WO2024033284A1 - Extracellular matrix substitute in a cellular microcompartment - Google Patents

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 capable of being used in the clinic for human and veterinary use, known as “GMP”. .

[0003] State of the art

[0004] Cell culture is a field which continues to arouse increasing interest since the discovery of induced pluripotent stem cells (iPS or iPSCs) by Professor Yamanaka.

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, making it possible to partially overcome the disadvantages of two-dimensional culture. [0006] Indeed, such systems are advantageously closer to natural in vivo systems, and can be used for numerous applications, in particular in cell therapies. The cells cultured in these systems can be of any type. These 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 being a highly promising technology, three-dimensional culture still suffers from certain drawbacks in order to be able to be used in the clinic. To do this, the cells and the three-dimensional culture systems from which the cells are derived must comply with regulations relating to Good Manufacturing Practices (GMP). However, most three-dimensional culture systems, such as cellular microcompartments, include an extracellular matrix, of animal origin and/or from cancer cell lines, incompatible with this regulation.

[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 making it possible to overcome the presence of such an extracellular matrix, while retaining the possibility of cells adhering and growing satisfactorily.

[0010] In the course of their work, the inventors surprisingly discovered that fibrin made it possible to obtain particularly promising results when used in cellular microcompartments, in that it makes it possible in particular to obtain cells in large quantity, rapid growth, but also large-scale production of cellular microcompartments. Such results thus make it possible to consider 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 layer in hydrogel 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 outer hydrogel layer,

- a fibrin mesh arranged between the external hydrogel layer and the at least one cell layer.

Advantageously, the fibrin mesh may or may not be segregated, that is to say 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 can be distributed inside the fibrin mesh and/or the fibrin mesh can be entangled in the outer hydrogel layer. Preferably, the fibrin mesh is entangled in the outer hydrogel layer.

[0015] When the fibrin mesh is entangled with the external hydrogel layer, the latter can form an interpenetrating network or not. When it forms an interpenetrating network, this can be an interpenetrated polymer network or “Interpenetrated Polymer Network” in English (IPN) 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 proteins. secreted, metabolites etc.

According to another object, the hydrogel layer preferably comprises alginate. Advantageously the cells constituting the layer of cells 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 microcompartment 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.

When the microcompartment comprises a lumen, the layer of cells, 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.

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.

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 cell therapy protocols. 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 medication. [0025] Furthermore, 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 process described below. Thus, the invention preferentially relates to a preparation process comprising the following steps: a) mixing cells, optionally previously incubated in a culture medium with a mixture of fibrinogen, b) encapsulating the mixture of step a) in a layer of hydrogel, c) cultivate the capsules obtained in step b) in a culture medium, d) optionally, cultivate the capsules resulting from step c) for at least 1 day, preferably from 3 to 50 days , and optionally recover the cellular microcompartments obtained, characterized in that a thrombin solution is added during step b) and/or c). Preferably, the method optionally comprises a step for rinsing the capsules from step c), between culture step c) and culture step d) for at least 1 day.

Preferably, encapsulation step b) comprises the following sub-steps: i. bringing the mixture from step a) into contact with a hydrogel solution to form at least one drop, and ii. collect 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 of step a) and possibly the thrombin solution .

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 is constituted by the mixture of step a) and the thrombin solution. Preferably, the mixture of step i) is carried out during co-injection by means of a microfluidic or millifluidic injector allowing the formation of the drop and bringing the thrombin into contact, resulting in the polymerization of fibrinogen into fibrin. When the thrombin solution is added during 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. Alternatively, the thrombin solution can be added after the formation of the microcompartment during step c) of culturing the capsules. The culture medium then includes a thrombin solution and this diffuses through the hydrogel layer allowing the polymerization of fibrinogen into fibrin.

According to a particularly preferred object, step i) consists of bringing the mixture of step a), the hydrogel solution, and an intermediate solution comprising said thrombin solution into contact. 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, of the mixture of 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 encapsulation step b) corresponding to the formation of the drop.

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 included 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. The microcompartment according to the invention is suitable for use in the clinic. Also, one aspect of the invention relates to the microcompartment or the set of microcompartments according to the invention for its use as a medicine.

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

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

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 represents a first embodiment of the invention, during which the thrombin solution is mixed with the sorbitol at the time of co-injection. 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 including sorbitol and thrombin at 0.02U. The ^first step concerns the co-injection of the different constituents forming a jet, breaking up into drops in the CaCl2 bath, stiffening the outer layer of the capsule. During a ^2nd step, the capsules are then resuspended in a rinsing medium. Finally, the capsules are resuspended in a final medium in flasks.

[0045] Figure 2 represents a second embodiment of the invention, during which the thrombin solution is added to the calcium bath, used to collect the drops form after the 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 ^first step concerns the co-injection of the different constituents forming a jet, breaking up into drops in the CaCl2 bath supplemented with the 0.02U thrombin solution, allowing the polymerization of fibrinogen and the stiffening of the outer layer of the capsule. , made of alginate. During a ^2nd step, the capsules are then resuspended in a rinsing medium. Finally, the capsules are resuspended in a final medium in flasks.

[0046] Figure 3 represents a first embodiment of the invention, during 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 ^first step concerns the co-injection of the different constituents forming a jet, breaking up into drops in the CaCl2 bath, stiffening the outer layer of the capsule, made up of alginate. During a ^2nd 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 represents neurospheres or neuronal micro-tissue in microcompartments of the prior art comprising matrigel.

[0052] Figure 9 represents a principal component analysis (in English “Principal Component Analysis” PCA), of the 1000 most variable genes between the iPSCs at day 0 and at day 7, day 24 after neural differentiation in capsules seeded with fibrinogen or matrigel. Dopaminergic progenitors matured in capsules are used as a positive control.

[0053] Detailed description of the invention

[0054] Definition

[0055] By “microcompartment” or “capsule” within the meaning of the invention, we also mean a partially or completely closed three-dimensional structure, containing several cells. This is formed from a matrix of polymer chains, for example alginate, swollen with a liquid and preferably water. The structure is notably made up of an external layer of stiffened hydrogel.

[0056] By “drop” within the meaning of the invention, we also mean a three-dimensional structure formed from at least one liquid solution comprising the constituents of a non-rigidified hydrogel (polymerization precursors, polymer chains not or partially crosslinked...), hydrogel precursor elements. Also, the drop constitutes a transient state between the co-injection of the different constituents and the microcompartment.

[0057] By “differentiated” cells within the meaning of the invention we mean cells which present a particular phenotype, as opposed to pluripotent stem cells which are not differentiated or progenitor cells which are in the process of differentiation.

[0058] By “human cells” within the meaning of the invention is meant human cells or immunologically humanized non-human mammalian cells. Even when this is not specified, the cells, stem cells, progenitor cells and tissues according to the invention are constituted or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0059] By “mutant cell” within the meaning of the invention, we mean a cell carrying at least one mutation.

[0060] By “progenitor cell” within the meaning of the invention, we mean a stem cell already engaged in cell differentiation but not yet differentiated.

[0061] By “embryonic stem cell” within the meaning of the invention is meant a pluripotent stem cell of a cell derived from the internal cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA3/4, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destruction of the embryo from which they are derived, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Possibly human embryonic stem cells may be excluded.

[0062] By “pluripotent stem cell” or “pluripotent cell” within the meaning of the invention, we mean a cell which has the capacity to form all the tissues present in the entire original organism, without however being able to form a entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. These may in particular be induced pluripotent stem cells (iPSC or hiPSC for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for “Multilineage-differentiating Stress Enduring”). [0063] By “induced pluripotent stem cell” within the meaning of the invention is meant a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are notably positive for markers of pluripotency, such as alkaline phosphatase staining and expression of NANOG, SOX2, OCT4 and SSEA3/4 proteins. 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] By “layer of cells” or “seat of cells” in the sense of the invention, we mean several cells forming a layer or a seat which can be structured around a light, it may for example be a tissue or a cellular micro-tissue or a three-dimensional pooled culture. The thickness of the layer of cells can 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 sense of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a set of similar cells of the same origin (most often coming from a common cell lineage, although they can find their origin by association of distinct cell lineages), grouped in clusters, network or bundle (fiber ). A tissue forms a functional whole, that is to say that its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs.

[0066] By “fibrin mesh” or “fibrin network” within the meaning of the invention, we mean several fibrin fibers entangled together constituting a mesh or a network. These are possibly entangled with the internal face of the external layer of the hydrogel microcompartment.

[0067] By “light” or “lumen” in the sense of the invention, we mean a volume of aqueous solution topologically surrounded by cells. Preferably its content is not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment.

[0068] Cellular microcompartment

The subject of the present invention is therefore 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 being understood that the microcompartment can also include cells suspended in the medium or possibly housed in the fibrin mesh.

[0070] Also, the cellular microcompartment advantageously comprises:

- at least one layer of cells,

- an outer hydrogel layer,

- a fibrin mesh arranged between the external hydrogel layer and said cell layer.

Preferably, the microcompartment is a three-dimensional microcompartment, delimited by the external hydrogel layer and inside said external layer, said microcompartment comprises the cells and a fibrin mesh. It can 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, a spheroid, a sphere or a teardrop.

[0073] Preferably the hydrogel used is biocompatible, that is to say it is not toxic to cells. The hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartment and allow their survival. According to one embodiment, the external hydrogel layer comprises at least alginate. It can consist exclusively of alginate. The alginate may in particular be a sodium alginate, composed of 80% a-L-guluronate and 20% p-D-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 makes it possible to protect the cells from the external environment, to limit the uncontrolled proliferation of cells, and their differentiation in the event of differentiation. 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, 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. Pluripotent stem cells may in particular be induced pluripotent stem cells (iPS), MUSE (“Multilineage-differentiating Stress Enduring”) cells found in the skin and bone marrow of adult mammals, or embryonic stem cells. (ES). According to one embodiment, the 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 multipotent human or animal 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 join that of multipotent cells and /or progenitors particular, typically by forced expression of transcription factors specific to the target cellular 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 preferentially been obtained from pluripotent stem cells or progenitor cells, in particular from human pluripotent stem cells or human progenitor cells, or possibly from non-pluripotent human cells whose transcriptional profile has been artificially modified to join that of particular differentiated cells, typically by forced expression of transcription factors specific to the target cellular phenotype. Preferably, the differentiated cells were obtained from pluripotent or multipotent stem cells or progenitors after contact with a solution capable of initiating the differentiation of said stem cells. According to one variant, the cellular content of the microcompartment comprises homogeneous or mixed cellular identities.

The differentiated cells may in particular be in the form of at least one layer of cells or in the form of a three-dimensional tissue or micro-tissue or in the form of several tissues or micro-tissues in the microcompartment. It may be a tissue or micro-tissue, compacted or not, with or without light.

[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.

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 hydrogel layer 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 cells, 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.

Preferably the microcompartment is obtained after at least 2 passes after encapsulation, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passes. 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, a re-encapsulation corresponds to a new passage and each encapsulation cycle corresponds to a 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 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 outer 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.

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 10,000. [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 the multiplication of cells in a satisfactory manner.

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

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

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

[0095] According to a particularly preferred object, 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, polymerization takes place within the newly formed drop or capsule.

The fibrin mesh may optionally comprise a mixture of proteins and extracellular compounds necessary for the culture of cells undergoing differentiation as well as isolated cells.

[0097] Advantageously, the encapsulation is carried out by means of a co-injection carried out with concentrically via a microfluidic injector forming a jet at the injector outlet consisting of the mixture of 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 outer layer of each microcompartment.

[0098] According to a first embodiment, the polymerization of the fibrinogen solution by thrombin takes place during encapsulation. The mixture of cells, the mixture of fibrinogen, 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, fibrinogen polymerization 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 during stiffening.

[0100] According to a third embodiment, the thrombin solution is not added to the calcium bath. From then on, the process of stiffening the hydrogel solution by the calcium bath completed, the microcompartments formed 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 polymerization of fibrinogen by 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, makes it possible to obtain a fibrin mesh within the capsule in which the cells will lodge or bind to the surface of the mesh to multiply. The fibrin mesh can either form a distinct network or form an interpenetrating network with at least one of the other constituents of the microcompartment, preferably the external hydrogel layer. [0103] The microcompartment according to the invention may also include 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 those skilled in the art.

[0105] According to another preferred object of the invention, the microcompartment comprises at least one lumen or one lumen. The at least one lumen 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 monolayer or spherical cell base surrounding the central lumen or lumen can also be called a cyst.

[0106] The light is preferentially generated, at the time of formation of the cyst, by the 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 external layer are organized around the lumen, more preferably they are organized successively around the lumen.

[0108] The conformation in the form of a cyst makes it possible to reduce the pressures experienced by the stem cells compared to 2D or aggregate cultures. This configuration also makes it possible to reduce cell mortality and increase the culture amplification factor. As a result, this makes it possible to reduce the number of passages and dissociation required; to reduce the culture time needed to reach the final number of cells needed.

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

[0110] The cellular microcompartment according to the invention is closed or partially closed, that is to say that the outer 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 may have any shape compatible with cell encapsulation. Preferably the microcompartment according to the invention is in a spherical or elongated shape. It can have the shape of an ovoid, a cylinder, a spheroid or a sphere. It may in particular be in the form of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0112] It is the external layer of the microcompartment, that is to say the hydrogel layer, which gives its size and shape to the microcompartment according to the invention. Preferably the smallest dimension of the microcompartment according to the invention is between 10 pm and 1 mm, preferably between 100 pm and 700 pm. It can be between 200 pm and 600 pm, in particular between 300 pm and 500 pm.

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

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

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

[0116] Also, the invention also targets a set or series of cellular microcompartments as described above 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 cellular microcompartments in three dimensions, each microcompartment comprising at least one outer layer of hydrogel and inside said outer 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 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 outer hydrogel layer 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, volume of cells, intermediate layer, light, 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 subject of the invention is 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 external layer of hydrogel and possibly an intermediate layer of isotonic aqueous solution allows uniform distribution of cells between the microcompartments. Furthermore, this hydrogel layer makes it possible to avoid fusions of microcompartments which are a major source of unfavorable variability for the phenotypic homogeneity of 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 process for preparing microcompartments according to the invention.

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

[0127] Advantageously, the method according to the invention may comprise additional steps. Thus, preferentially, 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 can for example be one or more inhibitor(s) of the RHO/ROCK (“Rho-associated protein kinase”) pathways, or any other apoptosis inhibitor known to man. of career. The apoptosis inhibitor must promote cell survival and cell adhesion to fibrin at the time of formation of the outer hydrogel layer.

[0129] The method according to the invention may comprise a step of dissociating the cells by chemical, enzymatic or mechanical dissociation, prior to or simultaneously implemented in the step of incubation of the cells, itself carried out prior to the step a) 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 clusters of cells. Preferably, the single cells represent less than 50% by number of all the encapsulated cells, more preferably the single cells are hPSC cells. In fact, it is preferable to encapsulate clusters of cells because this reduces the occurrence of mutagenesis phenomena.

[0131] Preferably, the steps subsequent to encapsulation are carried out with permanent or sequential stirring. This agitation 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 cellular oxygenation level; thus avoiding the phenomena of necrosis linked to hypoxia, or oxidative stress linked to hyperoxia. Consequently, it avoids an increase in cell death 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 inhibitor of apoptosis.

[0133] Preferably, encapsulation step b) comprises the following sub-steps: i. bringing the mixture from step a), that is to say the cells and the fibrinogen mixture, into contact with the hydrogel solution to form at least one drop, and ii. collect 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 i). [0134] Once the external hydrogel layer has been stiffened by the calcium bath, the microcompartment is formed. This can then be rinsed, in order to eliminate, for example, the apoptosis inhibitor.

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

[0136] According to one 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 in the form of a collinear and concentric flow said mixture of step a) and the hydrogel solution.

[0138] Advantageously, the addition of thrombin during simultaneous co-injection makes it possible to control the polymerization in that the thrombin/fibrinogen contact time can be controlled. On the other hand, the quantity of thrombin added is less significant, 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 process according to the invention comprises a step of rinsing the capsules obtained, the solution constituting the calcium bath is eliminated and replaced by a medium suitable for culturing the microcompartments according to the invention, preferably an isotonic solution , more preferably a culture medium containing an apoptosis inhibitor. This medium may, depending on another subject, comprise a thrombin solution. Again, the thrombin solution can diffuse through the stiffened microcompartment hydrogel shell and polymerize fibrinogen into fibrin, constituting the fibrin meshwork. Also, according to another object of the invention, the thrombin solution is added when of step c).

[0141] Particularly preferably, step b) of the process according to the invention is carried out by simultaneous co-injection of the hydrogel solution, of the mixture of 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” we mean a unit of enzymatic activity (i.e. the concentration for an enzyme) which represents the quantity of enzyme needed to process 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 in a 1:1 ratio. Also, within the capsule, when the concentration of thrombin, 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 a drop. 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 culturing the capsules is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15 cycles of cell division.

[0148] Preferably the microcompartment is obtained after at least 2 passages (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 reencapsulation of the cells after step (d), that is to say at least two encapsulation cycles. Preferably each encapsulation cycle corresponds to a passage. In this variant of the process (at least one re-encapsulation of the cells after step (d) the number of cell divisions of the entire process (for all of 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 can 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 which consists of dissociating the microcompartment or the series of microcompartments to obtain a suspension of cells or a suspension of clusters of cells; the elimination of the external hydrogel layer can be carried out in particular by hydrolysis, dissolution, drilling and/or rupture by any biocompatible means, that is to say non-toxic for the cells. For example, removal can be accomplished using phosphate buffer saline, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel includes alginate and/or laser microdissection, and

- a step of re-encapsulating all or part of the cells or clusters of cells in a hydrogel capsule.

[0152] Re-encapsulation is a means suitable for increasing the 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,

- resuspend 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, cultivate 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 cycle of cell division, 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, that the culture cellular is implemented by means of cellular microcompartment, tube or 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 with at least one layer of cells,

- an outer hydrogel layer,

- a fibrin mesh arranged between the external hydrogel layer and the at least one cell layer.

[0158] When the kit is intended to be used in tubes or fibers comprising the cells, the preparation process can be as follows: a. mix cells, possibly previously incubated in a culture medium, with a mixture of fibrinogen, b. coating the mixture from step (a) in a layer of hydrogel; vs. cultivate 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 implemented using a concentric flow. The concentric flow including:

- a central flow (i) comprising the mixture of cells and fibrinogen from step a),

- optionally an intermediate flow (ii) positioned more externally 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 flow (iii) compared to the intermediate flow 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 use in the clinic. 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 medicine. [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, a thrombin solution, a hydrogel solution, preferably alginate, an isotonic solution, preferably a solution of 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 to 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 Fibrinogen at 14 mg/mL. The microfluidic injector allowing the co-injection of the different solutions includes 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 thrombin solution at 0.02U/ml.

[0171] Once encapsulation has been achieved, 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 the fragmentation of the jet into The output of the microfluidic injector will be collected.

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

[0175] Once encapsulation has been achieved, 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 Fibrinogen at 14 mg/mL, this solution was injected into the corresponding line upstream of the microfluidic injector and encapsulation was carried out. The other two lines respectively comprising a 2% alginate solution, and an intermediate solution comprising the sorbitol solution.

[0179] Once encapsulation has been achieved, the capsules are collected in the solution constituting the CaCl2 bath and this solution was rinsed with a cell culture medium devoid of serum and supplemented with the 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 capsules in the clinic, for example in the context of differentiated cell production. 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 cells obtained therefore require compliance with GMP regulations. However, matrigel®, given its composition cannot be used under GMP conditions. This problem is resolved satisfactorily with the fibrin mesh as shown in the results below.

[0182] Protocol

[0183] As part of this test, the inventors used an iPS cell line which was generated according to the usual standards for two-dimensional iPS culture, then the cells Tl were separated from the flasks via the action of an enzyme, according to the knowledge of those skilled in the art, and taken up in culture medium suitable for the culture of iPS.

[0184] The iPS cells were mixed in a suitable culture medium, comprising a fibrinogen solution at 14 mg/ml, so as to obtain a cell density of the order of 3 M/ml. The thrombin solution was mixed in 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 of the order of 1.2*10 ^A 6.

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

[0186] From D1 to D5 after encapsulation, the capsules are checked visually. On D5, the appearance of the cells, the quantity of cells, their viability and pluripotency is 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 invention according to Example 1 (D). Phase contrast microscopy images were generated and the inventors observed the obtaining of capsules comprising at least one layer of cells, an external hydrogel layer and a fibrin mesh (C) and (D) in comparison with the prior art capsules 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 fibrin mesh-based capsules 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 the capsules based on fibrin mesh make it possible to obtain a cyst and therefore their uses to produce 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 assay, the inventors observed more cells positive for Oct4, Nanog, SSEA4 and SSEA5 (factors characteristic of iPS cells) in capsules comprising fibrin than those lacking exogenous extracellular matrix, thus demonstrating that fibrin-based capsules make it possible to maintain the pluripotency of cells and therefore viability.

[0193] The results thus demonstrate that fibrin presents itself as a satisfactory extracellular matrix substitute in the context of the invention, although presenting slightly inferior results to matrigel®. Indeed, fibrin is systematically superior to the use of capsules devoid of exogenous extracellular matrix and makes it possible to obtain good amplification, maintain pluripotency, and develop cysts in a satisfactory manner.

[0194] Thus, the use of fibrin, in particular the use of a fibrin mesh, makes it possible to overcome the disadvantages 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 as part of a protocol for differentiating iPS cells into neuronal tissue.

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

[0197] Protocol

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

[0199] In the context of this test, the method implemented 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] The capsules comprising the 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. Neuronal tissue at D24 expresses tyrosine hydroxylase (TH), a specific marker of neuronal cells constituting neuronal tissue.

[0204] Finally, the inventors have surprisingly observed that the capsules according to the invention comprise neuronal cells having a more mature phenotype than those included in the capsules based on matrigel® (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 on D24 cultured in the capsules of the invention and the capsules based on matrigel®. A positive control based on the addition of dopamine 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 the 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 capable of being used on a large scale, with a view to clinical use requiring large quantities of cells, and therefore of capsules producing them.

[0208] Protocol

[0209] The protocol is identical to that of Example 4, with the difference that a fibrinogen solution at 20 mg/ml is used, so as to obtain a cell density of the order of 0.85 M/ml. Finally, the concentration of the thrombin solution is 0.04U/ml. [0210] Three conditions were studied, namely a culture condition in a 2D flask, 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 checked visually. On D7, the appearance of the cells, their viability, pluripotency and the amplification factor are also observed. The results are presented in Table 1, below.

extracellulaire répond au problème technique de la présente invention, y compris en culture à grande échelle dans des bioréacteurs appropriés. Par conséquent, lesdites capsules selon l'invention sont particulièrement adaptées à une utilisation dans des applications cliniques. [0213] [Table 1]

extracellular addresses the technical problem of the present invention, including large-scale culture in appropriate bioreactors. Consequently, said capsules according to the invention are particularly suitable for use in clinical applications.

Claims

Claims [Claim 1] Cellular microcompartment comprising: a. at least one layer of cells, b. an outer hydrogel layer, c. a fibrin mesh arranged between the outer hydrogel layer and said cell layer. [Claim 2] Cellular microcompartment according to the preceding claim, in which the fibrin mesh is entangled with the external hydrogel layer. [Claim 3] Cellular microcompartment according to one of the preceding claims, characterized in that the external layer comprises alginate. [Claim 4] Cellular microcompartment according to one of the preceding claims, characterized in that the microcompartment is closed. [Claim 5] Microcompartment according to one of the preceding claims, characterized in that the microcompartment is a three-dimensional microcompartment. [Claim 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. [Claim 7] Cellular microcompartment according to one of the preceding claims, characterized in that the cells constituting the layer of cells are cells chosen from eukaryotic cells, pluripotent cells, and differentiated cells. [Claim 8] Cellular microcompartment according to one of the preceding claims, characterized in that the microcompartment comprises a lumen. [Claim 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. [Claim 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. [Claim 11] Set of microcompartments, characterized in that at least one microcompartment is a microcompartment according to one of the preceding claims. [Claim 12] Microcompartment according to one of claims 1 to 10 or set of microcompartments according to claim 11 for its use as a medicament. [Claim 13] Process for preparing a cellular microcompartment according to one of claims 1 to 10, comprising the following steps: a. mix cells, possibly previously incubated in a culture medium with a mixture of fibrinogen, b. encapsulating the mixture from step (a) in a hydrogel layer; vs. cultivate the capsules obtained in step (b) in a culture medium, optionally, cultivate the capsules obtained from step (c) for at least 1 day, preferably from 3 to 50 days, and optionally recover the cellular microcompartments obtained , characterized in that a thrombin solution is added during step (b) and/or (c). [Claim 14] Method according to the preceding claim, characterized in that step (b) comprises the following substeps: i. bringing the mixture from step (a) into contact with a hydrogel solution to form at least one drop, and ii. collect the at least one drop obtained in a calcium bath capable of stiffening the hydrogel solution to form the outer layer of each microcompartment, the internal part of each drop being constituted by the mixture of step (a). [Claim 15] Method according to the preceding claim, characterized in that the thrombin solution is added during step i) or ii). [Claim 16] Method according to one of claims 13 to 15, characterized in that step i) consists of bringing the mixture of step a), the hydrogel solution, and an intermediate solution comprising said thrombin solution. [Claim 17] Method according to one of claims 13 or 14, characterized in that the thrombin solution is added during step c). [Claim 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. [Claim 19] Method according to the preceding claim, characterized in that the concentration of fibrinogen is between 14 and 20 mg/mL. [Claim 20] Method according to one of claims 13 to 19, characterized in that the concentration of thrombin is between 0.001 and 2U/ml, preferably between 0.01 U/ml and 0.03 U/ml. [Claim 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, of the mixture of 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. [Claim 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. [Claim 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. [Claim 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).