Extracellular matrix substitutes in cell microcompartments

Abstract

The present invention relates to a clinically usable "GMP" compliant cell microcell comprising at least: a. A cellular layer, b. An outer hydrogel layer, and c. A fibrin web disposed between the outer hydrogel layer and the cellular layer. The invention also relates to a method for producing such a microcompartment and to the use of a kit for producing a cell microcompartment comprising a fibrinogen solution and a thrombin solution.

Description

Technical Field

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

[0002] Cell culture is an area that continues to attract increasing interest since Professor Yamanaka discovered induced pluripotent stem cells (iPS or iPSCs).

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

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

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

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

[0007] Given the advent of cell therapies based on this technology and the production of animal or plant cells for human or animal food consumption, there is a need to develop an alternative, one that makes it possible to eliminate the presence of this type of extracellular matrix while maintaining the possibility of satisfactory cell adhesion and growth.

[0008] In the context of their work, the inventors surprisingly discovered that fibrin made it possible to achieve particularly promising results in its use in cell microcompartments, as it made it possible, in particular, to obtain large numbers of cells with rapid growth and to mass-produce said cell microcompartments. These results therefore make it possible to envisage its clinical use in three-dimensional cell microcompartments for human and veterinary applications.

[0009] Therefore, in order to meet the requirements of not having any non-GMP extracellular matrix (such as ), the present invention proposes a three-dimensional culture system based on cell microcompartments, which include a fibrin mesh arranged between a hydrogel outer layer and at least one cell layer. Summary of the Invention

[0010] Therefore, the present invention relates to a novel cell microcompartment comprising:

[0011] - at least one cell layer,

[0012] - hydrogel outer layer,

[0013] - a fibrin network disposed between the hydrogel outer layer and the at least one cell layer.

[0014] Advantageously, the fibrin mesh may or may not be isolated, i.e., entangled with at least one of the other components of the cell microcompartment (such as the cells or the hydrogel outer layer). Thus, according to one embodiment, the cells may be distributed inside the fibrin mesh and / or the fibrin mesh may be entangled in the hydrogel outer layer. Preferentially, the fibrin mesh is entangled in the hydrogel outer layer.

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

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

[0017] Advantageously, the fibrin network may contain other molecules, such as growth factors, proteins, peptides, elements of the culture medium and / or elements originating from cellular activity (such as secreted proteins, metabolites, etc.).

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

[0019] Advantageously, the cells constituting the cell layer are selected from the group consisting of eukaryotic cells, pluripotent cells and differentiated cells.

[0020] According to another preferred object, the microcompartment according to the present invention comprises at least one of the following features:

[0021] - the microcompartment is closed, and / or

[0022] - the microcompartments are three-dimensional microcompartments, preferably hollow three-dimensional microcompartments, and / or

[0023] - the microcompartment is in the shape of an ovoid, cylinder, spheroid, sphere or teardrop, and / or

[0024] - The microcompartment comprises one or more lumens within said at least one cell layer.

[0025] When the microcompartment comprises an inner cavity, the cell layer, the fibrin network and the outer layer are organized around the inner cavity.Preferably, the cell layer, the fibrin network and the outer layer are organized continuously around the inner cavity.

[0026] According to a particularly preferred purpose, the fibrin contained in the microcompartment according to the invention is obtained from the polymerization of fibrinogen performed by thrombin. Advantageously, the polymerization of fibrinogen performed by thrombin is obtained during and / or after the encapsulation process.

[0027] According to another aspect, the invention also relates to a set of microcompartments comprising at least one microcompartment according to the invention.

[0028] In the context of the present invention, the microcompartment is particularly suitable for use in cell therapy protocols.In addition, another aspect relates to the microcompartment according to the invention or the set of microcompartments according to the invention for use as a medicament.

[0029] Furthermore, the microcompartments according to the invention or the set of microcompartments according to the invention can be obtained by any means known to a person skilled in the art.

[0030] According to a particularly preferred aspect, the microcompartment according to the present invention can be obtained according to the following preparation method. Therefore, the present invention preferably relates to a preparation method comprising the following steps:

[0031] a) mixing cells, optionally previously incubated in culture medium, with a mixture of fibrinogen,

[0032] b) encapsulating the mixture from step a) in a hydrogel layer,

[0033] c) culturing the capsule obtained in step b) in a culture medium,

[0034] d) optionally, culturing the capsule obtained from step c) for at least 1 day, preferentially 3 to 50 days, and optionally recovering the cell microcompartments obtained,

[0035] Characterized in that a thrombin solution is added during steps b) and / or c).

[0036] Preferentially, the method optionally comprises a step of washing the capsule obtained from step c) for at least 1 day between the culturing step c) and the culturing step d).

[0037] Preferably, the encapsulation step b) comprises the following sub-steps:

[0038] i. contacting the mixture of step a) with a hydrogel solution to form at least one droplet, and

[0039] ii. collecting said obtained droplets in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment, the inner part of each droplet consisting of the mixture of step a) and optionally the thrombin solution.

[0040] More preferably, the thrombin solution can be added during step i) or ii). When the thrombin solution is added during step i), the droplets obtained comprise an outer layer, and the inner part of each droplet consists of the mixture of step a) and the thrombin solution. Preferably, the mixture of step i) is injected during co-injection through a microfluidic or millifluidic syringe, which allows the formation of the droplets and brings the thrombin into contact, thereby causing the polymerization of fibrinogen into fibrin.

[0041] When the thrombin solution is added during step ii), it is present in the calcium bath, which is able to harden the hydrogel solution to form the outer layer of each microcompartment. The thrombin solution then diffuses through the hydrogel outer layer, allowing the polymerisation of fibrinogen into fibrin.

[0042] Alternatively, the thrombin solution may be added after the formation of the microcompartments during step c) of culturing the capsule.The culture medium then contains the thrombin solution, which diffuses through the hydrogel layer, allowing the polymerisation of fibrinogen into fibrin.

[0043] According to a particularly preferred embodiment, step i) consists of contacting the mixture of step a), the hydrogel solution and an intermediate solution comprising the thrombin solution. For the purposes of the present invention, the term "intermediate solution" is intended to mean a solution that does not contain molecules capable of hardening the hydrogel solution and / or does not contain calcium. According to a specific embodiment, the intermediate solution is an isotonic intermediate solution. Thus, the thrombin solution is added during the formation of the droplets.

[0044] In addition, 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 of step a), and optionally the intermediate solution; the co-injection is carried out concentrically via a microfluidic or millifluidic syringe to form a jet composed of a mixture of the solutions at the syringe outlet, and the jet is divided into droplets.

[0045] In the context of the present invention, the thrombin solution cannot be added before the encapsulation step b) corresponding to the formation of the droplets.

[0046] Preferably, the fibrinogen concentration is 5 mg / mL to 30 mg / mL, more preferably 10 mg / mL to 25 mg / mL. Even more preferably, the fibrinogen concentration is 14 mg / mL to 20 mg / mL.

[0047] Preferably, the thrombin concentration is 0.001 U / mL to 2 U / mL, more preferably 0.01 U / mL to 1 U / mL, 0.01 U / mL to 0.05 U / mL, 0.01 U / mL to 0.03 U / mL, even more preferably 0.02 U / mL.

[0048] Preferably, the final opening diameter of the microfluidic syringe is 50 μm to 800 μm, more preferably 50 μm to 300 μm, even more preferably 80 μm to 240 μm, and the flow rate of each of these solutions is 0.1 mL / h to 1000 mL / h, preferably 1 mL / h to 500 mL / h, more preferably 10 mL / h to 150 mL / h. Even more preferably, the opening of the microfluidic syringe is 100 μm or 215 μm, and the flow rate of each of these solutions is 23 mL / h to 100 mL / h.

[0049] The microcompartments according to the invention can be used in a clinical setting.In addition, one aspect of the present invention relates to a microcompartment or a microcompartment assembly according to the invention for use as a medicament.

[0050] Finally, according to another aspect, the present invention also relates to the use of a kit for preparing a microcompartment according to the invention, said kit comprising at least one fibrinogen solution and one thrombin solution. Therefore, the present invention also relates to the use of a kit comprising at least one fibrinogen solution and one thrombin solution for preparing a microcompartment according to the invention.

[0051] According to another object, the present invention also relates to a kit comprising at least one fibrinogen solution, a thrombin solution, a hydrogel solution (preferably alginate), an isotonic solution (preferably sorbitol solution), a calcium solution, and a suitable culture medium. According to a variant, the kit is a kit of parts.

[0052] Preferably, the fibrinogen solution and the thrombin solution are of human origin and comply with regulations relating to Good Manufacturing Practice (GMP).

[0053] Other features and advantages will become apparent from the detailed description of the invention, the examples and the following drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] [ Figure 1] shows a first embodiment of the present invention, during which the thrombin solution is mixed with sorbitol during co-injection. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate, CS: suspension of cells and culture medium with fibrinogen. IS: intermediate solution containing 0.02 U of sorbitol and thrombin. Step 1 involves the co-injection of the different components to form a jet, which separates into droplets in a CaCl2 bath and hardens the outer layer of the capsule. During step 2, the capsule is then resuspended in a flushing medium. Finally, the capsule is resuspended in the final culture medium in the flask.

[0055] [ Figure 2 ] shows a second embodiment of the invention, during which a thrombin solution is added to the calcium bath for collecting the droplets formed after the jet has been split at the syringe outlet. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate, CS: suspended cells and culture medium with fibrinogen. IS: intermediate solution, i.e., sorbitol. Step 1 involves the co-injection of the various components to form the jet, which is then split into droplets in a CaCl2 bath supplemented with 0.02 U of thrombin solution, allowing polymerization of the fibrinogen and hardening of the outer layer of the capsule composed of alginate. During step 2, the capsule is then resuspended in a flushing medium. Finally, the capsule is resuspended in the final culture medium in the flask.

[0056] [ Figure 3 ] shows a first embodiment of the invention, during which a thrombin solution is added to the final culture medium. The concentration of the fibrinogen solution is 14 mg / mL. A: 2% alginate, CS: suspended cells and culture medium as well as fibrinogen. IS: intermediate solution, i.e. sorbitol. The first step involves the co-injection of the different components to form a jet, which separates into droplets in a CaCl2 bath, hardening the outer layer of the capsule composed of alginate. During step 2, the capsule is then resuspended in a flushing medium. Finally, the capsule is resuspended in the final culture medium in the flask supplemented with 0.02 U of thrombin solution, thus allowing the polymerization of the fibrinogen.

[0057] [ Figure 4 ] are phase contrast microscopy images at D5, showing the capsule in the absence of exogenous extracellular matrix (A), the capsule in the presence of matrigel (B), and the capsule in the presence of [ Figure 2 ] embodiment of the capsule (C) according to the present invention and according to [ Figure 1 ] the capsule (D) according to the present invention of the embodiment.

[0058] [ Figure 5] show the results relating to the expansion of capsules according to the invention, capsules in the presence of matrigel and capsules in the absence of exogenous extracellular matrix.

[0059] [ Figure 6 ] show the results for capsules according to the invention, capsules in the presence of matrigel and capsules without exogenous extracellular matrix relative to the percentage of capsules containing cysts.

[0060] [ Figure 7 ] show results related to the multipotency of capsules according to the invention, capsules in the presence of matrigel and capsules in the absence of exogenous extracellular matrix.

[0061] [ Figure 8 ] are phase contrast microscopy images of neurons at D17 after encapsulation. Panel A shows a neurosphere or neural microtissue contained in a microcompartment according to the present invention, i.e., polymerized fibrin from 14 mg / mL fibrinogen, while Panel B shows a neurosphere or neural microtissue contained in a prior art microcompartment containing Matrigel.

[0062] [ Figure 9 ] shows the principal component analysis (PCA) of the 1000 most variable genes between iPSCs at d0 and d17, d24 after neural differentiation in capsules seeded with fibrinogen or matrigel. Mature dopaminergic progenitor cells in the capsule were used as a positive control. DETAILED DESCRIPTION

[0063] definition

[0064] For the purposes of the present invention, "microcompartment" or "capsule" also refers to a partially or completely enclosed three-dimensional structure containing a plurality of cells. This is formed by a matrix of polymer chains (e.g., alginate) that is expanded by liquid and preferentially by water. The structure is particularly composed of a hardened hydrogel outer layer.

[0065] For the purposes of the present invention, a "droplet" is also understood to mean a three-dimensional structure formed from at least one liquid solution of hydrogel precursor elements, said at least one liquid solution comprising the constituents of a non-hardening hydrogel (polymeric precursors, non-crosslinked polymer chains, etc.). Furthermore, the droplet constitutes a transitional state between the co-injection of the various components and the microcompartment.

[0066] For the purposes of the present invention, a "differentiated" cell means a cell with a specific phenotype, as opposed to an undifferentiated pluripotent stem cell or a progenitor cell undergoing differentiation.

[0067] For the purposes of the present invention, "human cells" means human cells or immunohumanized non-human mammalian cells. Even when this is not specified, the cells, stem cells, progenitor cells and tissues according to the present invention consist of or are obtained from human cells or immunohumanized non-human mammalian cells.

[0068] For the purposes of the present invention, the term "mutant cell" refers to a cell that carries at least one mutation.

[0069] For the purposes of the present invention, "progenitor cell" means a stem cell that has begun cell differentiation but has not yet differentiated.

[0070] For the purposes of the present invention, "embryonic stem cells" means pluripotent stem cells derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be evaluated 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 present invention are obtained without destroying the embryo from which they originated, for example using the techniques described by Chang et al. (Cell Stem Cell, 2008, 2 (2)): 113-117. Optionally, embryonic stem cells from humans can be excluded.

[0071] For the purposes of the present invention, "pluripotent stem cells" or "multipotent cells" means cells that have the ability to form all tissues present in the entire origin organism, but cannot form the entire organism itself. In the context of the present invention, human pluripotent stem cells may be referred to as hPSCs. These cells may particularly be induced pluripotent stem cells (iPSCs or human induced pluripotent stem cells hiPSCs), embryonic stem cells, or MUSE cells (for "tolerance to multilineage differentiation stress").

[0072] For the purposes of the present invention, "induced pluripotent stem cells" means pluripotent stem cells that have been induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are particularly positive for pluripotency markers, such as staining with alkaline phosphatase and expression of proteins NANOG, SOX2, OCT4, and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in 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).

[0073] For the purposes of the present invention, a "cell layer" or "cell nest" is understood to mean a plurality of cells forming a layer or nest that can be configured around a lumen, which can be, for example, a cell tissue or microtissue or a three-dimensional grouped culture. The thickness of the cell layer can be variable. The cell layer is organized in a three-dimensional form within the microcompartment.

[0074] For the purposes of this invention, "tissue" or "biological tissue" has the usual meaning of tissue in biology, that is, an intermediate level of organization between cells and organs. A tissue is a group of similar cells of the same origin (usually from a common cell line, although they may originate from a union of different cell lines) grouped into clusters, networks, or bundles (fibers). Tissues form functional assemblies, that is, their cells contribute to the same function. Biological tissues regenerate regularly and assemble together to form organs.

[0075] "Fibrin mesh" or "fibrin network" within the meaning of the present invention refers to a plurality of entangled fibrin fibers constituting a mesh or network. These are optionally entangled with the inner surface of the hydrogel outer layer of the microcompartments.

[0076] For the purposes of the present invention, "lumen" means a volume of aqueous solution surrounded by the cellular topology.Preferably, the contents of the lumen are not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0077] Cell microcompartments

[0078] Therefore, the present invention relates to a cell microcompartment comprising cells, an outer layer of hydrogel and a fibrin mesh. The microcompartment according to the present invention comprises at least one cell layer. It should be understood that the microcompartment may also comprise cells suspended in a culture medium or optionally contained in a fibrin mesh.

[0079] Thus, the cell microcompartment advantageously comprises:

[0080] - at least one cell layer,

[0081] - hydrogel outer layer,

[0082] - a fibrin network arranged between the outer hydrogel layer and the cell layer.

[0083] Preferably, the microcompartment is a three-dimensional microcompartment defined by an outer layer of hydrogel and inside said outer layer said microcompartment comprises cells and a fibrin network. It may be in the shape of an ovoid, a cylinder, a spheroid, a sphere or a teardrop.

[0084] Advantageously, the three-dimensional microcompartments are hollow, more preferably, the hollow microcompartments are in the form of ovoids, cylinders, spheroids, spheres or teardrops.

[0085] Preferably, the hydrogel used is biocompatible, that is, non-toxic to cells. The hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartments and enable their survival. According to one embodiment, the outer hydrogel layer contains at least alginate. It can be composed solely of alginate. The alginate can particularly be sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular weight of 100 kDa to 400 kDa and a total concentration of 0.5 wt % to 5 wt %. Advantageously, the hydrogel layer does not contain cells.

[0086] The hydrogel layer also makes it possible to protect the cells from the external environment, thus limiting their uncontrolled proliferation and, in the case of differentiation, their differentiation.

[0087] The cells present in the microcompartments can be any type of cells, in particular the cells are eukaryotic cells, advantageously mammalian cells. More preferably, the cells are human or animal cells.

[0088] In a specific embodiment, the microcompartment includes pluripotent stem cells. Pluripotent stem cells or multipotent cells refer to cells with the ability to form all tissues present in the entire original organism, but cannot form a complete organism in this way. Pluripotent stem cells can especially be induced pluripotent stem cells (iPS), MUSE ("tolerance to multilineage differentiation stress") cells or embryonic stem cells (ES) found in the skin and bone marrow of adult mammals. According to one embodiment, the microcompartment according to the present invention does not include embryonic stem cells (ES).

[0089] According to a particularly suitable variant of the invention, the microcompartments according to the invention comprise human or animal induced pluripotent stem cells.

[0090] In another specific embodiment, microcompartment according to the present invention comprises people or animal special-potent cells and / or people or animal progenitor cells that derive from these special-potent cells.Pluripotent cells and / or progenitor cells are preferentially obtained from pluripotent stem cells (particularly human pluripotent stem cells) or optionally from non-pluripotent human cells, and the transcriptional profiles of these pluripotent stem cells are usually artificially modified to match the transcriptional profiles of specific special-potent cells and / or progenitor cells by forcing the specific transcription factors of expression target cell phenotype.Preferentially, after contacting with the solution that can start described stem cell differentiation, special-potent cells and / or progenitor cells are obtained from pluripotent stem cells.

[0091] According to another variant, microcompartment according to the present invention comprises the people or animal cell of differentiation.Differentiated cells are preferentially obtained from pluripotent stem cells or progenitor cells (particularly people's pluripotent stem cells or people's progenitor cells, or optionally non-pluripotent human cells), and the transcriptional profiles of these pluripotent stem cells or progenitor cells are usually artificially modified to match the transcriptional profiles of specific differentiated cells by forcing the specific transcription factors of the target cell phenotype to express.Preferably, after contacting with the solution that can start the differentiation of the stem cell, differentiated cells are obtained from pluripotent cells or specialized cells or progenitor stem cells.According to a variant, the cell contents of microcompartment comprise homogeneous or mixed cell identifications.

[0092] The differentiated cells may in particular be in the form of at least one cell layer, or in the form of a three-dimensional tissue or microtissue, or in the form of a plurality of tissues or microtissues in microcompartments. It may be a dense or non-dense tissue or microtissue, with or without a lumen.

[0093] The microcompartment according to the present invention may include various types of cells. In particular, the microcompartment according to the present invention may include, for example, stem cells and / or multipotent cells and / or progenitor cells and / or differentiated cells induced to be pluripotent.

[0094] Advantageously, the microcompartments according to the invention are obtained after a plurality of cell division cycles.In fact, the cells comprised in the microcompartments according to the invention are cells obtained from at least one cell by amplification.

[0095] Furthermore, the cells present in the microcompartments according to the invention are obtained after at least two cell division cycles following encapsulation in the hydrogel outer layer of at least one cell.

[0096] Preferably, the cells present in the microcompartments according to the present invention are obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles after encapsulating at least 1 cell, preferably 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 100 cells in the outer hydrogel layer. For example, the cells present in the microcompartments are obtained after at least six cell division cycles after encapsulating at least 1 cell, preferably 1 to 50 cells, in the outer hydrogel layer.

[0097] Preferably, the microcompartments are obtained after at least 2 passages after encapsulation, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage may last, for example, at least 1 day, or 2 to 50 days, particularly 3 to 10 days.

[0098] Preferably, the microcompartments are obtained after at least one repackaging, more preferably 1 to 14 repackagings, in particular 2 to 7 repackagings. Very preferably, the repackaging corresponds to a new passage, and each packaging cycle corresponds to one passage.

[0099] Preferably, all of the cells initially encapsulated in the microcompartment prior to the first cell division cycle exhibit a volume that is 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.

[0100] Thus, according to one embodiment, the cells present in the microcompartments according to the invention are obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles after encapsulation of the cells in the outer hydrogel layer and exhibit a volume that is less than 50%, more preferentially less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0101] 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.

[0102] The microcompartment according to the invention comprises a plurality of cells, preferentially at least 20 cells, even more preferentially at least 100, at least 500, at least 1000, at least 10,000 cells.

[0103] In the context of the present invention, fibrin networks are particularly suitable as non-GMP extracellular matrices (such as ) and makes it possible to cope with the shortcomings of the prior art. In addition, the fibrin network makes it possible to achieve cell proliferation in a satisfactory manner.

[0104] Thus, the fibrin network advantageously forms a fibrin network within the capsule, potentially forming a fibrin gel or clot within the capsule. The network may or may not interpenetrate with at least one other component of the microcompartments, preferably with the outer layer of the microcompartments. When the network does not interpenetrate with, for example, the outer layer, it forms a separate network in which cells can be housed and proliferate.

[0105] Preferentially, the fibrin network is entangled with the outer layer of the hydrogel, more preferably with the inner surface of the outer layer of the hydrogel. In addition, the boundary between the fibrin network and the outer layer may not be very clear. Therefore, at least a portion of the fibrin network may be entangled with the inner surface of the outer layer, preferably with the alginate constituting it. Therefore, at least a portion of the network composed of fibrin is preferentially entangled with the outer layer of the hydrogel.

[0106] According to another specific embodiment, the fibrin network forms an interpenetrating polymer network (IPN) with the hydrogel outer layer.

[0107] According to particularly preferred purposes, fibrin is obtained from the polymerization of the fibrinogen carried out by a fibrinogen polymerization agent (advantageously said agent is thrombin) during and / or after encapsulation. In addition, the polymerization of the fibrinogen solution carried out by the thrombin solution occurs during and / or after encapsulation. When it occurs after encapsulation, this polymerization occurs in the newly formed droplet or capsule.

[0108] The fibrin network may optionally contain a mixture of extracellular proteins and compounds necessary for culturing the cells during differentiation, as well as the isolated cells.

[0109] Advantageously, encapsulation is carried out by concentric co-injection through a microfluidic syringe which forms at the syringe outlet a jet consisting of a mixture of the various useful solutions, said jet being divided into droplets. The droplets are then collected in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment.

[0110] According to a first embodiment, polymerization of the fibrinogen solution by thrombin occurs during encapsulation. The cell mixture, fibrinogen mixture, hydrogel solution, and thrombin solution are simultaneously contacted and co-injected concentrically via a microfluidic or millifluidic syringe, thereby forming a jet at the syringe outlet that is divided into droplets. Once the various solutions are brought into contact, polymerization is initiated and then carried out almost immediately.

[0111] According to a second embodiment, the droplets are collected in a calcium bath that can harden the hydrogel solution to form the outer layer of each microcompartment. In the absence of a thrombin solution during co-injection via a microfluidic syringe, polymerization of the fibrinogen is not initiated. Therefore, polymerization of the fibrinogen solution by the thrombin solution occurs after encapsulation. In accordance with this objective, the thrombin solution is added to the calcium bath, thereby allowing collection and formation of the microcompartments. Thus, the thrombin solution can diffuse through the hydrogel solution being hardened.

[0112] According to a third embodiment, the thrombin solution is not added to the calcium bath. Thus, once the hardening of the hydrogel solution by the calcium bath is complete, the formed microcompartments are rinsed and an isotonic solution is added, preferably a culture medium containing an apoptosis inhibitor. This isotonic solution is then supplemented with the thrombin solution. The thrombin solution can then diffuse through the hardened hydrogel shell, allowing the polymerization of fibrinogen by the thrombin.

[0113] According to a particularly preferred purpose, the thrombin solution is co-injected together with the cells, the fibrinogen solution, the culture medium containing the cells and the hydrogel solution. More preferably, the isotonic solution is also co-injected and contains the thrombin solution, advantageously, the isotonic solution is a sorbitol solution.

[0114] According to any of the three embodiments described above, the polymerization of fibrinogen by a fibrinogen polymerizing agent (such as thrombin) makes it possible to obtain a fibrin network within the capsule, wherein cells will be accommodated or bound to the surface of the network for proliferation. The fibrin network can form a distinct network with at least one of the other components of the microcompartment (preferably the outer layer of the hydrogel), or form an interpenetrating network.

[0115] The microcompartments according to the invention may also contain other elements, in particular culture medium.

[0116] According to the knowledge of a person skilled in the art, the culture medium is a culture medium suitable for the cells present in the microcompartments.

[0117] According to another preferred object of the present invention, the microcompartment comprises at least one lumen. This at least one lumen may contain a liquid, in particular a culture medium and / or a liquid secreted by the cells. Advantageously, the presence of this hollow portion allows the cells to have a small diffusion volume, the composition of which they can control, thereby promoting so-called autocrine / paracrine cell communication. This three-dimensional arrangement of cells in a monolayer or spherical cell mass surrounding a lumen or central cavity may also be referred to as an encapsulation.

[0118] During cyst formation, the lumen is preferentially created by cells that proliferate and develop on or within the fibrin network.

[0119] According to another preferred object, the cell layer, the fibrin network and the outer layer are organized around the inner cavity, more preferably they are organized continuously around the inner cavity.

[0120] The encapsulated form makes it possible to reduce the stress experienced by stem cells relative to 2D cultures or aggregates. This configuration also makes it possible to reduce cell mortality and increase culture expansion factors. This makes it possible to reduce the number of required passages and dissociations, and to reduce the culture time required to reach the desired number of final cells.

[0121] According to one embodiment, the microcompartment may comprise a plurality of capsules or tissues or microtissues.

[0122] The cell microcompartments according to the invention are closed or partially closed, that is to say the outer layer is closed or partially closed.Preferably, the microcompartments are closed.

[0123] The microcompartment according to the present invention may be in any three-dimensional form, i.e., it may have the shape of any object in space. The microcompartment may have any form compatible with cell encapsulation. Preferably, the microcompartment according to the present invention is spherical or elongated. It may have the shape of an ovoid, a cylinder, a spheroid or a sphere. In particular, it may be in the shape of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0124] It is the outer layer of the microcompartment (i.e. the hydrogel layer) that gives the microcompartment according to the invention its size and shape. Preferably, the minimum dimension of the microcompartment according to the invention is 10 μm to 1 μm, preferably 100 μm to 700 μm. It can be 200 μm to 600 μm, in particular 300 μm to 500 μm.

[0125] Its maximum dimension is preferably greater than 10 μm, more preferably 10 μm to 1 m, even more preferably 10 μm to 50 cm.

[0126] The microcompartments according to the invention can optionally be stored frozen. They then have to be thawed before use.

[0127] The invention also relates to a plurality of microcompartments together.

[0128] Therefore, the present invention also relates to a set or series of cell microcompartments as described above, comprising at least two cell microcompartments according to the present invention.

[0129] The present invention also relates to a microcompartment assembly or a series of at least two three-dimensional cell microcompartments, each microcompartment comprising at least one hydrogel outer layer and at least one cell layer within said outer layer, wherein at least one microcompartment is a microcompartment according to the present invention.

[0130] Preferably, the cells present in the microcompartments of the set of microcompartments according to the present invention are obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cell division cycles, after encapsulating at least one cell in each microcompartment in an outer hydrogel layer. The microcompartments present in the set of microcompartments may have one or more characteristics of a microcompartment according to the present invention (size, shape, number of cells, cell volume, middle layer, lumen, etc.).

[0131] The set of microcompartments according to the invention preferably comprises 2 to 10 16 A micro compartment.

[0132] Preferably, the series of microcompartments according to the invention is in a culture medium, in particular in an at least partially convective culture medium.

[0133] According to a particularly suitable embodiment, the present invention has as its object a series of cell microcompartments in a culture medium in a closed chamber such as a bioreactor, preferentially in a closed chamber such as a bioreactor.

[0134] The presence of an outer layer of hydrogel and a possible intermediate layer of isotonic aqueous solution allows the cells to be evenly distributed between the microcompartments. Furthermore, this hydrogel layer makes it possible to prevent the fusion of microcompartments, which is a major source of variability that is detrimental to the homogeneity of the cell phenotype.

[0135] method

[0136] Microcompartments may be obtained by any means known to those skilled in the art for the preparation of microcompartments or capsules.

[0137] According to another aspect, the present invention also relates to a method for producing a microcompartment according to the invention.

[0138] The method for preparing a microcompartment or a set of microcompartments according to the present invention comprises at least the following steps:

[0139] a. mixing the cells, optionally previously incubated in culture medium, with a mixture of fibrinogen,

[0140] b. encapsulating the mixture from step (a) in a hydrogel layer;

[0141] c. culturing the capsule obtained in step (b) in a culture medium,

[0142] d. culturing the capsule obtained from step (c) for at least 1 day, preferentially 3 to 50 days, and optionally recovering the cell microcompartments obtained,

[0143] Characterized in that a thrombin solution is added during steps b) and / or c).

[0144] Advantageously, the method according to the invention may comprise further steps. Thus, preferably, the cells are incubated before the step of mixing the cells with the fibrinogen mixture in a suitable culture medium. Said culture medium preferably contains at least one cytoprotective factor, more preferably at least one apoptosis inhibitor.

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

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

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

[0148] Preferably, post-encapsulation steps are performed under permanent or continuous stirring. This stirring is important because it maintains the homogeneity of the culture environment and prevents the formation of any diffusion gradients. For example, it allows for uniform control of the cellular oxygenation level; thus, avoiding necrosis associated with hypoxia or oxidative stress associated with hyperoxia. Thus, it avoids an increase in cell mortality and / or oxidative stress.

[0149] Preferentially, after the step of culturing the capsules obtained, the method comprises a step consisting of washing the capsules obtained from step (d), advantageously in order to eliminate cytoprotective factors, such as inhibitors of apoptosis.

[0150] Preferably, the encapsulation step b) comprises the following sub-steps:

[0151] i. contacting the mixture of step a) (ie, the mixture of cells and fibrinogen) with a hydrogel solution to form at least one droplet, and

[0152] ii. collecting said at least one droplet obtained in a calcium bath capable of hardening the hydrogel solution to form the outer layer of each microcompartment, the inner part of each droplet consisting of the mixture of step i).

[0153] Once the outer hydrogel layer has been hardened by the calcium bath, the microcompartments are formed. The microcompartments can then be flushed to remove, for example, apoptosis inhibitors.

[0154] When the thrombin solution is added during the encapsulation step b), it may be added during the mixing step i) or during the step ii) of collecting the droplets.

[0155] According to one object of the present invention, a thrombin solution is mixed with the mixture of step a), and the hydrogel solution (preferably thrombin) and the other solution are co-injected simultaneously. Preferably, step i) comprises contacting the mixture of step a), the hydrogel solution and an isotonic intermediate solution containing the thrombin solution, more preferably, the isotonic intermediate solution is a sorbitol solution.

[0156] Particularly advantageously, the step of mixing the mixture of step a) and the hydrogel is a step aimed at structuring said mixture of step a) and the hydrogel solution in the form of linear and concentric flows.

[0157] Advantageously, adding thrombin during simultaneous co-injection makes it possible to control polymerization since the thrombin / fibrinogen contact time can be controlled. On the other hand, the amount of thrombin added is less significant, preferentially 4 to 8 times greater, than when the thrombin solution is added after encapsulation.

[0158] According to another object of the present invention, a thrombin solution is added to the calcium bath, i.e., during step ii). Thus, the thrombin solution can diffuse through the hydrogel shell of the microcompartments during the hardening period and thus polymerize the fibrinogen into fibrin. Thus, a fibrin network is formed on or in the cells, and encapsulation is formed.

[0159] When the method according to the present invention comprises the step of flushing the capsule obtained, the solution constituting the calcium bath is removed and is used to culture a suitable culture medium for the microcompartments according to the present invention, preferably an isotonic solution, more preferably a culture medium containing an apoptosis inhibitor. According to another purpose, the culture medium may include a thrombin solution. Here, the thrombin solution can diffuse again through the hydrogel shell of the hardened microcompartments and polymerize fibrinogen into fibrin, thereby forming a fibrin network. In addition, according to another purpose of the present invention, a thrombin solution is added during step c).

[0160] In addition, in a particularly preferred manner, step b) of the method according to the present invention is carried out by simultaneous co-injection of the hydrogel solution, the mixture of step a) and optionally the intermediate solution; the co-injection is carried out concentrically via a microfluidic or millifluidic syringe, forming a jet composed of the mixture of the solutions at the outlet of the syringe, and the jet is divided into droplets.

[0161] When the thrombin solution is co-injected with other solutions, it preferentially mixes with the isotonic intermediate solution.

[0162] Preferably, the fibrinogen concentration is 5 mg / mL to 30 mg / mL, preferably 10 mg / mL to 25 mg / mL, more preferably 14 mg / mL to 20 mg / mL.

[0163] According to another purpose of the present invention, the concentration of thrombin is preferably 0.001U / mL to 2U / mL, more preferentially 0.01U / mL to 1U / mL, 0.01U / mL to 0.05U / mL, 0.01U / mL to 0.03U / mL, even more preferentially 0.02U / mL." U " should be interpreted as meaning enzyme activity unit (i.e. the concentration of enzyme), and this enzyme activity unit represents the amount of the necessary enzyme of the substrate of micromole in 1 minute. Should be understood that indicated concentration is the concentration in the mixture. In fact, advantageously, thrombin and other compositions are mixed according to the ratio of 1: 1. In addition, in capsule, when the concentration of thrombin before mixing was 0.01U / mL, the concentration in capsule was about 0.01U / mL.

[0164] The method according to the invention is implemented via a microfluidic syringe that allows the co-injection of various solutions and the formation of a jet divided into droplets. Preferably, the final opening diameter of the microfluidic syringe is 50 to 800 μm, more preferably 50 to 300 μm, even more preferably 80 to 240 μm, and the flow rate of each of the solutions is 0.1 to 1000 mL / h, preferably 1 to 500 mL / h, more preferably 10 to 150 mL / h.

[0165] The method according to the invention is preferably carried out in a closed chamber, such as a closed bioreactor or a flange.

[0166] The number of cell divisions in step (d) of culturing the capsule is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cell division cycles.

[0167] Preferably, the microcompartments are 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, 2 to 15 days, in particular 3 to 8 days.

[0168] In a preferred variant, the method according to the invention comprises at least one repackaging 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 repackaging of the cells after step (d)), the number of cell divisions of the entire method (for all passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0169] In the method according to the invention, there may be a plurality of resealings, preferably 1 to 100, in particular 1 to 10 resealings.

[0170] Each repackaging may include:

[0171] a step consisting of dissociating the microcompartment or the series of microcompartments so as to obtain a suspension of cells or a suspension of cell clusters; the outer layer of the hydrogel may be eliminated in particular by hydrolysis, dissolution, puncture and / or decomposition by any biocompatible means (that is to say, means that are non-toxic to the cells). This elimination may be achieved, for example, using phosphate-buffered saline, divalent ion chelators, enzymes such as alginate lyase (if the hydrogel contains alginate) and / or laser microdissection, and

[0172] - a step of re-encapsulating all or part of the cells or cell clusters in a hydrogel capsule.

[0173] Recapsulation is a useful means to increase the expansion of cells obtained from the pluripotency step and to reduce the risk of mutations.

[0174] According to a specific embodiment, repackaging comprises the following steps:

[0175] - Eliminate the hydrogel outer layer,

[0176] - resuspending the cells contained in the microcompartments so as to obtain single cells and / or at least one group or cluster of cells in an isotonic medium, preferably containing an inhibitor of apoptosis,

[0177] - encapsulating the mixture in a hydrogel layer;

[0178] - preferentially, culturing the obtained microcompartments in an isotonic solution containing an inhibitor of apoptosis, preferentially in a culture medium containing an inhibitor of apoptosis;

[0179] - preferentially, flushing the microcompartment, advantageously in order to eliminate the apoptosis inhibitor;

[0180] - culturing the microcompartments in an isotonic solution, preferably culture medium, for at least one cell division cycle, and

[0181] - Optionally recovering the cell microcompartments obtained.

[0182] use

[0183] The formation of fibrin as the extracellular matrix (especially The use of fibrinogen solutions and thrombin solutions as alternatives to thrombin is particularly suitable for three-dimensional cell culture, whether the cell culture is carried out by cell microcompartments, tubular bodies or fibers comprising the cells.

[0184] Therefore, the present invention also relates to the use of a kit intended for three-dimensional culture, said kit comprising a fibrinogen solution and a thrombin solution.

[0185] In particular, the present invention relates to the use of a kit comprising a fibrinogen solution and a thrombin solution for obtaining a cell microcompartment having: at least one cell layer,

[0186] - hydrogel outer layer,

[0187] - a fibrin network disposed between the outer hydrogel layer and at least one cell layer.

[0188] When the kit is intended to be implemented in tubular bodies or fibers comprising cells, the preparation method may be as follows:

[0189] a. mixing the cells, optionally previously incubated in culture medium, with a mixture of fibrinogen,

[0190] b. coating the mixture from step (a) in a hydrogel layer;

[0191] c. culturing the fibers or tubular bodies obtained in step (b) in a culture medium,

[0192] Characterized in that a thrombin solution is added during steps b) and / or c).

[0193] Advantageously, the coating step b) is carried out by a concentric flow. The concentric flow comprises:

[0194] - a central stream (i) comprising the mixture of cells and fibrinogen from step a),

[0195] - optionally an intermediate stream (ii), which is located further outwards from the central stream and which does not contain calcium and comprises an isotonic solution, for example an isotonic sorbitol solution which may optionally contain fibrinogen,

[0196] - for the outermost stream (iii) of the intermediate stream, the outermost stream comprises a hydrogel solution, such as an alginate solution, optionally comprising thrombin, and

[0197] - an even outer calcium-containing stream (iv) comprising an isotonic solution, such as an isotonic sorbitol solution which may optionally contain fibrinogen.

[0198] According to another aspect, the present 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.

[0199] Preferably, the fibrinogen solution and the thrombin solution are of human origin and comply with regulations relating to Good Manufacturing Practice (GMP).

[0200] Finally, the microcompartments are particularly suitable for use in a clinical setting.Furthermore, the present invention also relates to a microcompartment according to the invention or a set of microcompartments according to the invention for use as a medicament.

[0201] According to another aspect, the present invention relates to the use of a microcompartment according to one of the aforementioned objects for producing cells, tissues, preferentially for the large-scale production of such cells and / or tissues.

[0202] The microcompartments according to the invention can also be used to produce animal or plant cells for human or animal food consumption.Such use is particularly useful for producing substitutes for meat products (such as meat) in order to limit the consumption of meat products.

[0203] According to another aspect, the present 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, and a suitable culture medium. According to one variant, the kit is a kit of parts.

[0204] The invention will now be illustrated by non-limiting examples and results of compositions according to the invention.

[0205] Example

[0206] Example 1 - Capsule according to the first embodiment .

[0207] This example describes a first embodiment of the invention, also shown in [ Figure 1 ], in which a thrombin solution was added to a sorbitol solution and co-injected with a cell mixture in culture medium and a hydrogel solution via a microfluidic syringe.

[0208] Therefore, cells were mixed with cell culture medium and 14 mg / mL of fibrinogen. A microfluidic syringe capable of co-injecting various solutions included three lines upstream of the nozzle. The solution containing fibrinogen was injected into the line corresponding to the cells and encapsulated. The other two lines each contained a 2% alginate solution and an intermediate solution comprising a 0.02 U / mL sorbitol solution and a thrombin solution.

[0209] Once encapsulation has been performed, the droplets are collected in a CaCl2 bath to allow hardening of the alginate and formation of an alginate shell that forms microcompartments or capsules. The capsule-containing solution is then rinsed with serum-free cell culture medium.

[0210] Example 2 - Capsule according to the second embodiment .

[0211] This example describes a second embodiment of the invention, also shown in [ Figure 2 ], where a thrombin solution is added to a calcium bath, where newly formed droplets are collected after jet breakup at the outlet of a microfluidic syringe.

[0212] The cells were mixed with cell culture medium and 14 mg / mL fibrinogen, and the solution was injected into the corresponding line upstream of the microfluidic syringe and encapsulated. The other two lines contained a 2% alginate solution and an intermediate solution containing a sorbitol solution, respectively.

[0213] Once encapsulation has taken place, the capsules are collected in a solution that constitutes a CaCl2 bath and supplemented with 0.02 U of thrombin solution. The solution is rinsed with serum-free cell culture medium.

[0214] Example 3 - Capsule according to the second embodiment .

[0215] This example describes a third embodiment of the invention, also shown in [ Figure 3 ], in which a thrombin solution is added to the isotonic solution after washing the capsule obtained after hardening of the alginate by the action of a calcium bath.

[0216] The cells were mixed with cell culture medium and 14 mg / mL fibrinogen, and the solution was injected into the corresponding line upstream of the microfluidic syringe and encapsulated. The other two lines contained a 2% alginate solution and an intermediate solution containing a sorbitol solution, respectively.

[0217] Once encapsulation has been performed, the capsules are collected in a solution constituting a CaCl2 bath and the solution is rinsed with serum-free cell culture medium and supplemented with 0.02 U of thrombin solution.

[0218] Example 4 - Capsules according to the present invention and based on Comparison of capsules .

[0219] Compared to the microcompartments according to the present invention, in the presence of extracellular matrix (especially Comparative results were obtained between prior art microcompartments in the absence of exogenous extracellular matrix or extracellular matrix substitutes. This represents the most efficient solution for obtaining capsules, but it is not suitable for clinical capsule use, for example in the context of differentiated cell production. By way of example, capsules can produce large numbers of neuronal cells that can be injected into patients suffering from neurodegenerative diseases such as Alzheimer's disease. Therefore, the product directly obtained by means of the capsules according to the invention can be used in cell therapy contexts. Therefore, the capsules and cells obtained need to comply with GMP regulations. However, given their composition, It cannot be used under GMP conditions. As shown in the following results, the use of fibrin mesh solves this problem satisfactorily.

[0220] plan

[0221] In the context of this test, the inventors used iPS cell lines generated according to conventional standards for 2D iPS culture, and then enzymatically detached the cells from the flask and transferred them to a medium suitable for iPS culture according to the knowledge of those skilled in the art.

[0222] iPS cells were mixed in a suitable culture medium containing 14 mg / mL fibrinogen solution to achieve a cell density of approximately 3 M / mL. A thrombin solution was mixed in a sorbitol solution. The various solutions were then loaded via dedicated lines and co-injected simultaneously using a microfluidic syringe. The amount of encapsulated cells was approximately 1.2*10^6.

[0223] The same protocol was followed to obtain capsules without exogenous extracellular matrix and with capsule.

[0224] From D1 to D5 after encapsulation, the capsule was visually inspected. On D5, the appearance of the cells, the number of cells, their viability and pluripotency were observed.

[0225] result

[0226] The results are shown in [ Figure 4 ]、[ Figure 5 ]、[ Figure 6 ]and[ Figure 7 ]middle.

[0227] [ Figure 4 ] shows a capsule without exogenous extracellular matrix (A), containing The capsule of the invention (B), the capsule of the invention according to Example 2 (C) and the capsule of the invention according to Example 1 (D) were generated by phase contrast microscopy and compared with the results based on Comparing the prior art capsule (B) and the capsule without exogenous extracellular matrix (A), the inventors observed that capsules (C) and (D) were obtained comprising at least one cell layer, an outer hydrogel layer and a fibrin network.

[0228] The inventors then characterized the obtained capsules. [ Figure 5 ] showed that the capsule according to the present invention, in the presence The present inventors observed that compared with The expansion was better with fibrin than in the absence of exogenous extracellular matrix but in the presence of lower extracellular matrix. However, the results obtained demonstrate that capsules based on fibrin mesh allow good expansion of capsules, thus allowing their use for the production of cells.

[0229] [ Figure 6 ] showed that the capsule according to the present invention, in the presence The percentage of capsules containing no exogenous extracellular matrix compared to capsules containing cysts was calculated. The inventors observed the presence of at least one cyst in approximately 60% of the capsules according to the invention. These results demonstrate once again that capsules based on fibrin networks make it possible to obtain cysts and, therefore, are useful for generating cells.

[0230] [ Figure 7 ] showed that the capsule according to the present invention, in the presence The present inventors observed that more cells were positive for Oct 4, Nanog, SSEA4, and SSEA5 (characteristic factors of iPS cells) in capsules containing fibrin than in those without exogenous extracellular matrix, thus demonstrating that the fibrin-based capsule makes it possible to maintain the pluripotency of the cells and therefore their viability.

[0231] The results thus demonstrate that fibrin serves as a satisfactory extracellular matrix substitute in the context of the present invention, although showing slightly lower Indeed, fibrin is systematically larger than the use of capsules without exogenous extracellular matrix and makes it possible to obtain good expansion, maintenance of multipotency and encapsulation in a satisfactory manner.

[0232] Therefore, the use of fibrin, and in particular the use of a fibrin mesh, makes it possible to overcome the drawbacks of the prior art, allowing the use of this fibrin mesh-based three-dimensional microcompartment technology in a clinical setting in the context of cell therapy.

[0233] Example 5 - Pods according to the invention in the context of a protocol for differentiating iPS cells into neural tissue membrane .

[0234] This study aimed to use the microcompartments according to the invention in the context of a protocol for differentiating PS cells into neural tissue.

[0235] plan

[0236] Once the cell microcompartments comprising iPS cells according to the invention have been obtained, they will undergo cell differentiation in order to differentiate them into neural cells according to the desired phenotype.

[0237] In the context of this test, the methods implemented were 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 from Nolbrant et al. (“Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation,” Nature Protocols 2017).

[0238] The capsules, which contained iPS cells and then differentiated into neural tissue, were cultured for 24 days after encapsulation.

[0239] result

[0240] The results are presented in [ Figure 8 ]and[ Figure 9 ]. Thus, the present inventors observed the presence of neural tissue 24 days after encapsulation from iPS cells. Figure A is an image of a microcompartment according to the present invention based on polymerized fibrin from 14 mg / mL fibrinogen, and Figure B is an image of a microcompartment based on Microcompartments of the prior art.

[0241] Thus, the inventors were able to observe the presence of neural tissue, in particular neurospheres of similar size, whether in the presence of a fibrin-based capsule or a The nervous tissue at D24 did express tyrosine hydroxylase (TH), a specific marker of neuronal cells that constitute the nervous tissue.

[0242] Finally, the inventors surprisingly observed that the capsules according to the invention comprise a higher concentration of lipopolysaccharide than the capsules comprising the lipopolysaccharide based on These results were presented in [ Figure 9 The present inventors analyzed the presence of the capsule of the present invention and the The expression of certain genes in a cell population of neural tissue cultured in a capsule of the present invention was measured. A positive control based on the addition of dopaminergic neuron progenitor cells to the capsule was also added. The results showed that the neural tissue present in the capsule of the present invention had a distribution close to that of the positive control, thus demonstrating the presence of a more mature phenotype.

[0243] Thus, the results clearly demonstrate that fibrin meshes are particularly suitable as an alternative to non-GMP extracellular matrices in the context of three-dimensional culture of cells in microcompartments.

[0244] Example 6 - Large-scale production of capsules according to the invention

[0245] The aim of this study was to demonstrate that the capsules according to the invention are also suitable for large-scale use, for use in the clinic, since large numbers of cells and therefore capsules are needed to produce them.

[0246] plan

[0247] The protocol was the same as that of Example 4, except that a 20 mg / ml fibrinogen solution was used to obtain a cell density of approximately 0.85 M / ml. Finally, the concentration of the thrombin solution was 0.04 U / ml.

[0248] Three conditions were investigated in this study: cultivation in 2D flasks, cultivation in small-scale bioreactors (30 mL), and cultivation in large-scale bioreactors (500 mL).

[0249] result

[0250] From D1 to D5 after encapsulation, the capsule was visually inspected. At D7, cell appearance, viability, pluripotency, and expansion factors were observed. The results are presented in Table 1 below.

[0251] [Table 1]

[0252] Amplification Viability (FACS) Cysts at D7 Pluripotency (Oct4 / Nanog) Large scale x155 98.6 none 97.4％ Small scale x251 97.9 none 90％ flask x191 98.9 none 95.8％

[0253] The results demonstrate that the use of fibrin as an extracellular matrix substitute satisfies the technical problem of the present invention, including large-scale cultivation in suitable bioreactors.Thus, the capsule according to the present invention is particularly suitable for clinical applications.

Claims

1. A cell microcompartment, comprising: a. at least one cell layer, b. Hydrogel outer layer, c. a fibrin network disposed between the hydrogel outer layer and the cell layer.

2. The cell microcompartment according to the preceding claim, wherein the fibrin network is entangled with the hydrogel outer layer.

3. The cell microcompartment according to claim 1 , characterized in that The outer layer comprises alginate.

4. Cell microcompartment according to one of the preceding claims, characterized in that The cell microcompartments are closed.

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

6. Microcompartment according to one of the preceding claims, characterized in that The microcompartments are in the shape of ovoids, cylinders, spheroids, spheres or teardrops.

7. Cell microcompartment according to one of the preceding claims, characterized in that The cells constituting the cell layer are cells selected from the group consisting of eukaryotic cells, pluripotent cells and differentiated cells.

8. Cell microcompartment according to one of the preceding claims, characterized in that The microcompartment includes an inner cavity.

9. Cell microcompartment according to the preceding claim, characterized in that The cell layer, the fibrin network and the outer layer are continuously 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 and / or after encapsulation.

11. A group of microcompartments characterized by At least one microcompartment is a microcompartment according to one of the preceding claims.

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

13. A method for preparing a cell microcompartment according to one of claims 1 to 10, comprising the following steps: a. mixing the cells, optionally previously incubated in a culture medium, with a mixture of fibrinogen, b. encapsulating the mixture from step (a) in a hydrogel layer; c. culturing the capsule obtained in step (b) in a culture medium, Optionally, culturing the capsule obtained from step (c) for at least 1 day, preferentially 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).

14. The method according to the preceding claim, characterized in that Step (b) comprises the following sub-steps: i. contacting the mixture of step (a) with a hydrogel solution to form at least one droplet, and ii. collecting said at least one droplet obtained in a calcium bath capable of hardening said hydrogel solution to form said outer layer of each microcompartment, the inner part of each droplet consisting of said mixture of step (a).

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

16. The method according to claim 13, characterized in that Step i) consists of contacting said mixture of step a), said hydrogel solution and an intermediate solution comprising said thrombin solution.

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

18. The method according to claim 13, characterized in that The concentration of the fibrinogen is 5 mg / mL to 30 mg / mL, preferably 10 mg / mL to 25 mg / mL.

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

20. The method according to claim 13, characterized in that The concentration of the thrombin is 0.001 U / mL to 2 U / mL, preferably 0.01 U / mL to 0.03 U / mL.

21. The method according to claim 13, characterized in that Step b) is performed by simultaneous co-injection of the hydrogel solution, the mixture from step a) and optionally the intermediate solution; The co-injection is performed concentrically via a microfluidic or microfluidic syringe, forming a jet consisting of the mixture of the solutions at the syringe outlet, which jet breaks up into droplets.

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

23. Use of a kit intended for the preparation of microcompartments 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, characterised in that The fibrinogen solution and the thrombin solution are of human origin and comply with regulations related to Good Manufacturing Practice (GMP).

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