or plant cells for human or animal food consumption, it is necessary to develop substitutes, i.e. alternatives that allow the elimination of the presence of such extracellular matrices while preserving the possibility for cells to attach and grow satisfactorily.

[0008] In the context of these studies, the inventors have surprisingly discovered that fibrin makes it possible to obtain particularly promising results during its use in cell microcompartments, namely that fibrin not only makes it possible to obtain large amounts of cells with rapid proliferation, but also allows the large-scale production of said cell microcompartments, thus making it possible to envisage its clinical use in three-dimensional cell microcompartments for human and veterinary use.

[0009] Therefore, to meet this need for cell microcompartments that do not contain non-GMP extracellular matrices such as Matrigel®, the present invention proposes a three-dimensional culture system based on cell microcompartments that comprise a fibrin mesh placed between an outer layer of hydrogel and at least one cell layer. Summary of the Invention

[0010] Thus, the present invention provides a novel cellular microcompartment comprising: at least one cell layer; an outer layer of hydrogel; - a fibrin mesh disposed between an outer layer of hydrogel and at least one cell layer.

[0011] Advantageously, the fibrin mesh may be separated or not, i.e., intertwined with at least one of the other components of the cellular microcompartment, such as the cells or the outer layer of the hydrogel. Thus, according to one embodiment, the cells may be distributed inside the fibrin mesh and / or the fibrin mesh may be intertwined with the outer layer of the hydrogel. Preferentially, the fibrin mesh is intertwined with the outer layer of the hydrogel.

[0012] When the fibrin mesh intertwines with the outer layer of the hydrogel, the outer layer of the hydrogel may or may not form an interpenetrating network, which, if it does form an interpenetrating network, may be an interpenetrated polymer network (IPN).

[0013] If the fibrin mesh forms a distinct network, it is not intertwined with at least one of the other components of the cellular microcompartment.

[0014] Advantageously, the fibrin mesh may contain other molecules, such as growth factors, proteins, peptides, elements of the culture medium and / or elements resulting from cellular activity, such as secreted proteins, metabolic products, etc.

[0015] According to another object, the layer of hydrogel preferentially comprises alginate.

[0016] Advantageously, the cells constituting the cell layer are selected from eukaryotic cells, pluripotent cells and differentiated cells.

[0017] According to another preferred object, the microcompartments according to the invention comprise: The microcompartments are closed and / or the microcompartments are three-dimensional microcompartments, preferentially hollow three-dimensional microcompartments, and / or the microcompartments are ovoid, cylindrical, spheroidal, spherical or teardrop-shaped, and / or - the microcompartment comprises one or more lumens within said at least one cell layer.

[0018] When the microcompartment comprises a lumen, the cell layer, fibrin mesh, and outer layer are organized around the lumen. Preferentially, the cell layer, fibrin mesh, and outer layer are organized continuously around the lumen.

[0019] According to a particularly preferred object, the fibrin contained in the microcompartments according to the invention is obtained from the polymerization of fibrinogen by thrombin, advantageously obtained during and / or after encapsulation.

[0020] According to another aspect, the present invention also relates to a set of microcompartments comprising at least one microcompartment according to the invention.

[0021] In the context of the present invention, the microcompartments are particularly suitable for cell therapy protocols.Another aspect also relates to a microcompartment according to the invention or a set of microcompartments according to the invention for use as a medicament.

[0022] Furthermore, the microcompartments according to the invention or the set of microcompartments according to the invention can be obtained by any means known to the person skilled in the art.

[0023] According to a particularly preferred embodiment, the microcompartments according to the invention can be obtained according to the preparation method described below. The present invention therefore preferentially relates to a preparation method comprising the steps of: a) mixing cells, optionally pre-incubated in culture medium, with a mixture of fibrinogen; b) encapsulating the mixture from step a) in a hydrogel layer; c) culturing the capsules obtained in step b) in a culture medium; d) optionally culturing the capsules resulting from step c) for at least 1 day, preferentially between 3 and 50 days, and optionally recovering the resulting cell microcompartments, The present invention relates to a preparation method characterized in that a thrombin solution is added during steps b) and / or c).

[0024] Preferentially, the method optionally comprises, between the culturing steps c) and d), a step for rinsing the capsules resulting from step c) for at least one day.

[0025] Preferentially, the encapsulation step b) comprises: i. contacting the mixture of step a) with a solution of hydrogel to form at least one droplet; ii. collecting the resulting droplets, the inner part of each droplet consisting of the mixture of step a) and optionally a thrombin solution, in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0026] More preferentially, the thrombin solution can be added during step i) or ii). If the thrombin solution is added during step i), the resulting droplets comprise an outer layer and the interior of each droplet consists of the mixture of step a) and the thrombin solution. Preferentially, the mixing of step i) is carried out during co-injection by a microfluidic or millifluidic injector, allowing droplet formation and contact with thrombin, resulting in the polymerization of fibrinogen to fibrin.

[0027] When the thrombin solution is added during step ii), it is present in a calcium bath that can harden the hydrogel solution to form the outer layer of each microcompartment. The thrombin solution then diffuses through the outer layer of the hydrogel, allowing the polymerization of fibrinogen to fibrin.

[0028] Alternatively, the thrombin solution can be added after the formation of the microcompartments during step c) of culturing the capsules. The culture medium then contains the thrombin solution, which diffuses through the hydrogel layer and allows the polymerization of fibrinogen to fibrin.

[0029] According to a particularly preferred purpose, step i) consists in contacting the mixture of step a), the hydrogel solution, and an intermediate solution containing the thrombin solution. For the purposes of the present invention, the term "intermediate solution" is intended to mean a solution that does not contain molecules capable of hardening the hydrogel solution and / or does not contain calcium. According to a particular embodiment, the intermediate solution is an isotonic intermediate solution. Thus, the thrombin solution is added during the formation of the droplets.

[0030] Also, in a particularly preferred manner, step b), and more preferentially sub-step i), is carried out by simultaneous co-injection of the hydrogel solution, the mixture of step a) and, optionally, the intermediate solution, said co-injection being carried out concentrically via a microfluidic or millifluidic injector forming at the injector outlet a jet consisting of the mixture of said solutions, said jet breaking up into droplets.

[0031] In the context of the present invention, the thrombin solution cannot be added before the encapsulation step b) corresponding to the formation of the droplets.

[0032] Preferentially, the fibrinogen concentration is between 5 and 30 mg / mL, more preferentially between 10 and 25 mg / mL, and even more preferentially, the fibrinogen concentration is between 14 and 20 mg / mL.

[0033] Preferentially, the thrombin concentration is between 0.001 U / mL and 2 U / mL, more preferentially between 0.01 U / mL and 1 U / mL, between 0.01 U / mL and 0.05 U / mL, between 0.01 U / mL and 0.03 U / mL, and even more preferentially 0.02 U / mL.

[0034] Preferentially, the final opening diameter of the microfluidic injector is 50 to 800 μm, more preferentially 50 to 300 μm, even more preferentially 80 to 240 μm, and the flow rate of each of the solutions is 0.1 to 1000 mL / h, preferentially 1 to 500 mL / h, even more preferentially 10 to 150 mL / h. Even more preferably, the opening of the microfluidic injector is 100 μm or 215 μm, and the flow rate of each of the solutions is 23 mL / h to 100 mL / h.

[0035] The microcompartments according to the invention can be used in a clinical setting.An aspect of the invention also relates to microcompartments or assemblies of microcompartments according to the invention for use as a pharmaceutical.

[0036] Finally, according to another aspect, the present invention also relates to the use of a kit intended for the preparation of microcompartments according to the invention, said kit comprising at least one fibrinogen solution and a thrombin solution. The present invention therefore also relates to the use of a kit comprising at least a fibrinogen solution and a thrombin solution for the preparation of microcompartments according to the invention.

[0037] According to another object, the present invention also relates to a kit comprising at least one fibrinogen solution, a thrombin solution, a hydrogel solution, preferentially alginate, an isotonic solution, preferentially a sorbitol solution, a calcium solution, a suitable culture medium. According to one variant, the kit is a kit of parts.

[0038] Preferentially, the fibrinogen and thrombin solutions are of human origin and comply with Good Manufacturing Practice (GMP) regulations.

[0039] Other features and advantages will become apparent from the following detailed description of the invention, the examples, and the drawings. [Brief explanation of the drawings]

[0040] [Figure 1]This shows a first embodiment of the present invention, during which a thrombin solution is mixed with sorbitol during co-injection. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate; CS: suspended cells and culture medium and fibrinogen; IS: intermediate solution containing sorbitol and 0.02 U of thrombin. The first step involves the co-injection of the different components, which forms a jet and breaks into droplets in a CaCl2 bath, hardening the outer layer of the capsule. Then, during the second step, the capsule is resuspended in rinsing medium. Finally, the capsule is resuspended in the final medium in a flask. [Figure 2] This figure shows a second embodiment of the invention, during which a thrombin solution is added to the calcium bath used to collect the droplets formed after the splitting of the jet at the injector outlet. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate; CS: suspended cells and culture medium and fibrinogen; IS: intermediate solution, i.e., sorbitol. The first step involves the co-injection of the various components, which forms a jet and splits it into droplets in a CaCl2 bath supplemented with 0.02 U of thrombin solution, allowing the polymerization of fibrinogen and the hardening of the outer layer of the capsule, made of alginate. Then, during the second step, the capsule is resuspended in rinsing medium. Finally, the capsule is resuspended in final medium in a flask. [Figure 3] This shows a first embodiment of the present invention, during which a thrombin solution is added to the final medium. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate; CS: suspended cells and culture medium and fibrinogen; IS: intermediate solution, i.e., sorbitol. The first step involves the co-injection of the different components, forming a jet that breaks into droplets in a CaCl2 bath and hardens the outer layer of the capsule, made of alginate. Then, during the second step, the capsules are resuspended in rinsing medium. Finally, the capsules are resuspended in a flask in final medium supplemented with 0.02 U of thrombin solution, allowing the fibrinogen to polymerize. [Figure 4]5A and 5B are phase contrast microscopy images showing capsules in the absence of exogenous extracellular matrix (A), in the presence of Matrigel (B), capsules of the invention according to the embodiment of FIG. 2 (C), and capsules of the invention according to the embodiment of FIG. 1 (D) at day 5. [Figure 5] 1 shows results for the amplification of capsules according to the invention, capsules in the presence of Matrigel, and capsules in the absence of exogenous extracellular matrix. [Figure 6] The results are shown for the percentage of capsules containing cysts for capsules according to the invention, capsules in the presence of Matrigel, and capsules without exogenous extracellular matrix. [Figure 7] Figure 1 shows the results regarding pluripotency for capsules according to the invention, capsules in the presence of Matrigel, and capsules in the absence of exogenous extracellular matrix. [Figure 8] Phase contrast microscopy images 17 days after encapsulation of neurons: Panel A shows neurospheres or neural microtissues contained in microcompartments according to the invention, i.e., fibrin polymerized from 14 mg / mL fibrinogen, and Panel B shows neurospheres or neural microtissues in prior art microcompartments containing Matrigel. [Figure 9] Principal component analysis (PCA) of the 1000 most variable genes between iPSCs at days 0, 17, and 24 after neural differentiation in capsules seeded with fibrinogen or Matrigel is shown. Mature dopaminergic progenitor cells in capsules are used as a positive control. DETAILED DESCRIPTION OF THE INVENTION

[0041] definition For the purposes of the present invention, "microcompartment" or "capsule" also refers to a partially or completely closed three-dimensional structure containing a plurality of cells, formed from a matrix of polymer chains, such as alginate, swollen by a liquid and preferentially by water. This structure in particular consists of a hardened outer layer of hydrogel.

[0042] For the purposes of the present invention, "droplet" is also understood to mean a three-dimensional structure formed from at least one liquid solution containing the components of a non-hardened hydrogel (polymerized precursors, non-crosslinked polymer chains, etc.) of hydrogel precursor elements. Droplets also constitute a transitional state between the co-injection of various components and microcompartments.

[0043] For purposes of the present invention, a "differentiated" cell refers to a cell that has a particular phenotype, as opposed to an undifferentiated pluripotent stem cell or a progenitor cell that has undergone differentiation.

[0044] For the purposes of the present invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even if this is not specified, the cells, stem cells, progenitor cells, and tissues according to the present invention consist of or are obtained from human cells or immunologically humanized non-human mammalian cells.

[0045] For purposes of the present invention, the term "mutated cell" refers to a cell that has at least one mutation.

[0046] For the purposes of the present invention, "progenitor cells" refers to stem cells that have already begun to differentiate but have not yet differentiated.

[0047] For purposes of the present invention, "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4, NANOG, and SOX2, as well as surface markers such as SSEA3 / 4, Tra-1-60, and Tra-1-81. Embryonic stem cells used in connection with the present invention can be obtained without destroying the embryo from which they are derived, for example, using the techniques described in Chang et al. (Cell Stem Cell, 2008, 2(2)):113-117). Optionally, embryonic stem cells from humans can be excluded.

[0048] For the purposes of the present invention, "pluripotent stem cells" or "pluripotent cells" refer to cells that have the potential to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. These may be, inter alia, induced pluripotent stem cells (iPSCs, or in the case of human induced pluripotent stem cells, hiPSCs), embryonic stem cells, or MUSE cells ("multilineage-differentiating stress enduring").

[0049] For the purposes of the present invention, "induced pluripotent stem cells" refers to pluripotent stem cells that have been induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4, and SSEA3 / 4, among others. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007, 318(5858):1917-1920), Takahashi et al. (Cell, 207, 131(5):861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1):101-106).

[0050] For the purposes of the present invention, a "cell layer" or "cell sheet" is understood to mean a plurality of cells forming a layer or sheet that can be structured around a lumen, which can be, for example, a cell tissue or microtissue or a three-dimensional grouping culture. The thickness of the cell layer can be variable. This cell layer is organized three-dimensionally in microcompartments.

[0051] For the purposes of this invention, "tissue" or "biological tissue" has the general meaning of tissue in biology, i.e., an intermediate level of organization between cells and organs. Tissues are sets of similar cells of the same origin (generally derived from a common cell line, but which can result from the association of different cell lines) grouped into clusters, networks, or bundles (fibers). Tissues form functional assemblies, i.e., their cells contribute to the same function. Biological tissues regenerate regularly and assemble together to form organs.

[0052] A "fibrin mesh" or "fibrin network" within the meaning of the present invention refers to a plurality of intertwined fibrin fibers that make up a mesh or network, which are optionally intertwined with the inner surface of the outer hydrogel layer of the microcompartments.

[0053] For purposes of the present invention, "lumen" means the volume of aqueous solution topologically surrounded by a cell, the contents of which are preferentially not in diffusional equilibrium with the volume of convective liquid present outside the microcompartment.

[0054] Cellular microcompartments Thus, the present invention relates to a cell microcompartment comprising cells, an outer layer of hydrogel, and a fibrin mesh. The microcompartment according to the present invention comprises at least one cell layer. It is understood that the microcompartment may also comprise cells suspended in a medium or optionally contained in a fibrin mesh.

[0055] Thus, the cell microcompartments advantageously comprise: at least one cell layer; an outer layer of hydrogel; a fibrin mesh disposed between the outer layer of hydrogel and the cell layer.

[0056] Preferentially, the microcompartments are three-dimensional microcompartments defined by and inside an outer layer of hydrogel, the microcompartments containing cells and a fibrin mesh, and may be ovoid, cylindrical, spheroidal, spherical, or teardrop-shaped.

[0057] Advantageously, the three-dimensional microcompartments are hollow, and more preferentially the hollow microcompartments are ovoid, cylindrical, spheroidal, spherical or teardrop-shaped in shape.

[0058] Preferentially, the hydrogel used is biocompatible, i.e., non-toxic to cells. The hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartments and enable them to survive. According to one embodiment, the outer hydrogel layer comprises at least alginate. The outer hydrogel layer may consist exclusively of alginate. The alginate may in particular be sodium alginate composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular weight of 100 to 400 kDa and a total concentration of 0.5 to 5% by weight. Advantageously, the hydrogel layer does not contain cells.

[0059] The hydrogel layer also makes it possible to protect the cells from the external environment and to limit the uncontrolled proliferation of the cells and (in the case of differentiation) their differentiation.

[0060] The cells present in the microcompartments can be of any type, in particular they are eukaryotic cells, advantageously mammalian cells, more preferentially they are human or animal cells.

[0061] In certain embodiments, the microcompartments comprise pluripotent stem cells. Pluripotent stem cells or pluripotent cells refer to cells that have the ability to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Pluripotent stem cells may be, in particular, induced pluripotent stem cells (iPS), MUSE ("multi-lineage stress-resistant") cells found in the skin and bone marrow of adult mammals, or embryonic stem cells (ES). According to one embodiment, the microcompartments according to the invention do not comprise embryonic stem cells (ES).

[0062] According to a particularly preferred variant of the invention, the microcompartments according to the invention contain human or animal induced pluripotent stem cells.

[0063] In another particular embodiment, the microcompartments according to the invention comprise human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells. The multipotent and / or progenitor cells are preferentially obtained from pluripotent stem cells, in particular human pluripotent stem cells, or optionally from non-pluripotent human cells that have been artificially modified to match the transcriptional profile of the particular multipotent and / or progenitor cells, typically by forcing the expression of specific transcription factors for the target cell phenotype. Preferentially, the multipotent and / or progenitor cells are obtained from pluripotent stem cells after contact with a solution capable of initiating differentiation of the stem cells.

[0064] According to another variant, the microcompartments according to the invention comprise differentiated human or animal cells. The differentiated cells are preferentially obtained from pluripotent stem or progenitor cells, in particular human pluripotent stem or progenitor cells, or optionally from non-pluripotent human cells artificially modified so that their transcriptional profile matches that of a particular differentiated cell, typically by forcing the expression of transcription factors specific to the target cell phenotype. Preferentially, the differentiated cells are obtained from pluripotent or multipotent, or progenitor, stem cells after contact with a solution capable of initiating differentiation of said stem cells. According to one variant, the cellular content of the microcompartments comprises homogeneous or mixed cellular identity.

[0065] The differentiated cells may be in the form of at least one cell layer, or in the form of a three-dimensional tissue or microtissue, or in the form of multiple tissues or microtissues in a microcompartment, in particular. The differentiated cells may be in the form of a compacted or non-compacted tissue or microtissue, with or without a lumen.

[0066] Microcompartments according to the invention may contain multiple types of cells, in particular microcompartments according to the invention may contain, for example, stem cells and / or multipotent cells and / or progenitor cells and / or differentiated cells induced to pluripotency.

[0067] Advantageously, the microcompartments according to the invention are obtained after several cell division cycles. Indeed, the cells contained in the microcompartments according to the invention are cells obtained by amplification from at least one cell.

[0068] Also, the cells present in the microcompartments according to the invention were obtained after at least two cell division cycles following encapsulation of at least one cell in the outer layer of the hydrogel.

[0069] Preferentially, the cells present in the microcompartments according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles after encapsulation in the outer hydrogel layer of at least one cell, preferentially 1-5, 1-10, 1-15, 1-20, 1-30, 1-40, 1-50, 1-60, 1-100 cells. For example, the cells present in the microcompartments have been obtained after at least 6 cell division cycles after encapsulation in the outer hydrogel layer of at least one cell, preferentially 1-50 cells.

[0070] Preferentially, the microcompartments are obtained after at least 2, more preferentially at least 3, 4, 5, 6, 7, 8, 9 or 10 passages after encapsulation, each passage lasting for example at least 1 day, or between 2 and 50 days, in particular between 3 and 10 days.

[0071] Preferentially, the microcompartments are obtained after at least one reencapsulation, more preferentially after 1 to 14 reencapsulations, in particular after 2 to 7 reencapsulations. Very preferentially, each reencapsulation corresponds to a new passage, each encapsulation cycle corresponding to a passage.

[0072] Preferentially, all of the cells initially encapsulated in the microcompartment prior to the first cell division cycle occupy less than 50% of the volume of the microcompartment in which they are encapsulated, more preferentially less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0073] Thus, according to one embodiment, the cells present in the microcompartments according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles after encapsulation in the outer hydrogel layer of cells occupying a volume of less than 50% of the volume of the microcompartment in which they are encapsulated, more preferentially less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0074] Preferentially, in the microcompartments according to the invention, the cells occupy more than 50% by volume of the volume of the microcompartment, and even more preferentially more than 60%, 70%, 75%, 80%, 85%, 90% by volume of the volume of the microcompartment.

[0075] The microcompartments according to the invention comprise a plurality of cells, preferentially at least 20 cells, even more preferentially at least 100, at least 500, at least 1000, at least 10,000 cells.

[0076] In the context of the present invention, fibrin mesh is particularly suitable as an alternative to non-GMP extracellular matrices such as Matrigel®, making it possible to address the shortcomings of the prior art, and also making it possible to achieve cell proliferation in a satisfactory manner.

[0077] Thus, the fibrin mesh advantageously forms a fibrin network within the capsule, possibly constituting a fibrin gel or mass in the capsule. This mesh may or may not interpenetrate with at least one of the other components of the microcompartment, preferentially with the outer layer of the microcompartment. If the mesh is not interpenetrating with, for example, the outer layer, it forms a separate network in which cells can reside and grow.

[0078] Preferentially, the fibrin mesh is intertwined with the outer layer of the hydrogel, more preferentially with the inner surface of the outer layer of the hydrogel. Also, the demarcation between the fibrin mesh and the outer layer does not have to be completely clear. Thus, at least a portion of the fibrin mesh can be intertwined with the inner surface of the outer layer, preferentially with the alginate constituting the outer layer. Thus, at least a portion of the fibrin mesh is preferentially intertwined with the outer layer of the hydrogel.

[0079] According to another particular embodiment, the fibrin mesh forms an interpenetrating polymer network (IPN) with the outer layer of the hydrogel.

[0080] According to a particularly preferred purpose, fibrin is obtained during and / or after encapsulation from the polymerization of fibrinogen by a fibrinogen polymerization agent, advantageously thrombin, and the polymerization of the fibrinogen solution by the thrombin solution occurs during and / or after encapsulation, in which case the polymerization occurs within the newly formed droplets or capsules.

[0081] The fibrin mesh may optionally contain a mixture of extracellular proteins and compounds necessary for culturing differentiating cells as well as isolated cells.

[0082] Advantageously, encapsulation is performed by co-injection, carried out concentrically through a microfluidic injector that forms at the injector outlet a jet consisting of a mixture of different useful solutions, which jet breaks into droplets that are then collected in a calcium bath that allows the hydrogel solution to harden and form the outer layer of each microcompartment.

[0083] According to a first embodiment, polymerization of the fibrinogen solution by thrombin occurs during encapsulation. The cell mixture, fibrinogen mixture, hydrogel solution, and thrombin solution are simultaneously contacted and co-injected concentrically through a microfluidic or millifluidic injector, which forms a jet at the injector outlet and splits into droplets. As soon as the various solutions are brought into contact, polymerization begins, which then occurs almost instantaneously.

[0084] According to a second embodiment, the droplets are collected in a calcium bath, which allows the hydrogel solution to harden and form the outer layer of each microcompartment. In the absence of thrombin solution during co-injection via the microfluidic injector, fibrinogen polymerization is not initiated. Therefore, polymerization of the fibrinogen solution by the thrombin solution occurs after encapsulation. To this end, thrombin solution is added to the calcium bath to allow collection and formation of the microcompartments. Thus, the thrombin solution can diffuse through the hardening hydrogel solution.

[0085] According to the third embodiment, no thrombin solution is added to the calcium bath. Thus, once the hardening process of the hydrogel solution in the calcium bath is complete, the formed microcompartments are rinsed and an isotonic solution, preferably a culture medium containing an apoptosis inhibitor, is added. This isotonic solution is then supplemented with thrombin solution. The thrombin solution can then diffuse through the hardened hydrogel shell, allowing fibrinogen to polymerize with thrombin.

[0086] According to a particularly preferred purpose, the thrombin solution is co-injected simultaneously with the cells, the fibrinogen solution, the cell-containing culture medium and the hydrogel solution. More preferentially, an isotonic solution is also co-injected, which comprises a thrombin solution, advantageously the isotonic solution being a sorbitol solution.

[0087] According to any of the three embodiments described above, the polymerization of fibrinogen by a fibrinogen polymerization agent such as thrombin makes it possible to obtain a fibrin mesh within the capsule, in which cells are housed or attached to the surface of the mesh and grow. The fibrin mesh can form a distinct network or a network interpenetrating with at least one of the other constituents of the microcompartment, preferentially the outer layer of the hydrogel.

[0088] The microcompartments according to the invention may also contain other elements, in particular culture medium.

[0089] The culture medium is a medium suitable for the cells present in the microcompartments according to the knowledge of the person skilled in the art.

[0090] According to another preferred object of the present invention, the microcompartment comprises at least one lumen. The at least one lumen may contain a liquid, in particular a culture medium and / or a liquid secreted by the cells. Advantageously, the presence of this hollow space allows the cells to have a small diffusion volume in which they can control the composition, facilitating what is called autocrine / paracrine cellular communication. This three-dimensional arrangement of a monolayer or spherical cell sheet surrounding a lumen or central lumen may also be called a cyst.

[0091] The lumen is preferentially generated by cells that grow and develop on or within the fibrin mesh during cyst formation.

[0092] According to another preferred object, the cell layer, the fibrin mesh and the outer layer are organized around the lumen, and more preferentially they are organized continuously around the lumen.

[0093] The cyst-shaped conformation allows stem cells to be subjected to less pressure than in 2D cultures or aggregates. This configuration also reduces cell death and increases the rate of culture expansion. As a result, this reduces the number of passages and dissociations required, and shortens the culture time required to reach the desired final cell number.

[0094] According to one embodiment, the microcompartment may comprise multiple cysts or tissues or microtissues.

[0095] The cell microcompartments according to the invention are closed or partially closed, i.e. the outer layer is closed or partially closed. Preferentially, the microcompartments are closed.

[0096] The microcompartments according to the invention can have any three-dimensional shape, i.e., the shape of any object in space. The microcompartments can have any shape suitable for cell encapsulation. Preferentially, the microcompartments according to the invention have a spherical or elongated shape. The microcompartments according to the invention can have an ovoid, cylindrical, spheroidal or spherical shape. The microcompartments according to the invention can in particular have the shape of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0097] The outer layer of the microcompartment, i.e. the hydrogel layer, gives the microcompartment according to the invention its size and shape. Preferentially, the minimum dimension of the microcompartment according to the invention is between 10 μm and 1 mm, preferentially between 100 μm and 700 μm. The minimum dimension of the microcompartment according to the invention may be between 200 μm and 600 μm, in particular between 300 μm and 500 μm.

[0098] Its largest dimension is preferentially greater than 10 μm, more preferentially between 10 μm and 1 m, even more preferentially between 10 μm and 50 cm.

[0099] Microcompartments according to the present invention may optionally be frozen for storage and must then be thawed before use.

[0100] The present invention also relates to a combination of multiple microcompartments.

[0101] Therefore, the present invention also relates to a set or series of cellular microcompartments as described above, comprising at least two cellular microcompartments according to the present invention.

[0102] The present invention also relates to an assembly or series of at least two three-dimensional cellular microcompartments, each microcompartment comprising at least one outer layer of hydrogel and at least one cell layer inside the outer layer, wherein at least one microcompartment is a microcompartment according to the present invention.

[0103] Preferably, the cells present in the microcompartments of a set of microcompartments according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles following encapsulation in an outer hydrogel layer of at least one cell per microcompartment. The microcompartments present in this set of microcompartments may have one or more characteristics (size, shape, cell number, cell volume, middle layer, lumen, etc.) of the microcompartments according to the invention.

[0104] The set of microcompartments according to the invention is preferentially comprised of 2 to 10 16 It contains microcompartments.

[0105] Preferably, the series of microcompartments according to the invention are in a culture medium, in particular in an at least partially convective culture medium.

[0106] According to a particularly preferred embodiment, the object of the present invention is a series of cell microcompartments in a closed chamber (e.g. a bioreactor), preferentially in a culture medium in a closed chamber (e.g. a bioreactor).

[0107] The presence of an outer layer of hydrogel and possibly an intermediate layer of isotonic aqueous solution allows for a uniform distribution of cells among the microcompartments. Furthermore, this hydrogel layer makes it possible to prevent the microcompartments from fusing, which is a major source of undesirable variability in the phenotypic homogeneity of the cells.

[0108] method The microcompartments may be obtained by any means known to those skilled in the art for preparing microcompartments or capsules.

[0109] According to another aspect, the present invention also relates to a method for preparing the microcompartments according to the invention.

[0110] The method for preparing a microcompartment or a set of microcompartments according to the invention comprises at least a. mixing the cells, optionally pre-incubated in culture medium, with a mixture of fibrinogen; b. encapsulating the mixture from step (a) in a hydrogel layer; c. culturing the capsules obtained in step (b) in a culture medium; d. Optionally, culturing the capsules resulting from step (c) for at least 1 day, preferentially 3 to 50 days, and optionally recovering the resulting cell microcompartments; A thrombin solution is added during steps b) and / or c).

[0111] Advantageously, the method according to the invention may comprise an additional step: thus, preferentially, the cells are incubated before the step of mixing the cells with the mixture of fibrinogen in a suitable culture medium, preferentially comprising at least one cytoprotective factor, and more preferentially at least one inhibitor of apoptosis.

[0112] The inhibitor of apoptosis can be, for example, one or more inhibitors of the RHO / ROCK (Rho-associated protein kinase) pathway, or any other inhibitor of apoptosis known to those skilled in the art. The inhibitor of apoptosis must be able to promote cell survival, cell adhesion to fibrin during the formation of the outer hydrogel layer.

[0113] The method according to the invention may comprise a step of dissociation of the cells by chemical, enzymatic or mechanical dissociation, carried out before or simultaneously with the cell incubation step which itself takes place before the mixing step a). This step is particularly important in the case of adherent cells.

[0114] The encapsulated cells are suspended in the form of single cells and / or cell clusters. Preferably, single cells account for less than 50% of the total number of encapsulated cells, and more preferentially, the single cells are hPSC cells. Indeed, it is preferred to encapsulate clusters of cells to reduce the occurrence of mutagenesis.

[0115] Preferentially, the steps following encapsulation are carried out under permanent or continuous agitation. This agitation is important because it maintains the homogeneity of the culture environment and prevents the formation of any diffusion gradients. For example, this agitation allows for homogeneous control of the cellular oxygenation level, thus avoiding necrosis associated with hypoxia or oxidative stress associated with hyperoxia. This agitation therefore avoids an increase in cell mortality and / or oxidative stress.

[0116] Preferentially, after the step of culturing the capsules obtained, the method comprises a step consisting of rinsing the capsules resulting from step (d), advantageously so as to eliminate cytoprotective factors such as inhibitors of apoptosis.

[0117] Preferentially, the encapsulation step b) comprises: i. a substep of contacting the mixture of step a), i.e. the mixture of cells and fibrinogen, with a solution of hydrogel to form at least one droplet; ii. collecting at least one of the obtained droplets, the inner part of each droplet consisting of the mixture of step i), in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0118] The outer hydrogel layer is hardened by a calcium bath to form the microcompartments, which can then be rinsed to remove, for example, inhibitors of apoptosis.

[0119] If the thrombin solution is added during the encapsulation step b), the thrombin solution may be added during the mixing step i) or the droplet collection step ii).

[0120] According to one object of the invention, a thrombin solution is mixed with the mixture of step a) and with the hydrogel solution, preferentially the thrombin is co-injected simultaneously with the other solutions. Preferentially, step i) consists of contacting the mixture of step a), the hydrogel solution and an isotonic intermediate solution containing said thrombin solution, more preferentially the isotonic intermediate solution being a sorbitol solution.

[0121] Particularly advantageously, the step of mixing the mixture and the hydrogel in step a) is a step aimed at structuring the mixture and the hydrogel solution in step a) in the form of linear and concentric flows.

[0122] Advantageously, the addition of thrombin during simultaneous co-injection allows for controlled polymerization in that the thrombin / fibrinogen contact time can be controlled, while the amount of thrombin added is preferentially 4-8 times less compared to the addition of thrombin solution after encapsulation.

[0123] According to another object of the present invention, a thrombin solution is added to the calcium bath during step ii), so that the thrombin solution can diffuse through the hydrogel shell of the microcompartments during hardening, thus polymerizing fibrinogen into fibrin. In this way, a fibrin mesh is formed on or within which cells can grow to form cysts.

[0124] When the method according to the invention includes a step of rinsing the obtained capsules, the solution constituting the calcium bath is removed and replaced by a culture medium suitable for culturing the microcompartments according to the invention, preferentially an isotonic solution, more preferentially containing an inhibitor of apoptosis. According to another object of the invention, this medium may contain a thrombin solution. Here again, the thrombin solution is able to diffuse through the hydrogel shell of the hardened microcompartments and polymerize fibrinogen to fibrin, constituting a fibrin mesh. According to another object of the invention, the thrombin solution is added during step c).

[0125] Also, in a particularly preferred manner, step b) of the method according to the invention is carried out by simultaneous co-injection of the hydrogel solution, the mixture of step a) and, optionally, the intermediate solution, said co-injection being carried out concentrically via a microfluidic or millifluidic injector which forms at the injector outlet a jet consisting of the mixture of said solutions, said jet breaking up into droplets.

[0126] If the thrombin solution is co-injected with other solutions, it is preferentially mixed with an isotonic intermediate solution.

[0127] Preferentially, the fibrinogen concentration is between 5 and 30 mg / mL, preferentially between 10 and 25 mg / mL, more preferentially between 14 and 20 mg / mL.

[0128] According to another object of the present invention, the concentration of thrombin is preferentially between 0.001 U / mL and 2 U / mL, more preferentially between 0.01 U / mL and 1 U / mL, between 0.01 U / mL and 0.05 U / mL, between 0.01 U / mL and 0.03 U / mL, and even more preferentially between 0.02 U / mL. "U" is understood to mean the enzyme activity unit (i.e., the enzyme concentration) that represents the amount of enzyme required to process micromole of substrate per minute. It is understood that the indicated concentrations are those in the mixture. In practice, advantageously, thrombin is mixed with the other components in a 1:1 ratio. Also, if the concentration of thrombin in the capsule before mixing is 0.01 U / mL, the concentration in the capsule will be approximately 0.01 U / mL.

[0129] The method according to the invention is carried out via a microfluidic injector that allows the co-injection of various solutions and the formation of a jet that breaks up into droplets. Preferentially, the final orifice diameter of the microfluidic injector is between 50 and 800 μm, more preferentially between 50 and 300 μm, even more preferentially between 80 and 240 μm, and the flow rate of each of the solutions is between 0.1 and 1000 mL / h, preferentially between 1 and 500 mL / h, more preferentially between 10 and 150 mL / h.

[0130] The method according to the invention is preferentially carried out in a closed chamber such as a closed bioreactor or flange.

[0131] The number of cell divisions in step (d) of culturing the capsules is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cell division cycles.

[0132] Preferentially, the microcompartments are obtained after at least two passages (a passage corresponding to a complete cycle of steps (a), (b) and (c), optionally (c)), more preferentially after at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage may last, for example, from 2 to 15 days, in particular from 3 to 8 days.

[0133] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the cells after step (d), i.e. at least two encapsulation cycles. Preferentially, each encapsulation cycle corresponds to a passage. In this variant of the method (at least one re-encapsulation of the cells after step (d)), the number of cell divisions over 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.

[0134] In the method according to the invention there may be multiple reencapsulations, preferentially 1 to 100, in particular 1 to 10 reencapsulations.

[0135] Each re-encapsulation - a step consisting of dissociating the microcompartment or a series of microcompartments to obtain a suspension of cells or a suspension of cell clusters, the outer layer of the hydrogel being eliminated, in particular by hydrolysis, dissolution, perforation and / or destruction by any biocompatible means, i.e., means that are not toxic to cells. For example, elimination can be achieved using phosphate-buffered saline, divalent ion chelators, enzymes, such as alginate lyase if the hydrogel contains alginate, and / or laser microdissection, and This can be achieved using a process of re-encapsulating all or part of the cells or cell clusters into hydrogel capsules.

[0136] Reencapsulation is a preferred means to increase cell expansion resulting from the pluripotency process and reduce the risk of mutations.

[0137] According to a particular embodiment, the reencapsulation comprises: - eliminating the outer layer of the hydrogel; - resuspending the cells contained in the microcompartments so as to obtain single cells and / or at least one set or cluster of cells in an isotonic culture medium, preferentially containing an inhibitor of apoptosis; - encapsulating the mixture in a hydrogel layer; - preferentially culturing the microcompartments obtained in an isotonic solution containing an inhibitor of apoptosis, preferentially in a culture medium containing an inhibitor of apoptosis; - Preferentially, rinsing the microcompartments so as to advantageously eliminate inhibitors of apoptosis; - culturing the microcompartments in an isotonic solution, preferentially a culture medium, for at least one cell division cycle; - optionally recovering the resulting cell micro-compartments.

[0138] use The use of fibrinogen and thrombin solutions that allow the formation of fibrin as a substitute for the extracellular matrix, in particular Matrigel®, is particularly suitable for three-dimensional cell culture, regardless of whether the cell culture is carried out by cell-containing microcompartments, tubes, or fibers.

[0139] Therefore, the present invention also relates to the use of a kit intended for three-dimensional culture, said kit comprising a fibrinogen solution and a thrombin solution.

[0140] In particular, the present invention provides a method for producing a cellular matrix comprising at least one cell layer and an outer layer of hydrogel; - Use of a kit comprising a fibrinogen solution and a thrombin solution to obtain cellular microcompartments having an outer layer of hydrogel and a fibrin mesh disposed between at least one cell layer.

[0141] If the kit is intended to be carried out on a tube or fiber containing cells, the preparation method may include: a. mixing the cells, optionally pre-incubated in culture medium, with a mixture of fibrinogen; b. coating the mixture from step (a) onto a hydrogel layer; c. culturing the fibers or tubes obtained in step (b) in a culture medium; The thrombin solution is added during steps b) and / or c).

[0142] Advantageously, the coating step b) is carried out by concentric flow, which is - a central flow (i) containing the mixture of cells and fibrinogen of step a), - an intermediate stream (ii) located further outside the central stream, which is optionally calcium-free and contains an isotonic solution, for example an isotonic sorbitol solution, which may optionally contain fibrinogen; an outermost flow (iii) relative to the intermediate flow, comprising a solution of a hydrogel, for example an alginate solution, optionally containing thrombin; and a further outer, calcium-free fluid (iv) comprising an isotonic fluid, for example an isotonic sorbitol solution, which may optionally contain fibrinogen.

[0143] According to another aspect, the invention relates to the use of a kit intended for the preparation of microcompartments according to the invention, said kit comprising a fibrinogen solution and a thrombin solution.

[0144] Preferentially, the fibrinogen and thrombin solutions are of human origin and comply with Good Manufacturing Practice (GMP) regulations.

[0145] Finally, the microcompartments are particularly suitable for use in a clinical environment.The present invention also relates to a microcompartment according to the invention or a set of microcompartments according to the invention for use as a medicament.

[0146] According to another aspect, the invention relates to the use of microcompartments according to one of the aforementioned purposes for the production of cells, tissues, preferentially for the large-scale production of such cells and / or tissues.

[0147] The microcompartments according to the invention can also be used for the production of animal or plant cells for human or animal food consumption, which is particularly useful for creating substitutes for meat products, such as meat, in order to limit the consumption of meat products.

[0148] According to another aspect, the present invention also relates to a kit comprising at least one fibrinogen solution, a thrombin solution, a hydrogel solution, preferentially alginate, an isotonic solution, preferentially a sorbitol solution, a calcium solution, a suitable culture medium. According to one variant, the kit is a kit of parts.

[0149] The invention will now be illustrated by non-limiting examples and results of compositions according to the invention. [Example]

[0150] Example 1 - Capsule according to the first embodiment. This example describes a first embodiment of the present invention, also shown in Figure 1, in which a thrombin solution is added to a sorbitol solution and co-injected with a mixture of cells in culture medium and a hydrogel solution via a microfluidic injector.

[0151] Therefore, the cells were mixed with cell culture medium and 14 mg / mL fibrinogen. The microfluidic injector, which allows for the co-injection of various solutions, contains three lines upstream of the nozzle. This solution containing fibrinogen was injected into the line corresponding to the cells, and encapsulation was performed. The other two lines contained a 2% alginate solution and an intermediate solution containing a sorbitol solution and 0.02 U / mL thrombin solution, respectively.

[0152] Once encapsulation occurred, the droplets were collected in a CaCl bath to allow the alginate to harden and form an alginate shell that formed the microcompartments or capsules. The solution containing the capsules was then rinsed with serum-free cell culture medium.

[0153] Example 2 - Capsules according to the second embodiment. This example describes a second embodiment of the invention, also shown in FIG. 2, in which a thrombin solution is added to a calcium bath and newly formed droplets are collected after jet fragmentation at the outlet of the microfluidic injector.

[0154] The cells were mixed with cell culture medium and 14 mg / mL fibrinogen, and this solution was injected into the corresponding upstream line of the microfluidic injector for encapsulation. The other two lines contained intermediate solutions, including a 2% alginate solution and a sorbitol solution, respectively.

[0155] Once encapsulated, the capsules are collected in a solution comprising a CaCl bath and supplemented with 0.02 U of thrombin solution, which is then rinsed with serum-free cell culture medium.

[0156] Example 3 - Capsules according to the second embodiment. This example describes a third embodiment of the invention, also shown in FIG. 3, in which a thrombin solution is added to the isotonic solution after rinsing the capsules obtained after hardening of the alginate by the action of a calcium bath.

[0157] The cells were mixed with cell culture medium and 14 mg / mL fibrinogen, and this solution was injected into the corresponding upstream line of the microfluidic injector for encapsulation. The other two lines contained intermediate solutions, including a 2% alginate solution and a sorbitol solution, respectively.

[0158] Once encapsulation was achieved, the capsules were collected in a solution comprising a CaCl2 bath, which was rinsed with serum-free cell culture medium and supplemented with 0.02 U of thrombin solution.

[0159] Example 4 - Comparison of capsules according to the invention with Matrigel®-based capsules. Comparisons were made between microcompartments of the prior art compared with microcompartments of the present invention in the presence of extracellular matrix, particularly Matrigel®, or in the absence of exogenous extracellular matrix or extracellular matrix substitutes. Matrigel® is the most efficient solution for obtaining cysts, but it is not suitable for clinical use in the production of differentiated cells. For example, the capsules can produce large amounts of neurons that can be injected into patients with neurodegenerative diseases, such as Alzheimer's disease. Therefore, the product directly obtained by the capsules of the present invention can be used in cell therapy. The capsules and cells thus obtained must comply with GMP regulations. However, Matrigel®, given its composition, cannot be used under GMP conditions. This problem is fully resolved by the fibrin mesh, as shown in the results below.

[0160] protocol In connection with this study, the inventors used iPS cell lines generated according to the usual standards for two-dimensional iPS culture, and then detached the cells from the flask via the action of enzymes and transferred them to a culture medium suitable for culturing iPS, according to the knowledge of those skilled in the art.

[0161] The iPS cells were mixed in a suitable culture medium containing a solution of 14 mg / mL fibrinogen to obtain a cell density of approximately 3 μM / mL. Thrombin solution was mixed in a sorbitol solution. The various solutions were then loaded via dedicated lines and co-injected simultaneously by a microfluidic injector. The amount of encapsulated cells was approximately 1.2 μM. * It is 10^6.

[0162] The same protocol was followed to obtain capsules without exogenous extracellular matrix and capsules containing Matrigel®.

[0163] Visually check the capsules from day 1 to day 5 after encapsulation. On day 5, observe the appearance of the cells, the amount of cells, their viability, and pluripotency.

[0164] result The results are presented in Figures 4, 5, 6, and 7.

[0165] 4 shows a capsule without exogenous extracellular matrix (A), a capsule with Matrigel® (B), a capsule according to the invention according to Example 2 (C), and a capsule according to the invention according to Example 1 (D). Phase contrast microscopy images were generated and the inventors observed that, compared to the prior art capsule based on Matrigel® (B) and the capsule without exogenous extracellular matrix (A), capsules (C) and (D) are obtained that contain at least one cell layer, an outer layer of hydrogel, and a fibrin mesh.

[0166] The inventors then characterized the resulting capsules. Figure 5 shows the results for the amplification of capsules according to the present invention, capsules in the presence of Matrigel®, and capsules in the absence of exogenous extracellular matrix. The inventors observed better amplification with fibrin than in the absence of exogenous, but lower, extracellular matrix compared to Matrigel®. However, the results obtained demonstrate that capsules based on a fibrin mesh allow for good capsule amplification and their use to produce cells.

[0167] The results in Figure 6 show the percentage of capsules containing cysts for capsules according to the invention, capsules in the presence of Matrigel®, and capsules without exogenous extracellular matrix. The inventors observed the presence of at least one cyst in approximately 60% of capsules according to the invention. Here again, the results obtained demonstrate that capsules based on a fibrin mesh make it possible to obtain cysts and therefore use them to produce cells.

[0168] The results in Figure 7 show the results regarding pluripotency for capsules according to the present invention, capsules in the presence of Matrigel®, and capsules in the absence of exogenous extracellular matrix. In this study, the inventors observed more cells positive for Oct4, Nanog, SSEA4, and SSEA5 (characteristic factors of iPS cells) in capsules containing fibrin than in those without exogenous extracellular matrix, thus demonstrating that fibrin-based capsules make it possible to maintain the pluripotency of cells and therefore their viability.

[0169] The results therefore demonstrate that fibrin presents a satisfactory alternative to extracellular matrix in the context of the present invention, albeit with slightly lower results than Matrigel®. In fact, fibrin is systematically superior to the use of capsules without exogenous extracellular matrix, allowing for good expansion, maintaining pluripotency, and allowing cysts to develop in a satisfactory manner.

[0170] Therefore, the use of fibrin, and in particular the use of a fibrin mesh, makes it possible to overcome the drawbacks of the prior art and allows the use of this three-dimensional microcompartment technology based on a fibrin mesh in a clinical setting related to cell therapy.

[0171] Example 5 - Capsules according to the invention relating to a protocol for differentiating iPS cells into neural tissue. This study aims to use microcompartments according to the invention in the context of a protocol for differentiating PS cells into neural tissue.

[0172] protocol Once the cell microcompartments according to the invention containing iPS cells are obtained, they undergo cell differentiation so as to differentiate them into neural cells according to the desired phenotype.

[0173] In connection with this study, the methods implemented will be adapted from Krik et al. ("Dopamine neurons derived from human ES cells efficiently engrafted in animal models of Parkinson's disease", Nature 2011 Nov 6;480(7378):547-51) and Nolbrant et al. ("Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation", Nature Protocols 2017).

[0174] The capsules containing iPS cells differentiated into neural tissue are then cultured for 24 days after encapsulation.

[0175] result The results are presented in Figures 8 and 9. Thus, we observed the presence of neural tissue 24 days after encapsulation from iPS cells. Panel A is an image representing a microcompartment according to the invention based on fibrin polymerized from 14 mg / mL fibrinogen, and panel B represents a prior art microcompartment based on Matrigel®.

[0176] Thus, we were able to observe the presence of neural tissue, especially neurospheres, of similar size in both fibrin-based and Matrigel®-based capsules. The neural tissue at 24 days indeed expresses tyrosine hydroxylase (TH), a specific marker of neural cells that constitute the neural tissue.

[0177] Finally, the inventors surprisingly observed that capsules according to the present invention contained neural cells with a more mature phenotype than those contained in capsules based on Matrigel® (outside the present invention). These results are presented in Figure 9. The inventors analyzed the expression of certain genes present in the cell populations of neural tissue cultured in capsules according to the present invention and in capsules based on Matrigel® at 24 days. A positive control based on the addition of dopaminergic neuronal precursors to the capsules was also included. The results show that the neural tissue present in capsules according to the present invention had a profile similar to that of the positive control, demonstrating the presence of a more mature phenotype.

[0178] Thus, these results clearly demonstrate that fibrin mesh is particularly suitable as an alternative to non-GMP extracellular matrix in the context of three-dimensional culture in cell microcompartments.

[0179] Example 6 - Large scale production of capsules according to the present invention The aim of this study is to demonstrate that the method is suitable for large-scale, even clinical, use, as it requires large amounts of cells and, consequently, the capsules that produce them.

[0180] protocol The protocol is identical to that of Example 4, except that a 20 mg / ml fibrinogen solution is used to obtain a cell density of approximately 0.85 M / ml. Finally, the concentration of the thrombin solution is 0.04 U / ml.

[0181] Three conditions were investigated in this study: cultivation in 2D flasks, cultivation in a small-scale bioreactor (30 mL), and cultivation in a large-scale bioreactor (500 mL).

[0182] result The capsules were visually checked from day 1 to day 5 after encapsulation. On day 7, the cells were observed for appearance, viability, pluripotency, and expansion rate. The results are presented in Table 1 below.

[0183] [Table 1]

[0184] The results demonstrate that the use of fibrin as an extracellular matrix substitute satisfies the technical problems of the present invention, including large-scale cultivation in suitable bioreactors. The capsules according to the present invention are therefore particularly suitable for use in clinical applications.

Claims

1. 1. A cell microcompartment comprising: a. at least one cell layer; b. an outer layer of hydrogel; c) a fibrin mesh disposed between the outer layer of the hydrogel and the cell layer.

2. The cellular microcompartment of claim 1 , wherein the fibrin mesh is intertwined with the outer layer of the hydrogel.

3. The cell microcompartment of claim 1 or 2, characterized in that the outer layer comprises alginate.

4. Cell microcompartments according to any one of claims 1 to 3, characterized in that they are closed.

5. The microcompartment according to any one of claims 1 to 4, wherein the microcompartment is a three-dimensional microcompartment.

6. The microcompartment according to any one of claims 1 to 5, characterized in that the microcompartment has an ovoid, cylindrical, spheroidal, spherical or teardrop shape.

7. The cell microcompartment according to any one of claims 1 to 6, wherein the cells constituting the cell layer are cells selected from eukaryotic cells, pluripotent cells, and differentiated cells.

8. The cell microcompartment according to any one of claims 1 to 7, characterized in that the microcompartment comprises a lumen.

9. The cellular microcompartment of claim 8 , wherein the cell layer, the fibrin mesh, and the outer layer are continuously organized around the lumen.

10. Microcompartments according to any one of claims 1 to 9, characterized in that the fibrin is obtained from the polymerization of fibrinogen by thrombin during and / or after encapsulation.

11. A set of microcompartments, characterized in that at least one microcompartment is a microcompartment according to any one of claims 1 to 10.

12. A micro-compartment according to any one of claims 1 to 10 or a set of micro-compartments according to claim 11 for its use as a medicament.

13. A method for preparing cellular microcompartments according to any one of claims 1 to 10, comprising: a. mixing the cells, optionally pre-incubated in culture medium, with a mixture of fibrinogen; b. encapsulating the mixture from step (a) in a hydrogel layer; c. Cultivating the capsules obtained in step (b) in a culture medium; Optionally, culturing the capsules resulting from step (c) for at least 1 day, preferentially between 3 and 50 days, and optionally recovering the resulting cell microcompartments, A method characterized in that a thrombin solution is added during steps (b) and / or (c).

14. Step (b) i. the substep of contacting the mixture of step (a) with a solution of hydrogel to form at least one droplet; ii. The method of claim 13, further comprising the substep of collecting at least one of the obtained droplets, the inner portion of each droplet consisting of the mixture of step (a), in a calcium bath capable of hardening the hydrogel solution to form an outer layer of each microcompartment.

15. 15. The method of claim 14, wherein the thrombin solution is added during step i) or ii).

16. 16. The method according to any one of claims 13 to 15, characterized in that step i) consists of contacting the mixture of step a), the hydrogel solution and an intermediate solution comprising the thrombin solution.

17. 15. The method according to claim 13 or 14, characterized in that the thrombin solution is added during step c).

18. Method according to any one of claims 13 to 17, characterized in that the concentration of fibrinogen is between 5 and 30 mg / mL, preferentially between 10 and 25 mg / mL.

19. 19. The method according to claim 18, wherein the concentration of fibrinogen is 14 to 20 mg / mL.

20. Method according to any one of claims 13 to 19, characterized in that the concentration of thrombin is between 0.001 and 2 U / mL, preferentially between 0.01 U / mL and 0.03 U / mL.

21. step b) is carried out by simultaneous co-injection of said hydrogel solution, said mixture from step a), and optionally said intermediate solution; 21. The method according to any one of claims 13 to 20, characterized in that the co-injection is performed concentrically via a microfluidic or microfluidic injector forming a jet of the mixture of the solutions at the injector outlet, the jet breaking up into droplets.

22. 22. The method of claim 21, characterized in that the final opening diameter of the microfluidic injector is between 50 and 800 μm, preferentially between 80 and 240 μm, and the flow rate of each of the solutions is between 0.1 and 1000 mL / h, preferentially between 10 and 150 mL / h.

23. Use of a kit intended for the preparation of microcompartments according to any one of claims 1 to 10, said kit comprising a fibrinogen solution and a thrombin solution.

24. 24. The use according to claim 23, wherein the fibrinogen solution and the thrombin solution are of human origin and comply with Good Manufacturing Practice (GMP) regulations.

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