Extracellular matrix substitute in a cellular microcompartment

Abstract

The invention relates to a cellular microcompartment that can be used clinically, termed "GMP", comprising at least: a. a cell layer, b. an external hydrogel layer, and c. a fibrin mesh placed between the external hydrogel layer and said cell layer. The invention also relates to a method for preparing the microcompartment, and also to the use of a kit comprising a fibrinogen solution and a thrombin solution, for preparing a cellular microcompartment.

Description

EXTRACELLULAR MATRIX SUBSTITUTE IN A CELLULAR MICROCOMPARTMENT

[0001] Technical field

[0002] The invention relates to the field of three-dimensional cell culture and concerns, in particular, cellular microcompartments for the production of cells and tissues suitable for clinical use for human and veterinary use, known as "GMP".

[0003] State of the art

[0004] Cell culture is a field that continues to attract growing interest since the discovery of induced pluripotent stem cells (iPS or iPSCs) by Prof. Yamanaka.

[0005] Historically, cells, including induced pluripotent stem cells, were cultured in two dimensions. Due to the limitations of two-dimensional cell culture, three-dimensional culture systems have been developed in recent years, partially overcoming the disadvantages of two-dimensional culture.

[0006] Indeed, such systems are advantageously closer to natural in vivo systems, and can be used for many applications, particularly in cell therapies. The cells cultured in these systems can be of any type. They can be differentiated cells with different phenotypes, progenitor cells, or stem cells.

[0007] A particularly suitable technology is that described in patent application WO 2018 / 096277 which consists of three-dimensional microcompartments for the culture of stem cells.

[0008] Although a highly promising technology, three-dimensional culture still suffers from certain drawbacks in order to be used in the clinic. To achieve this, the cells and the three-dimensional culture systems from which the cells are derived must comply with Good Manufacturing Practices (GMP) regulations. However, most three-dimensional culture systems, such as cell microcompartments, include an extracellular matrix, of animal origin and / or derived from cancer cell lines, which is incompatible with these regulations.

[0009] To consider the advent of cell therapies based on this technology but also the production of animal or plant cells for food consumption human or animal, there is therefore a need to develop an alternative, a substitute allowing us to do away with the presence of such an extracellular matrix, while retaining the ability for cells to adhere and grow satisfactorily.

[0010] As part of their work, the inventors surprisingly discovered that fibrin allowed particularly promising results to be obtained when used in cellular microcompartments, in that it allows in particular to obtain cells in large quantities, rapid growth, but also large-scale production of cellular microcompartments. Such results thus make it possible to envisage its clinical use in three-dimensional cellular microcompartments for human and veterinary use.

[0011] Also, to meet this need for a cellular microcompartment devoid of non-GMP extracellular matrix such as Matrigel®, the invention proposes a three-dimensional culture system based on a cellular microcompartment comprising a fibrin mesh arranged between an external hydrogel layer and at least one layer of cells.

[0012] Summary of the invention

[0013] Thus, the invention relates to a new cellular microcompartment comprising: - at least one layer of cells, - an external hydrogel layer, - a fibrin mesh arranged between the outer hydrogel layer and the at least one cell layer.

[0014] Advantageously, the fibrin mesh may or may not be segregated, i.e. entangled with at least one of the other constituents of the cellular microcompartment, such as the cells or the external hydrogel layer. Also, according to one embodiment, the cells may be distributed within the fibrin mesh and / or the fibrin mesh may be entangled in the external hydrogel layer. Preferably, the fibrin mesh is entangled in the external hydrogel layer.

[0015] When the fibrin mesh is entangled with the outer hydrogel layer, it can form an interpenetrating network or not. When it forms an interpenetrating network, it can be an interpenetrated polymer network (IPN)

[0016] When the fibrin mesh forms a distinct network, it is not entangled with at least one of the other constituents of the cellular microcompartment.

[0017] Advantageously, the fibrin mesh may comprise other molecules, for example growth factors, proteins, peptides, elements of the culture medium and / or resulting from the activity of the cells, for example secreted proteins, metabolites, etc.

[0018] According to another object, the hydrogel layer preferably comprises alginate.

[0019] Advantageously, the cells constituting the cell layer are chosen from eukaryotic cells, pluripotent cells and differentiated cells.

[0020] According to another preferred object, the microcompartment according to the invention comprises at least one of the following characteristics: - The micro-compartment is closed, and / or - the microcompartment is a 3-dimensional microcompartment, preferably a hollow 3-dimensional microcompartment, and / or - the microcompartment in the shape of an ovoid, a cylinder, a spheroid, a sphere or a teardrop, and / or - the microcompartment comprises one or more lumens inside said at least one layer of cells.

[0021] When the microcompartment comprises a lumen, the cell layer, the fibrin mesh and the outer layer are organized around the lumen. Preferably, the cell layer, the fibrin mesh and the outer layer are successively organized around the lumen.

[0022] According to a particularly preferred object, the fibrin included in the microcompartment according to the invention is obtained from the polymerization of fibrinogen by thrombin. Advantageously, the polymerization of fibrinogen by thrombin is obtained during encapsulation and / or after encapsulation.

[0023] According to another aspect, the invention also relates to a set of microcompartments, comprising at least one microcompartment according to the invention.

[0024] In the context of the invention, the microcompartment is particularly suitable in the context of a cell therapy protocol. Also, another aspect relates to the microcompartment according to the invention or the set of microcompartments according to the invention for its use as a medicament.

[0025] On the other hand, the microcompartment according to the invention or the set of microcompartments according to the invention can be obtained by any means known to those skilled in the art.

[0026] According to a particularly preferred aspect, the microcompartment according to the invention can be obtained according to the preparation method described below. Thus, the invention preferably relates to a preparation method comprising the following steps: a) mixing cells, optionally previously incubated in a culture medium with a fibrinogen mixture, 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, preferably from 3 to 50 days, and optionally recovering the cellular microcompartments obtained, characterized in that a thrombin solution is added during step b) and / or c).

[0027] Preferably, the method optionally comprises a step for rinsing the capsules resulting from step c), between step c) of culture and step d) of culture for at least 1 day.

[0028] Preferably, encapsulation step b) comprises the following sub-steps: i. bringing the mixture from step a) and a hydrogel solution into contact to form at least one drop, and ii. collecting said drop obtained in a calcium bath capable of stiffening the hydrogel solution to form the external layer of each microcompartment, the internal part of each drop being constituted by the mixture from step a) and optionally the thrombin solution.

[0029] More preferably, the thrombin solution can be added during step i) or ii). When the thrombin solution is added during step i), the drop obtained comprises an external layer and the internal part of each drop consists of the mixture from step a) and the thrombin solution. Preferably, the mixing from step i) is carried out during the co-injection by means of a microfluidic or millifluidic injector allowing the formation of the drop and the contacting of the thrombin resulting in the polymerization of the fibrinogen into fibrin.

[0030] When the thrombin solution is added in step ii), the thrombin solution is present in the calcium bath capable of stiffening the hydrogel solution to form the outer layer of each microcompartment. The thrombin solution then diffuses through the outer hydrogel layer allowing the polymerization of fibrinogen into fibrin.

[0031] Alternatively, the thrombin solution can be added after the formation of the microcompartment during step c) of capsule culture. The culture medium then comprises a thrombin solution and this diffuses through the hydrogel layer allowing the polymerization of fibrinogen into fibrin.

[0032] According to a particularly preferred object, step i) consists of bringing into contact the mixture from step a), the hydrogel solution, and an intermediate solution comprising said thrombin solution. By "intermediate solution", within the meaning of the invention, is meant a solution devoid of molecules capable of stiffening the hydrogel solution, and / or devoid of calcium. According to a particular embodiment, the intermediate solution is an isotonic intermediate solution. The thrombin solution is thus added during the formation of the drop.

[0033] Also, in a particularly preferred manner, step b), more preferably sub-step i), is carried out by simultaneous co-injection of the hydrogel solution, the mixture from step a) and optionally of said intermediate solution; said co-injection is carried out concentrically via a microfluidic or millifluidic injector forming a jet at the injector outlet consisting of the mixture of said solutions, said jet breaking up into drops.

[0034] In the context of the invention, the thrombin solution cannot be added before step b) of encapsulation corresponding to the formation of the drop.

[0035] Preferably, the concentration of fibrinogen is between 5 and 30 mg / mL, more preferably between 10 and 25 mg / mL. Even more preferably, the concentration of fibrinogen is between 14 and 20 mg / mL.

[0036] Preferably, the concentration of thrombin is between 0.001U / mL and 2U / ml, more preferably between 0.01U / mL and 1U / ml, between 0.01U / mL and 0.05U / ml, between 0.01U / ml and 0.03U / ml, even more preferably 0.02U / ml.

[0037] Preferably, the final opening diameter of the microfluidic injector is between 50 and 800 pm, more preferably between 50 and 300 pm, even more preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 0.1 and 1000 mL / h, preferably between 1 and 500 mL / h, more preferably between 10 and 150 mL / h. Even more preferably, the opening of the microfluidic injector is 100 pm or 215 pm and the flow rate of each of the solutions is between 23 mL / h and 100 mL / h.

[0038] The microcompartment according to the invention is suitable for clinical use. Also, one aspect of the invention relates to the microcompartment or microcompartment assembly according to the invention for its use as a medicament.

[0039] Finally, according to another aspect, the invention also relates to the use of a kit intended for the preparation of a microcompartment according to the invention, said kit comprising at least one fibrinogen solution and one thrombin solution. The invention therefore also relates to the use of a kit comprising at least one fibrinogen solution and one thrombin solution for the preparation of a microcompartment according to the invention.

[0040] According to another subject, the invention also relates to a kit comprising at least one fibrinogen solution, one thrombin solution, one hydrogel solution, preferably alginate, one isotonic solution, preferably one sorbitol solution, one calcium solution, one suitable culture medium. According to a variant, said kit is a kit-of-part.

[0041] Preferably, the fibrinogen solution and the thrombin solution are of human origin and comply with the regulations relating to Good Manufacturing Practices (GMP).

[0042] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow.

[0043] Brief description of the Figures

[0044] Figure 1 shows a first embodiment of the invention, in which the thrombin solution is mixed with sorbitol at the time of co-injection. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate, CS: Cell suspension and culture medium and fibrinogen. IS: Intermediate solution comprising sorbitol and thrombin at 0.02U. The l ère step involves the co-injection of the different constituents forming a jet, breaking up into drops in the CaCI2 bath, stiffening the external layer of the capsule. During a 2 ème step, the capsules are then resuspended in a rinsing medium. Finally, the capsules are resuspended in a final medium in flasks.

[0045] Figure 2 shows a second embodiment of the invention, in which the thrombin solution is added to the calcium bath used to collect the drops. form after fragmentation of the jet at the injector outlet. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate, CS: Cells in suspension and culture medium and fibrinogen. IS: Intermediate solution, i.e. sorbitol. The l ère This step involves the co-injection of the different constituents forming a jet, breaking up into drops in the CaCI2 bath supplemented with the 0.02U thrombin solution, allowing the polymerization of the fibrinogen and the stiffening of the external layer of the capsule, made of alginate. During a 2 ème step, the capsules are then resuspended in a rinsing medium. Finally, the capsules are resuspended in a final medium in flasks.

[0046] Figure 3 shows a first embodiment of the invention, in which the thrombin solution is added to the final culture medium. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate, CS: Cells in suspension and culture medium and fibrinogen. IS: Intermediate solution, i.e. sorbitol. The l ère step concerns the co-injection of the different constituents forming a jet, breaking up into drops in the CaCI2 bath, stiffening the external layer of the capsule, made of alginate. During a 2 ème step, the capsules are then resuspended in a rinsing medium. Finally, the capsules are resuspended in a final medium supplemented with the 0.02U thrombin solution in flasks, allowing the polymerization of fibrinogen.

[0047] Figure 4 is a phase contrast microscopy image at D5, representing capsules in the absence of exogenous extracellular matrix (A), in the presence of matrigel (B), capsules according to the invention according to the embodiment of [Figure 2] (C), and capsules according to the invention according to the embodiment of [Figure 1] (D).

[0048] Figure 5 represents the results relating to the amplification of the capsules according to the invention, of the capsules in the presence of matrigel, and of the capsules in the absence of exogenous extracellular matrix.

[0049] Figure 6 represents the results relating to the percentage of capsules comprising a cyst for the capsules according to the invention, the capsules in the presence of matrigel and the capsules devoid of exogenous extracellular matrix.

[0050] Figure 7 represents the results relating to pluripotency for the capsules according to the invention, the capsules in the presence of matrigel and in the absence of exogenous extracellular matrix.

[0051] Figure 8 is a phase contrast microscopy image at D17 post- encapsulation of neurons. Panel A represents neurospheres or neuronal micro-tissue included in the microcompartments according to the invention, namely fibrin polymerized from fibrinogen at 14 mg / ml, and panel B neurospheres or neuronal micro-tissue in microcompartments of the prior art comprising matrigel.

[0052] Figure 9 represents a Principal Component Analysis (PCA) of the 1000 most variable genes between iPSCs at day 0 and day 17, day 24 after neural differentiation in capsules seeded with fibrinogen or matrigel. Capsule-matured dopaminergic progenitors are used as a positive control.

[0053] Detailed description of the invention

[0054] Definition

[0055] For the purposes of the invention, the term "microcompartment" or "capsule" also means a partially or completely closed three-dimensional structure containing several cells. This is formed from a matrix of polymer chains, for example alginate, swollen by a liquid, preferably water. The structure is notably made up of a rigidified external hydrogel layer.

[0056] For the purposes of the invention, the term "drop" also means a three-dimensional structure formed from at least one liquid solution comprising the constituents of a non-rigidified hydrogel (polymerization precursors, non- or partially crosslinked polymer chains, etc.), hydrogel precursor elements. Also, the drop constitutes a transitional state between the co-injection of the different constituents and the microcompartment.

[0057] For the purposes of the invention, “differentiated” cells are understood to mean cells which have a particular phenotype, as opposed to pluripotent stem cells which are not differentiated or progenitor cells which are in the process of differentiating.

[0058] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention consist of or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0059] For the purposes of the invention, the term “mutant cell” means a cell carrying at least one mutation.

[0060] For the purposes of the invention, the term "progenitor cell" means a stem cell already engaged in cell differentiation but not yet differentiated.

[0061] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, embryonic stem cells of human beings can be excluded.

[0062] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. In particular, they may be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0063] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles 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).

[0064] For the purposes of the invention, the term "cell layer" or "cell base" means several cells forming a layer or base that can be structured around a lumen; this may be, for example, a cellular tissue or micro-tissue or a three-dimensional grouped culture. The thickness of the cell layer may be variable. This layer is organized in three dimensions in the microcompartment.

[0065] By "tissue" or "biological tissue" within the meaning of the invention, we mean the common meaning of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a collection of similar cells of the same origin (most often from a common cell lineage, although they can originate from the association of distinct cell lineages), grouped into clusters, networks, or bundles (fibers). A tissue forms a functional whole, meaning that its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs.

[0066] For the purposes of the invention, the term "fibrin mesh" or "fibrin network" means several fibrin fibers entangled with each other constituting a mesh or a network. These are optionally entangled with the internal face of the external layer of the hydrogel microcompartment.

[0067] By "light" or "lumen" within the meaning of the invention, we mean a volume of aqueous solution topologically surrounded by cells. Preferably, its contents are not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment.

[0068] Cellular microcompartment

[0069] The present invention therefore relates to a cellular microcompartment comprising cells, an external hydrogel layer and a fibrin mesh. The microcompartment according to the invention comprises at least one layer of cells. It is understood that the microcompartment may also comprise cells suspended in the medium or possibly housed in the fibrin mesh.

[0070] Also, the cellular microcompartment advantageously includes: - at least one layer of cells, - an external hydrogel layer, - a fibrin mesh arranged between the external hydrogel layer and said cell layer.

[0071] Preferably, the microcompartment is a three-dimensional microcompartment, delimited by the external hydrogel layer and within said external layer, said microcompartment comprises the cells and a fibrin mesh. It may be in the form of an ovoid, a cylinder, a spheroid, a sphere or a teardrop.

[0072] Advantageously, the three-dimensional microcompartment is hollow, more preferably, the hollow microcompartment is in the form of an ovoid, a cylinder, spheroid, sphere or teardrop.

[0073] Preferably, the hydrogel used is biocompatible, i.e. it is not toxic to the cells. The hydrogel layer must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival. According to one embodiment, the external hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be a sodium alginate, composed of 80% α-L-guluronate and 20% pD-mannuronate, with an average molecular mass of 100 to 400 kDa and a total concentration of between 0.5 and 5% by mass. Advantageously, the hydrogel layer is devoid of cells.

[0074] The hydrogel layer also helps protect cells from the external environment, limit uncontrolled cell proliferation, and their differentiation in the event of differentiation.

[0075] The cells present in the microcompartment can be any type of cell, in particular the cells are eukaryotic cells, advantageously they are mammalian cells. More preferably, the cells are human or animal cells.

[0076] In a particular embodiment, the microcompartment comprises pluripotent stem cells. A pluripotent stem cell, or pluripotent cell, is understood to mean a cell that has the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such. The pluripotent stem cells may in particular be induced pluripotent stem (iPS) cells, MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells. According to one embodiment, the microcompartment according to the invention does not comprise embryonic stem (ES) cells.

[0077] According to a particularly suitable variant of the invention, the microcompartment according to the invention comprises human or animal induced pluripotent stem cells.

[0078] In another particular embodiment, the microcompartment according to the invention comprises human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells. The multipotent and / or progenitor cells have preferably been obtained from pluripotent stem cells, in particular human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile has been artificially modified to match that of multipotent cells and / or progenitors. particular, typically by forced expression of transcription factors specific to the target cell phenotype. Preferably, the multipotent and / or progenitor cells were obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells.

[0079] According to another variant, the microcompartment according to the invention comprises differentiated human or animal cells. The differentiated cells have preferably been obtained from pluripotent stem cells or progenitor cells, in particular human pluripotent stem cells or human progenitor cells, or possibly from non-pluripotent human cells whose transcriptional profile has been artificially modified to match that of particular differentiated cells, typically by forced expression of transcription factors specific to the target cell phenotype. Preferably, the differentiated cells have been obtained from pluripotent or multipotent stem cells or progenitor cells after contact with a solution capable of initiating the differentiation of said stem cells.Alternatively, the cellular contents of the microcompartment comprise homogeneous or mixed cellular identities.

[0080] In particular, differentiated cells may be present as at least one layer of cells or as a three-dimensional tissue or microtissue or as several tissues or microtissues in the microcompartment. It may be a compacted or non-compacted tissue or microtissue, with or without a lumen.

[0081] The microcompartment according to the invention may comprise several types of cells. In particular, the microcompartment according to the invention may comprise, for example, stem cells induced to pluripotency and / or multipotent cells and / or progenitor cells and / or differentiated cells.

[0082] Advantageously, the microcompartment according to the invention is obtained after several cycles of cell division. Indeed, the cells included in the microcompartment according to the invention are cells obtained by amplification, from at least one cell.

[0083] Also, the cells present in the microcompartment according to the invention were obtained after at least two cycles of cell division after encapsulation in an external layer of hydrogel of at least one cell.

[0084] Preferably, the cells present in the microcompartment according to the invention were obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division after encapsulation in an external hydrogel layer of at least 1 cell, preferably between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 1 and 30, between 1 and 40, between 1 and 50, between 1 and 60, between 1 and 100 cells. For example, the cells present in the microcompartment were obtained after at least six cycles of cell division after encapsulation in an external hydrogel layer of at least 1 cell, preferably between 1 and 50 cells.

[0085] Preferably the microcompartment is obtained after at least 2 passages after encapsulation, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage can last for example at least 1 day, or between 2 and 50 days, in particular between 3 and 10 days.

[0086] Preferably, the microcompartment is obtained after at least one re-encapsulation, more preferably between 1 and 14 re-encapsulations, in particular between 2 and 7 re-encapsulations. Very preferably, one re-encapsulation corresponds to a new passage and each encapsulation cycle corresponds to one passage.

[0087] Preferably, all of the cells initially encapsulated in the microcompartment before the first cycle of cell division represent a volume 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.

[0088] Thus, according to one embodiment, the cells present in the microcompartment 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 cycles of cell division, after encapsulation in an external layer of hydrogel of cell(s) representing a volume 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.

[0089] Preferably, in the microcompartment according to the invention, the cells represent more than 50% by volume relative to the volume of the microcompartment, even more preferably more than 60%, 70%, 75%, 80%, 85%, 90% by volume relative to the volume of the microcompartment.

[0090] The microcompartment according to the invention comprises several cells, preferably at least 20 cells, even more preferably at least 100, at least 500, at least 1000, at least 10000.

[0091] In the context of the invention, the fibrin mesh is particularly suitable as a substitute for non-GMP extracellular matrices such as Matrigel®, and makes it possible to overcome the drawbacks of the prior art. Also, the fibrin mesh makes it possible to obtain satisfactory cell multiplication.

[0092] The fibrin mesh thus advantageously forms a fibrin network within the capsule, which may possibly constitute a fibrin gel or clot in the capsule. This mesh may be either interpenetrated with at least one of the other constituents of the microcompartment or not, preferably with the outer layer of the microcompartment. When the mesh is not interpenetrated, for example with the outer layer, it forms a distinct network in which the cells can lodge and multiply.

[0093] Preferably, the fibrin mesh is entangled with the outer hydrogel layer, more preferably the inner face of the outer hydrogel layer. Also, the delimitation between the fibrin mesh and the outer layer may not be perfectly clear. Consequently, at least a portion of the fibrin mesh may be entangled with the inner face of the outer layer, preferably with the alginate composing it. Thus, at least a portion of the mesh made of fibrin is preferentially entangled with the outer hydrogel layer.

[0094] According to another particular embodiment, the fibrin mesh forms an interpenetrated network, or “Interpenetrated Polymer Network” in English (IPN) with the external hydrogel layer.

[0095] According to a particularly preferred subject, the fibrin is obtained from the polymerization of fibrinogen by a fibrinogen polymerization agent, advantageously said agent is thrombin, during encapsulation and / or after encapsulation. Also, the polymerization of the fibrinogen solution by the thrombin solution takes place during encapsulation and / or after it. When it takes place after encapsulation, the polymerization takes place within the newly formed drop or capsule.

[0096] The fibrin mesh may optionally include a mixture of proteins and extracellular compounds necessary for the culture of differentiating cells as well as isolated cells.

[0097] Advantageously, the encapsulation is carried out by means of a co-injection carried out from concentrically via a microfluidic injector forming a jet at the injector outlet consisting of the mixture of the different useful solutions, said jet breaking up into drops. The drops are then collected in a calcium bath capable of stiffening the hydrogel solution to form the external layer of each microcompartment.

[0098] According to a first embodiment, the polymerization of the fibrinogen solution by thrombin takes place during encapsulation. The cell mixture, the fibrinogen mixture, the hydrogel solution and the thrombin solution are brought into contact simultaneously and co-injected concentrically via a microfluidic or millifluidic injector forming the jet at the injector outlet, splitting into drops. As soon as the different solutions come into contact, polymerization is initiated, which is then carried out almost instantly.

[0099] According to a second embodiment, the drops are collected in the calcium bath capable of stiffening the hydrogel solution to form the outer layer of each microcompartment. In the absence of thrombin solution during co-injection via the microfluidic injector, the polymerization of the fibrinogen is not initiated. Thus, the polymerization of the fibrinogen solution by the thrombin solution takes place after encapsulation. According to this object, the thrombin solution is added to the calcium bath allowing the collection and formation of the microcompartment. The thrombin solution can thus diffuse through the hydrogel solution being stiffened.

[0100] According to a third embodiment, the thrombin solution is not added to the calcium bath. Once the process of stiffening the hydrogel solution by 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 is then supplemented with a thrombin solution. The thrombin solution can then diffuse through the stiffened hydrogel shell, allowing the polymerization of fibrinogen by the thrombin.

[0101] According to a particularly preferred object, the thrombin solution is co-injected simultaneously with the cells, the fibrinogen solution, the culture medium comprising the cells and the hydrogel solution. More preferably, an isotonic solution is also co-injected and this comprises the thrombin solution, advantageously, the isotonic solution is a sorbitol solution.

[0102] According to any of the three embodiments described above, the Polymerization of fibrinogen by a fibrinogen polymerization agent, such as thrombin, provides a fibrin mesh within the capsule in which cells will lodge or bind to the surface of the mesh to multiply. The fibrin mesh can either form a separate network or form an interpenetrating network with at least one of the other constituents of the microcompartment, preferably the outer hydrogel layer.

[0103] The microcompartment according to the invention may also comprise other elements, in particular a culture medium.

[0104] The culture medium is a medium adapted to the cells present in the microcompartment according to the knowledge of the person skilled in the art.

[0105] According to another preferred object of the invention, the microcompartment comprises at least one light or lumen. The at least one light may contain a liquid, in particular culture medium and / or a liquid secreted by the cells. Advantageously, the presence of this hollow part allows the cells to have a small diffusive volume whose composition they can control, promoting cellular communication. This three-dimensional arrangement in a spherical cell monolayer or base surrounding the central light or lumen may also be called a cyst.

[0106] Light is preferentially generated, at the time of cyst formation, by cells which multiply and develop on or within the fibrin mesh.

[0107] According to another preferred object, the cell layer, the fibrin mesh and the outer layer are organized around the lumen, more preferably they are organized successively around the lumen.

[0108] The cyst-like conformation reduces the pressures on stem cells compared to 2D or aggregate cultures. This configuration also reduces cell mortality and increases the culture amplification factor. As a result, this reduces the number of passages and dissociations required; and reduces the culture time required to reach the final cell number required.

[0109] According to one embodiment, the microcompartment may comprise several cysts or tissue or microtissue.

[0110] The cellular microcompartment according to the invention is closed or partially closed, that is to say that the external layer is closed or partially closed. Preferably the microcompartment is closed.

[0111] The microcompartment according to the invention can be in any three-dimensional form, that is to say it can have the shape of any object in space. The microcompartment can have any shape compatible with the encapsulation of cells. Preferably, the microcompartment according to the invention is in a spherical or elongated 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.

[0112] 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 smallest dimension of the microcompartment according to the invention is between 10 μm and 1 mm, preferably between 100 μm and 700 μm. It may be between 200 μm and 600 μm, in particular between 300 μm and 500 μm.

[0113] Its largest dimension is preferably greater than 10 pm, more preferably between 10 pm and 1 m, even more preferably between 10 pm and 50 cm.

[0114] The microcompartment according to the invention can optionally be frozen for storage. It will then have to be defrosted before use.

[0115] The invention also relates to several microcompartments together.

[0116] Also, the invention also relates to a set or series of cellular microcompartments as described previously comprising at least two cellular microcompartments according to the invention.

[0117] The invention also relates to a set or series of microcompartments of at least two three-dimensional cellular microcompartments, each microcompartment comprising at least one external hydrogel layer and inside said external layer at least one layer of cells, in which at least one microcompartment is a microcompartment according to the invention.

[0118] Preferably, the cells present in the microcompartments of the 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 cycles of cell division after encapsulation in an external layer of hydrogel of at least 1 cell per microcompartment. The microcompartment(s) present in this set of microcompartments may have one or more characteristics of a microcompartment according to the invention (size, shape, number of cells, cell volume, intermediate layer, lumen, etc.).

[0119] The set of microcompartments according to the invention preferably comprises between 2 and 10 16 microcompartments.

[0120] Preferably, the series of microcompartments according to the invention is in a culture medium, in particular in an at least partially convective culture medium.

[0121] According to a particularly suitable embodiment, the invention relates to a series of cellular microcompartments in a closed enclosure, such as a bioreactor, preferably in a culture medium in a closed enclosure, such as a bioreactor.

[0122] The presence of an outer hydrogel layer and possibly an intermediate layer of isotonic aqueous solution allows for a uniform distribution of cells between the microcompartments. Furthermore, this hydrogel layer helps prevent microcompartment fusions, which are a major source of variability that is unfavorable for the phenotypic homogeneity of the cells.

[0123] Process

[0124] The microcompartment can be obtained by any means known to those skilled in the art for preparing microcompartments or capsules.

[0125] According to another aspect, the invention also relates to a method for preparing microcompartments according to the invention.

[0126] The method for preparing a microcompartment or a set of microcompartments according to the invention comprises at least the following steps: a. mixing cells, optionally previously incubated in a culture medium, with a fibrinogen mixture, b. encapsulating the mixture from step (a) in a hydrogel layer; c. cultivating the capsules obtained in step (b) in a culture medium, d. optionally, cultivating the capsules resulting from step (c) for at least 1 day, preferably from 3 to 50 days, and optionally, recovering the cell microcompartments obtained, characterized in that a thrombin solution is added during step b) and / or c).

[0127] Advantageously, the method according to the invention may comprise additional steps. Thus, preferably, the cells are incubated prior to the step of mixing the cells with the fibrinogen mixture in a suitable culture medium. Said culture medium preferably comprises at least one cytoprotective factor, more preferably at least one apoptosis inhibitor.

[0128] The apoptosis inhibitor may, for example, be one or more inhibitors of the RHO / ROCK (“Rho-associated protein kinase”) pathways, or any other apoptosis inhibitor known to those skilled in the art. The apoptosis inhibitor must promote cell survival and cell adhesion to fibrin at the time of formation of the external hydrogel layer.

[0129] The method according to the invention may comprise a step of dissociating the cells by chemical, enzymatic or mechanical dissociation, carried out before or simultaneously with the step of incubating the cells, itself carried out before step a) of mixing. This step is particularly important in the case of adherent cells.

[0130] The encapsulated cells are suspended in the form of single cells and / or cell clusters. Preferably, the single cells represent less than 50% in number of the total encapsulated cells, more preferably the single cells are hPSC cells. Indeed, it is preferable to encapsulate cell clusters because this reduces the occurrence of mutagenesis phenomena.

[0131] Preferably, the steps following encapsulation are carried out under permanent or sequential stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusive gradient. For example, it allows homogeneous control of the level of cellular oxygenation; thus avoiding the phenomena of necrosis linked to hypoxia, or oxidative stress linked to hyperoxia. Consequently, it avoids an increase in cell mortality and / or oxidative stress.

[0132] Preferably, after the step of culturing the capsules obtained, the method comprises a step which consists of rinsing the capsules resulting from step (d), advantageously so as to eliminate the cytoprotective factor, such as the apoptosis inhibitor.

[0133] Preferably, encapsulation step b) comprises the following sub-steps: i. bringing the mixture from step a), i.e. the cells and the fibrinogen mixture, into contact with the hydrogel solution to form at least one drop, and ii. collecting the at least one drop obtained in a calcium bath capable of stiffening the hydrogel solution to form the external layer of each microcompartment, the internal part of each drop being constituted by the mixture from step i).

[0134] Once the outer hydrogel layer has been stiffened by the calcium bath, the microcompartment is formed. This can then be rinsed, for example to remove the apoptosis inhibitor.

[0135] Where the thrombin solution is added in step b) of encapsulation, it can be added in step i) of mixing or ii) of collecting the drop.

[0136] According to an object of the invention, the thrombin solution is mixed with the mixture of step a), and the hydrogel solution, preferably the thrombin, is co-injected simultaneously with the other solutions. Preferably, step i) consists of bringing into contact the mixture of step a), the hydrogel solution, and an isotonic intermediate solution comprising said thrombin solution, more preferably the isotonic intermediate solution is a sorbitol solution.

[0137] Particularly advantageously, the step of mixing the mixture of step a) and the hydrogel is a step which aims to structure said mixture of step a) and the hydrogel solution in the form of a collinear and concentric flow.

[0138] Advantageously, the addition of thrombin during simultaneous co-injection allows polymerization to be controlled in that the thrombin / fibrinogen contact time can be controlled. On the other hand, the amount of thrombin added is less, preferably by a factor of 4 to 8, compared to the addition of the thrombin solution after encapsulation.

[0139] According to another object of the invention, the thrombin solution is added to the calcium bath, i.e. during step ii). The thrombin solution can thus diffuse through the hydrogel shell of the microcompartment being stiffened and thus polymerize the fibrinogen into fibrin. A fibrin mesh is thus formed on or in which the cells will multiply and form a cyst.

[0140] When the method according to the invention comprises a step of rinsing the capsules obtained, the solution constituting the calcium bath is removed and replaced by a medium suitable for the culture of the microcompartments according to the invention, preferably an isotonic solution, more preferably a culture medium containing an apoptosis inhibitor. This medium may, according to another object, comprise a thrombin solution. Here again, the thrombin solution can diffuse through the hydrogel shell of the stiffened microcompartment and polymerize the fibrinogen into fibrin, constituting the fibrin mesh. Also, according to another object of the invention, the thrombin solution is added during from step c).

[0141] Particularly preferably, step b) of the method according to the invention is carried out by simultaneous co-injection of the hydrogel solution, the mixture from step a) and optionally of said isotonic intermediate solution; said co-injection is carried out concentrically via a microfluidic injector forming a jet at the injector outlet consisting of the mixture of said solutions, said jet breaking up into drops.

[0142] When the thrombin solution is co-injected with the other solutions, it is preferentially mixed with the isotonic intermediate solution.

[0143] Preferably, the concentration of fibrinogen is between 5 and 30 mg / mL, preferably 10-25 mg / mL, more preferably between 14 and 20 mg / mL.

[0144] According to another object of the invention, the concentration of thrombin is preferably between 0.001U / mL and 2U / ml, more preferably between 0.01U / mL and 1U / ml, between 0.01U / mL and 0.05U / ml, between 0.01U / ml and 0.03U / ml, even more preferably 0.02U / ml. By "U" is meant a unit of enzymatic activity (i.e. the concentration for an enzyme) which represents the quantity of enzyme necessary to treat one micromole of substrate in 1 minute. It being understood that the concentration indicated is that in the mixture. Indeed, advantageously the thrombin is mixed with the other constituents according to a ratio of 1:1. Also, within the capsule, when the thrombin concentration, before mixing, is 0.01U / ml, the concentration in the capsule is of the order of 0.01U / ml.

[0145] The method according to the invention is implemented via a microfluidic injector allowing the co-injection of the different solutions and allowing the formation of a jet splitting into drops. Preferably, the final opening diameter of the microfluidic injector is between 50 and 800 pm, more preferably between 50 and 300 pm, even more preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 0.1 and 1000 mL / h, preferably between 1 and 500 mL / h more preferably between 10 and 150 mL / h.

[0146] The method according to the invention is preferably implemented in a closed enclosure such as a closed bioreactor or a flask.

[0147] The number of cell division cycles in step (d) of capsule culture is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cell division cycles.

[0148] Preferably the microcompartment is obtained after at least 2 passes (one passage corresponds to a complete cycle of steps (a), (b), and (c), optionally (c), more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage can last for example between 2 and 15 days, in particular between 3 and 8 days.

[0149] 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. 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 of the entire method (for all the passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0150] In a method according to the invention there may be several re-encapsulations, preferably between 1 and 100, in particular between 1 and 10 re-encapsulations.

[0151] Each re-encapsulation may include: - a step of dissociating the microcompartment or series of microcompartments to obtain a cell suspension or a cell cluster suspension; the removal of the external hydrogel layer may be achieved in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, the removal may be achieved using a saline phosphate buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection, and - a step of re-encapsulation of all or part of the cells or clusters of cells in a hydrogel capsule.

[0152] Re-encapsulation is a suitable means for increasing cellular amplification obtained from the pluripotent stage, and reducing the risks of mutation.

[0153] According to a particular embodiment, the re-encapsulation comprises the following steps: - remove the outer hydrogel layer, - resuspending the cells which were contained in the microcompartment so as to obtain single cells and / or at least one set or cluster of cells in an isotonic medium, preferably a culture medium containing an apoptosis inhibitor, - encapsulate the cell suspension in a hydrogel layer; - preferably, culturing the microcompartments obtained in an isotonic solution containing an apoptosis inhibitor, preferably a culture medium containing an apoptosis inhibitor; - preferably, rinse the microcompartments, advantageously, so as to eliminate the apoptosis inhibitor; - cultivate the microcompartments in an isotonic solution, preferably a culture medium, for at least one cell division cycle, and - optionally recover the cellular microcompartments obtained.

[0154] Use

[0155] The use of a fibrinogen solution and a thrombin solution allowing the formation of fibrin as a substitute for the extracellular matrix, in particular matrigel® is particularly suitable for three-dimensional cell culture, whether the cell culture is carried out by means of a cell microcompartment, a tube or a fiber comprising the cells.

[0156] Thus, the invention also relates to the use of a kit intended for three-dimensional culture, said kit comprising a fibrinogen solution and a thrombin solution.

[0157] In particular, the invention relates to the use of a kit comprising a fibrinogen solution and a thrombin solution, for obtaining cellular microcompartments at least one layer of cells, - an external hydrogel layer, - a fibrin mesh arranged between the outer hydrogel layer and the at least one cell layer.

[0158] When the kit is intended to be implemented in tubes or fibers comprising the cells, the preparation method may be as follows: a. mixing cells, optionally previously incubated in a culture medium, with a fibrinogen mixture, b. coating the mixture from step (a) in a layer of hydrogel; c. cultivating the fibers or tubes obtained in step (b) in a culture medium, characterized in that the thrombin solution is added during step b) and / or c).

[0159] Advantageously, coating step b) is carried out by means of a concentric flow. The concentric flow comprising: - a central flow (i) comprising the mixture of cells and fibrinogen from step a), - optionally an intermediate flow (ii) positioned more external to the central flow devoid of calcium and comprising an isotonic solution, for example an isotonic sorbitol solution which may optionally comprise fibrinogen, - a more external stream (iii) with respect to the intermediate stream comprising a hydrogel solution, for example an alginate solution, optionally comprising thrombin, and - even more externally, a flow (iv) devoid of calcium and comprising an isotonic solution, for example an isotonic sorbitol solution which may optionally comprise fibrinogen.

[0160] According to another aspect, the invention relates to the use of a kit intended for the preparation of a microcompartment according to the invention, said kit comprising a fibrinogen solution and a thrombin solution.

[0161] Preferably, the fibrinogen solution and the thrombin solution are of human origin and comply with the regulations relating to Good Manufacturing Practices (GMP).

[0162] Finally, the microcompartment is particularly suitable for clinical use. Also, the invention also relates to a microcompartment according to the invention or set of microcompartments according to the invention for its use as a medicament.

[0163] According to another aspect, the invention relates to the use of the microcompartment according to any of the preceding objects, for the production of cells, tissues, preferably for the production of such cells and / or tissues on a large scale.

[0164] The microcompartment according to the invention can also be used for the production of animal or plant cells for human or animal food consumption. This use is particularly useful for creating substitutes for meat products such as meat, with the aim of limiting the consumption of meat products.

[0165] According to another aspect, the invention also relates to a kit comprising at least one fibrinogen solution, one thrombin solution, one hydrogel solution, preferably alginate, one isotonic solution, preferably one sorbitol, a calcium solution, a suitable culture medium. According to one variant, said kit is a kit of part.

[0166] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results.

[0167] Examples

[0168] Example 1 - Capsule according to a first embodiment.

[0169] This example describes a first embodiment of the invention, also shown in [Figure 1], in which the thrombin solution is added into the sorbitol solution and co-injected via the microfluidic injector with the mixture of cells in the culture medium and with the hydrogel solution.

[0170] Thus, the cells were mixed with cell culture medium and 14 mg / mL fibrinogen. The microfluidic injector allowing the co-injection of the different solutions comprises three lines upstream of the nozzle. This solution comprising fibrinogen was injected into the line corresponding to the cells and encapsulation was carried out. The other two lines respectively comprising a 2% alginate solution, and an intermediate solution comprising the sorbitol solution and the 0.02U / ml thrombin solution.

[0171] Once encapsulation is complete, the drops are collected in the CaCl2 bath, allowing the alginate to stiffen and the alginate shell to form the microcompartment or capsule. This solution, including the capsules, was then rinsed with a serum-free cell culture medium.

[0172] Example 2 - Capsule according to a second embodiment.

[0173] This example describes a second embodiment of the invention, also shown in [Figure 2], in which the thrombin solution is added to the calcium bath, in which the newly formed drop after fragmentation of the jet at the outlet of the microfluidic injector will be collected.

[0174] The cells were mixed with cell culture medium and 14 mg / mL Fibrinogen, this solution was injected into the corresponding line upstream of the microfluidic injector and encapsulation was performed. The other two lines respectively included a 2% alginate solution, and an intermediate solution including the sorbitol solution.

[0175] Once the encapsulation is carried out, the capsules are collected in the solution constituting the CaCl2 bath and supplemented with the 0.02U Thrombin solution. This solution was rinsed with serum-free cell culture medium.

[0176] Example 3 - Capsule according to a third embodiment.

[0177] This example describes a third embodiment of the invention, also shown in [Figure 3], in which the thrombin solution is added to an isotonic solution after rinsing the capsules obtained after the stiffening of the alginate by the action of the calcium bath.

[0178] The cells were mixed with cell culture medium and 14 mg / mL Fibrinogen, this solution was injected into the corresponding line upstream of the microfluidic injector and encapsulation was performed. The other two lines respectively included a 2% alginate solution, and an intermediate solution including the sorbitol solution.

[0179] Once encapsulation is complete, the capsules are collected in the CaCl2 bath solution and this solution was rinsed with serum-free cell culture medium supplemented with 0.02U Thrombin solution.

[0180] Example 4 - Comparative results of capsules according to the invention and capsules based on matriaol®

[0181] Comparative results were carried out between microcompartments of the prior art in the presence of extracellular matrix, in particular matrigel®, or in the absence of exogenous extracellular matrix or extracellular matrix substitute, in comparison with the microcompartments according to the invention. Although matrigel® represents the most efficient solution for obtaining cysts, it is not suitable for the use of the capsules in the clinic, for example in the context of the production of differentiated cells. For example, the capsules can produce large quantities of neuronal cells capable of being injected into patients suffering from neurodegenerative diseases, for example Alzheimer's disease. Thus, the product directly obtained by the capsules according to the invention can be used in the context of cell therapy. The capsules and the cells obtained therefore require compliance with GMP regulations.However, given its composition, matrigel® cannot be used under GMP conditions. This problem is satisfactorily resolved with the fibrin mesh as shown in the results below.

[0182] Protocol

[0183] In this test, the inventors used an iPS cell line that was generated according to the usual standards of two-dimensional iPS culture, and then the cells They were detached from the flasks via the action of an enzyme, according to the knowledge of the person skilled in the art, and taken up in culture medium suitable for iPS culture.

[0184] The iPS cells were mixed in a suitable culture medium, including a 14 mg / ml fibrinogen solution, to obtain a cell density of around 3 M / ml. The thrombin solution was mixed in a sorbitol solution. The different solutions were then loaded via the dedicated lines and co-injected simultaneously using a microfluidic injector. The quantity of encapsulated cells is around 1.2*10 A 6.

[0185] The same protocol was implemented to obtain capsules devoid of exogenous extracellular matrix and capsules with matrigel®.

[0186] From D1 to D5 after encapsulation, the capsules are visually checked. On D5, the appearance of the cells, the quantity of cells, their viability and pluripotency are observed.

[0187] Results

[0188] The results presented in [Figure 4], [Figure 5], [Figure 6], and [Figure 7],

[0189] [Figure 4] represents capsules devoid of exogenous extracellular matrix (A), capsules comprising matrigel® (B), capsules according to the invention according to example 2 (C), and capsules according to the invention according to example 1 (D). Phase contrast microscopy images were generated and the inventors observed the production of capsules comprising at least one layer of cells, an external hydrogel layer and a fibrin mesh (C) and (D) in comparison with the capsules of the prior art based on matrigel® (B), and capsules without exogenous extracellular matrix (A).

[0190] The inventors then characterized the capsules obtained. The results in [Figure 5] represent the results relating to the amplification of the capsules according to the invention, of the capsules in the presence of matrigel®, and of the capsules in the absence of exogenous extracellular matrix. The inventors observed better amplification with fibrin compared to the absence of exogenous extracellular matrix but lower compared to matrigel®. However, the results obtained demonstrate that the capsules based on fibrin mesh allow good amplification of the capsules allowing their use to produce cells.

[0191] The results in [Figure 6] represent the percentage of capsules comprising a cyst, whether for the capsules according to the invention, the capsules in the presence of matrigel® and the capsules devoid of exogenous extracellular matrix. The inventors observed the presence of at least one cyst in approximately 60% of the capsules according to the invention. Here again, the The results obtained demonstrate that fibrin mesh capsules make it possible to obtain a cyst and therefore their uses for producing cells.

[0192] The results in [Figure 7] represent the results relating to pluripotency for the capsules according to the invention, the capsules in the presence of matrigel® and in the absence of exogenous extracellular matrix. In this test, the inventors observed more cells positive for Oct4, Nanog, SSEA4 and SSEA5 (factors characteristic of iPS cells) in the capsules comprising fibrin than those without exogenous extracellular matrix, thus demonstrating that the fibrin-based capsules make it possible to maintain the pluripotency of the cells and therefore viability.

[0193] The results thus demonstrate that fibrin is a satisfactory extracellular matrix substitute in the context of the invention, although its results are slightly inferior to matrigel®. Indeed, fibrin is systematically superior to the use of a capsule devoid of exogenous extracellular matrix and allows good amplification to be obtained, pluripotency to be preserved, and cysts to develop satisfactorily.

[0194] Thus, the use of fibrin, in particular the use of a fibrin mesh, makes it possible to overcome the drawbacks of the prior art, allowing the use of this three-dimensional microcompartment technology based on fibrin mesh, in the clinic in the context of cell therapies.

[0195] Example 5 - Capsule according to the invention in the context of a protocol for differentiating iPS cells into neuronal tissue.

[0196] This study aims to use the microcompartment according to the invention within the framework of a protocol for differentiation into neuronal tissue from iPS cells.

[0197] Protocol

[0198] Once the cellular microcompartments according to the invention are obtained comprising the iPS cells, these will undergo cellular differentiation so as to differentiate them into neuronal cells according to the desired phenotype.

[0199] In this trial, the method used is adapted from Kriks et al. (“Dopamine neurons derived from human ES cells efficiently engraft 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).

[0200] Capsules containing iPS cells then differentiated into neuronal tissue are cultured for 24 days after encapsulation.

[0201] Results

[0202] The results are presented in [Figure 8] and [Figure 9]. The inventors thus observed the presence of neuronal tissue 24 days after encapsulation from iPS cells. Panel A is an image representing the microcompartments according to the invention based on fibrin polymerized from fibrinogen at 14 mg / ml, and panel B represents microcompartments of the prior art based on matrigel®.

[0203] The inventors were thus able to observe the presence of neuronal tissue, in particular neurospheres having a similar size whether in the fibrin-based capsules or the matrigel®-based capsules. The neuronal tissue at D24 clearly expresses tyrosine hydroxylase (TH), a specific marker of neuronal cells constituting neuronal tissue.

[0204] Finally, the inventors surprisingly observed that the capsules according to the invention comprise neuronal cells with a more mature phenotype than those included in the matrigel®-based capsules (Outside the Invention). These results are presented in [Figure 9]. The inventors analyzed the expression of certain genes present in the cell population of neuronal tissue at D24 cultured in the capsules of the invention and the matrigel®-based capsules. A positive control based on the addition of dopaminergic neuron progenitors in capsules was also added. The results show that the neuronal tissue present in the capsules of the invention has a profile close to that of the positive control, demonstrating the presence of a more mature phenotype.

[0205] Also, the results clearly demonstrate that a fibrin mesh is particularly suitable as a substitute for non-GMP extracellular matrix in the context of three-dimensional culture in cellular microcompartments.

[0206] Example 6 - Production of capsules according to the invention on a large scale

[0207] This study aims to demonstrate that the capsule according to the invention is also suitable for use on a large scale, with a view to clinical use requiring large quantities of cells, and consequently of capsules producing them.

[0208] Protocol

[0209] The protocol is identical to that of example 4, except that a 20 mg / ml fibrinogen solution is used, so as to obtain a cell density of around 0.85 M / ml. Finally, the concentration of the thrombin solution is 0.04 U / ml.

[0210] Three conditions were studied, namely a 2D flask culture condition, a culture condition in a small bioreactor (small scale, 30mL), and a culture condition in a large bioreactor (large scale, 500mL).

[0211] Results

[0212] From D1 to D5 after encapsulation, the capsules are visually checked. On D7, the appearance of the cells, their viability, pluripotency and amplification factor are also observed. The results are presented in Table 1 below.

[0213] [Table 1] extracellular addresses the technical problem of the present invention, including in large-scale culture in suitable bioreactors. Therefore, said capsules according to the invention are particularly suitable for use in clinical applications.

Claims

Claims

1. A cellular microcompartment comprising: a. at least one cell layer, b. an outer hydrogel layer, c. a fibrin mesh arranged between the outer hydrogel layer and said cell layer.

2. A cellular microcompartment according to the preceding claim, wherein the fibrin mesh is entangled with the outer hydrogel layer.

3. Cellular microcompartment according to one of the preceding claims, characterized in that the outer layer comprises alginate.

4. Cellular microcompartment according to one of the preceding claims, characterized in that the microcompartment is closed.

5. Microcompartment according to one of the preceding claims, characterized in that the microcompartment is a three-dimensional microcompartment.

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

7. Cellular microcompartment according to one of the preceding claims, characterized in that the cells constituting the cell layer are cells chosen from eukaryotic cells, pluripotent cells, and differentiated cells.

8. Cellular microcompartment according to one of the preceding claims, characterized in that the microcompartment comprises a lumen.

9. Cellular microcompartment according to the preceding claim, characterized in that the cell layer, the fibrin mesh and the external layer are successively organized around the lumen.

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

11. Set of microcompartments, characterized in that at least one microcompartment is a microcompartment according to one of the preceding claims.

12. Microcompartment according to one of claims 1 to 10 or set of microcompartments according to claim 11 for its use as a medicament.

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

14. Method according to the preceding claim, characterized in that step (b) comprises the following sub-steps: i. bringing the mixture of step (a) and a hydrogel solution into contact to form at least one drop, and ii. collecting the at least one drop obtained in a calcium bath capable of stiffening the hydrogel solution to form the external layer of each microcompartment, the internal part of each drop being constituted by the mixture of step (a).

15. Method according to the preceding claim, characterized in that the thrombin solution is added during step i) or ii).

16. Method according to one of claims 13 to 15, characterized in that step i) consists of bringing into contact the mixture of step a), the hydrogel solution, and an intermediate solution comprising said thrombin solution.

17. Method according to one of claims 13 or 14, characterized in that the thrombin solution is added during step c).

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

19. Method according to the preceding claim, characterized in that the concentration of fibrinogen is between 14 and 20 mg / mL.

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

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

22. Method according to the preceding claim, characterized in that the final opening diameter of the microfluidic injector is between 50 and 800 pm, preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 0.1 and 1000 mL / h, preferably between 10 and 150 mL / h.

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

24. Use according to the preceding claim, characterized in that the fibrinogen solution and the thrombin solution are of human origin and comply with the regulations relating to Good Manufacturing Practices (GMP).

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