Large cellular microcompartments comprising several cysts

Abstract

The invention relates to a three-dimensional cellular microcompartment or a set of three-dimensional cellular microcompartments of ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an outer hydrogel layer defining an inner part (14), said inner part comprising at least:- extracellular matrix elements, and- at least two cysts, each cyst being formed by at least one layer of human or animal cells organized in three dimensions around a lumen, the smallest radius or the average radius of the inner part being at least 100 µm. The invention also relates to a method for preparing such a microcompartment or set of microcompartments.

Description

Title of the invention: Large cellular microcompartments comprising several cysts Technical field

[0001] The invention relates to the culture of epithelial-type cells, such as pluripotent stem cells, in three dimensions. Prior art

[0002] Ex vivo cell culture is an area of increasing interest. Cultured cells can be of any type. They can include differentiated cells with different phenotypes, progenitor cells, and stem cells. A significant advance in cell culture techniques is the introduction of three-dimensional culture systems.

[0003] Three-dimensional cultures are indeed advantageously closer to natural in vivo systems, and can be used for numerous applications, particularly in the development of therapies. A particularly suitable technology is that described in application WO2018 / 096277, which consists of three-dimensional cellular microcompartments for the culture of stem cells.

[0004] However, despite their effectiveness, existing 3D culture systems still have limitations in terms of cell yield and growth rate to come even closer to in vivo expansion rates and cycle duration while ensuring the maintenance of a stable epithelial phenotype.

[0005] The objective of the invention is to propose a three-dimensional cell culture solution meeting all of these needs and overcoming the drawbacks and limitations of the prior art for an even more quantitative culture that is always at least as qualitative. Summary of the invention

[0006] By working on the development of cellular microcompartments for the 3D culture of epithelial cells or cells having an epithelial-type morphology and capable of forming cysts, such as pluripotent stem cells, the inventors have developed a system making it possible to increase the maximum number of cells contained in a microcompartment organized around a lumen (cyst) while retaining an epithelial phenotype.

[0007] According to the invention, maintaining a low seeding of cells makes it possible to increase the amplification factor between the seeding of the cells in the microcompartment and the harvesting of the microcompartment containing the amplified cells. However, in existing systems, microcompartments comprising some cells at seeding (1 to 3 in particular) die or restart their growth with a latency rate which harms the yield of the culture and increases the necessary duration of encapsulated culture.

[0008] According to the invention, these problems are linked in particular to microcompartments that are too small, and in particular to microcompartments whose internal part volume is too small.

[0009] The invention also relates to a three-dimensional cellular microcompartment with an external layer and an internal part, the internal part of which has dimensions large enough to allow a significant growth rate and a large quantity of cells at the time of harvesting the microcompartment, starting from a low seeding of cells at the start.

[0010] In particular, the invention relates to a three-dimensional microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an external hydrogel layer defining an internal part, said internal part comprising at least: - extracellular matrix elements, and - at least two cysts, each cyst being formed by at least one layer of cells organized in three dimensions around a lumen, the smallest radius of the internal part of the microcompartment being at least 100 pm, preferably at least 200 pm.

[0011] The cells of each layer of cells organized in three dimensions around a lumen are cells capable of forming a cyst, that is to say polarized cells with a basal face capable of forming tight junctions and of expressing podocalyxin on the apical face (facing the lumen of the cyst). These are in particular epithelial cells or cells having an epithelial-type morphology, human or animal. The cells of each layer of cells organized in three dimensions around a lumen are preferentially chosen from induced pluripotent stem cells (iPSCs) and the following cells: glandular epithelium cells (e.g., mammary or salivary), renal epithelium cells, intestinal epithelium cells (enterocytes), skin epithelium cells (keratinocytes), retinal pigment epithelium cells, epicardium cells, endocardium cells.

[0012] Advantageously, such an arrangement, in particular the presence of at least two cysts, makes it possible to increase the maximum number of cells contained in a microcompartment while retaining an epithelial phenotype around a lumen (cyst). The size of the microcompartment according to the invention is chosen to allow the growth of several cysts while preserving a diffusion distance compatible with the physiology of the cells.

[0013] The invention also relates to a set of three-dimensional cellular microcompartments comprising at least one cellular microcompartment according to the invention, preferably in liquid suspension in a bioreactor.

[0014] The microcompartments according to the invention can be useful for different applications and in particular in the prevention and / or treatment of pathologies.

[0015] The cellular microcompartments according to the invention can be obtained in particular by implementing a specific preparation method comprising the following steps: - (a) incubating cells in a culture medium, preferably in a culture medium containing at least one cytoprotective factor, in particular an inhibitor of apoptosis and / or Rho / A kinases, - (b) mixing the cells from step (a) with extracellular matrix elements, in particular a biological or synthetic extracellular matrix, - (c) encapsulating the cell suspension in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an external hydrogel layer defining an internal part, the smallest radius of said internal part being at least 100 pm; (d) culturing the microcompartments obtained in an isotonic rinsing buffer, preferably for less than 30 minutes, then in a culture medium, preferably in a culture medium containing at least one cytoprotective factor, in particular an inhibitor of apoptosis and / or Rho / A kinases; - (e) preferentially rinsing the microcompartments, so as to eliminate the cytoprotective factor (inhibitor of apoptosis and / or Rho / A kinases), preferentially within 48 hours after encapsulation, even more preferentially within 24 hours; - (f) culturing the microcompartments for at least two cell division cycles (amplification), preferably between 1 and 60 days, between 1 and 30 days, between 1 and 20 days, even more preferably between 2 and 30 days, between 2 and 20 days, between 3 and 30 days, between 3 and 20 days, in particular between 4 and 7 days, in particular between 5 and 7 days, in a culture medium devoid of cytoprotective factor, and - (g) optionally recover the cellular microcompartments obtained.

[0016] The method according to the invention makes it possible to obtain microcompartments according to the invention with at least two cysts.

[0017] Other characteristics and advantages will emerge from the detailed description of the invention and the examples which follow. Brief description of the figures

[0018] [Fig.1a] is a diagram of a microcompartment according to the invention comprising several cysts of induced pluripotent stem cells. This diagram is a representation of the microcompartment shown in the photograph of [Fig.1b].

[0019] [Fig. 1b] is a phase contrast microscopy image of a microcompartment according to the invention.

[0020] [[Fig.2a] is a diagram of a series of microcompartments according to the invention.

[0021] [Fig.2b] is a phase contrast microscopy image of a series of microcompartments according to the invention.

[0022] [Fig.3a] is a diagram of a bioreactor containing a series of microcompartments according to the invention.

[0023] [Fig.3b] is an image of a bioreactor containing a series of microcompartments according to the invention.

[0024] [Fig.4a] is a diagram of the fusion of two cysts in a microcompartment according to the invention.

[0025] [Fig.4b] is an image of the fusion of two cysts in a microcompartment according to the invention.

[0026] [Fig.5] is a representation of the results of tests on the amplification of induced pluripotent stem cells in microcompartments according to the invention. Detailed description of the invention

[0027] Definitions

[0028] For the purposes of the invention, the term "alginate" means linear polysaccharides formed from [3-D-mannuronate and aL-guluronate, salts and derivatives thereof.

[0029] By “hydrogel capsule” or “hydrogel microcompartment” within the meaning of the invention, we mean a three-dimensional structure formed from a matrix of polymer chains, swollen by a liquid and preferably water.

[0030] For the purposes of the invention, “differentiated” cells are understood to mean cells which have a particular phenotype, as opposed to pluripotent stem cells which are not differentiated or progenitor cells which are in the process of differentiating.

[0031] For the purposes of the invention, the term "epithelial cells" or "epithelial-type cells" means human or animal cells associated with each other by means of intercellular junctions structuring a simple epithelium (cuboidal, prismatic, squamous, or pseudostratified, i.e. a layer of closely juxtaposed or joined cells, the majority of whose cells contact the apical face and the basolateral face of the layer).

[0032] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention are constituted or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0033] For the purposes of the invention, the term “mutant cell” means a cell carrying at least one mutation.

[0034] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in cell differentiation but not yet differentiated.

[0035] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, embryonic stem cells of human beings can be excluded.

[0036] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire organism of origin, without being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPS in the present document. In particular, they may 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").

[0037] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106).

[0038] For the purposes of the invention, the term “layer of cells” means a monolayer of cells or epithelial layer.

[0039] For the purposes of the invention, the term “cyst” means a three-dimensional arrangement in a monolayer of cells or in an epithelial layer, spherical, surrounding a central lumen.

[0040] By "Feret diameter" of a microcompartment (or of a part of a microcompartment) according to the invention, we mean the distance "d" between two tangents to said microcompartment (or to said part), these two tangents being parallel, such that the entire projection of said microcompartment (or of said part) is between these two parallel tangents. A Ferret diameter of the internal part of the microcompartment is measured between two interfaces of the internal part and the external layer of the microcompartment, i.e. the distance "d" between two tangents to said internal part, these two tangents being parallel, such that the entire projection of said internal part is between these two parallel tangents.

[0041] By “variable thickness” of a layer, we mean within the meaning of the invention the fact that the layer for the same microcompartment does not have the same thickness everywhere.

[0042] By “microcompartment” or “capsule” within the meaning of the invention, we mean a partially or totally closed three-dimensional structure, containing several cells.

[0043] By “convective culture medium” within the meaning of the invention is meant a culture medium animated by internal movements.

[0044] For the purposes of the invention, the term "mutation" means a genetic or epigenetic mutation, preferably a functional mutation. It may in particular be a specific modification of the genetic sequence, a structural variant, an epigenetic modification, or a modification of mitochondrial DNA.

[0045] By "functional mutation" within the meaning of the invention, is meant a transmissible genetic or epigenetic modification which confers a gain or loss of function or potential loss of function to the mutant cell concerned. This is preferably a mutation leading to a modification of the phenotype of the mutant cell concerned. Very preferably it is a change in the sequence of the genome and / or the epigenome which alters the therapeutic potential of a population of cells, either by increasing the risk associated with the therapy produced or by reducing the benefit provided by the therapy produced.

[0046] By "largest dimension" of a microcompartment or of a cluster of cells or of a layer of cells within the meaning of the invention, we mean the value of the largest Feret diameter of said microcompartment.

[0047] By "smallest dimension" of a microcompartment or of a layer of cells within the meaning of the invention, we mean the value of the smallest Feret diameter of said microcompartment.

[0048] By "tissue" or "biological tissue" within the meaning of the invention, we mean the common meaning of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a set of similar cells of the same origin (most often from a common cell lineage, although they can find their origin by association of distinct cell lineages), grouped in clusters, networks or bundles (fibers). A tissue forms a functional whole, that is to say its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs.

[0049] By "light" or "lumen" within the meaning 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.

[0050] By "smallest radius" of the internal part of a microcompartment according to the invention, we mean half the value of the smallest Ferret diameter of the internal part of the microcompartment.

[0051] By "average radius" of the internal part of a microcompartment according to the invention, we mean the average of the radii of the smallest compartment, each radius corresponding to half the value of a Ferret diameter of the internal part of the microcompartment.

[0052] Cellular microcompartments

[0053] The subject of the invention is therefore a three-dimensional microcompartment 10 of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an external layer 12 of hydrogel defining an internal part 14, said internal part 14 comprising at least: - extracellular matrix elements 16, and - at least two cysts, each cyst being formed by at least one layer of cells 18 organized in three dimensions around a lumen 20.

[0054] Preferably, the microcompartment is characterized in that the smallest radius of the internal part 14 is at least 100 pm, preferably at least 200 pm, in particular between 200 and 400 pm.

[0055] The microcompartment according to the invention comprises an external hydrogel layer. Preferably, the hydrogel used is biocompatible, that is to say it is not toxic to the cells. The external hydrogel layer must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival. According to one embodiment, the external hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be a sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, with a mass average molecular weight of 100 to 400 kDa and a total concentration of between 0.5 and 5% by mass. The outer hydrogel layer is devoid of cells.

[0056] The external hydrogel layer makes it possible in particular to protect the cells from the external environment, to limit the uncontrolled proliferation of the cells, and their differentiation in the event of differentiation.

[0057] The average thickness of the external layer 12 can be variable. It is preferably between 5 and 100 um, more preferably between 20 and 60 um. The ratio between the smallest radius of the internal part 14 and this thickness is preferably between 2 and 10.

[0058] The presence of an external layer of hydrogel and extracellular matrix elements (preferably an extracellular matrix) allows a uniform distribution of the cells between the microcompartments. Furthermore, this external layer of hydrogel makes it possible to avoid fusions of microcompartments which are a major source of variability unfavorable for the phenotypic homogeneity of the cells.

[0059] The internal part 14, inside the external hydrogel layer, therefore comprises at least: - extracellular matrix elements 16, preferably in the form of an isotonic solution comprising extracellular matrix elements, in particular a biological or synthetic extracellular matrix, such as for example Matrigel®, and - at least two cysts, each cyst being formed by at least one layer of cells 18 organized in three dimensions around a lumen 20.

[0060] The extracellular matrix elements preferably comprise peptide or peptidomimetic sequences capable of binding to integrins. They are preferably located in a layer between the cell cysts and the external hydrogel layer 12. These extracellular matrix elements have preferably been added during the manufacture of the microcompartment and / or they have been added to the microcompartment a posteriori and / or they have been secreted or induced by the other constituents of the microcompartment.

[0061] The extracellular matrix elements preferably comprise a mixture of proteins and extracellular compounds necessary for the culture and amplification of cells. Preferably, the extracellular matrix elements comprise structural proteins, such as collagen, laminins, entactin, vitronectin, as well as growth factors, such as TGF-beta and / or EGF. According to a variant, the internal part 14 may consist of or comprise Matrigel® and / or Geltrex® and / or a hydrogel-type matrix of plant origin such as modified alginates or of synthetic origin or of copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) type Mebiol®. The presence of extracellular matrix elements promotes the adhesion of the initially encapsulated cells and contributes to obtaining and maintaining in the microcompartment at least two sources of separate cells that do not meet to each form a cyst.

[0062] According to a preferred embodiment, the internal part 14 comprises an extracellular matrix.

[0063] If the microcompartment comprises in the internal part 14, extracellular matrix, it may be extracellular matrix secreted by the cells present in the microcompartment and / or extracellular matrix added at the time of preparation / manufacture of the microcompartment.

[0064] According to a variant, the solution containing extracellular matrix elements, in particular if it is a natural (biological) or synthetic extracellular matrix, can form a gel.

[0065] At the surface of the solution containing the extracellular matrix elements (preferably a natural or synthetic extracellular matrix) in contact with a layer of cells, the solution containing the extracellular matrix elements (preferably a natural or synthetic extracellular matrix) may optionally contain one or more cells.

[0066] Preferably, the solution containing the extracellular matrix elements (preferably a natural or synthetic extracellular matrix) has a Young's modulus of between 0.05 and 3 kDa. The Young's modulus can be measured by any method known to those skilled in the art, in particular by measuring the rheology of gels of the same composition as the intermediate layer or by AFM (atomic force microscopy).

[0067] A solution containing the extracellular matrix elements (preferably a natural or synthetic extracellular matrix) with such Young's modulus values makes it possible to improve the maintenance of the cellular phenotype and the genomic integrity of the cells contained in this intermediate layer during cell divisions.

[0068] In addition, such Young's modulus (elasticity) values promote the adhesion of the initially encapsulated cells and contribute to obtaining and maintaining in the microcompartment at least two separate sources of cells which do not meet to each form a cyst. The viscosity of the solution containing the extracellular matrix elements (preferably a synthetic or natural extracellular matrix) also promotes the adhesion of the initially encapsulated cells and contributes to obtaining and maintaining in the microcompartment at least two separate sources of cells which do not meet to each form a cyst.

[0069] The internal part 14 may also comprise liquid zones without extracellular matrix elements. These liquid zones result from the equilibration by diffusion of the liquids present during the culture (initial seeding and possible change / renewal of media). This liquid zone is preferably mainly composed of culture media. Advantageously, the external layer of the microcompartment retains (at least partially) close to the cells, the factors and elements secreted by them, which reinforces the paracrine and autocrine effects within the microcompartment.

[0070] The internal part 14 of the microcompartment according to the invention comprises at least two cysts, each cyst being formed by at least one layer of cells 18 organized in three dimensions around a lumen 20.

[0071] Each cyst is formed by at least one layer of cells organized in three dimensions around a lumen, these human or animal cells preferably excluding human embryonic stem cells. These cells are cells capable of forming a cyst, that is to say polarized cells with a basal face capable of forming tight junctions and of expressing podocalyxin on the apical face (facing the lumen of the cyst). In a preferred embodiment, each cyst is formed by at least one layer of epithelial cells or cells having an epithelial-like morphology. In a preferred embodiment, the cells of each layer 18 are epithelial cells or cells having an epithelial-like morphology and capable of forming a cyst.

[0072] Preferably, each cyst is formed by at least one layer of human or animal cells, organized in three dimensions around a lumen, chosen from induced pluripotent stem cells (iPSCs) and the following cells: glandular epithelium cells (e.g. mammary or salivary), renal epithelium cells, intestinal epithelium cells (enterocytes), skin epithelium cells (keratinocytes), retinal pigment epithelium cells, epicardium cells, endocardium cells.

[0073] A pluripotent stem cell, or pluripotent cell, is 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 be, in particular, induced pluripotent stem (IPS) cells, MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells.

[0074] Preferably, the cells constituting each cyst are polarized. The polarity of these cells inside each cyst can be demonstrated by the proteins TJP-1 or ZO-1, both located on the inner / apical face of the pluripotent cell layer, which adjoins the lumen.

[0075] If the cells encapsulated in the microcompartment are intended to be used in cell therapy in humans, the cells may be immunocompatible with the person intended to receive them to avoid any risk of rejection.

[0076] The cells present in the microcompartment carry few, if any, functional mutations.

[0077] The microcompartment according to the invention can contain at least 80, preferably at least 800, at least 1000, at least 5000, in particular at least 8000 cells, these cells being organized in the form of at least two cysts.

[0078] Each cyst formed by at least one layer of cells within the microcompartment is hollow, that is to say it has a light or lumen. The light is preferentially generated, at the time of cyst formation, by the secretion of podocalyxin by the cells.

[0079] The lumen of each cyst 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. Furthermore, the internal part 14 is in equilibrium with the medium external to the microcompartment but can advantageously be enriched by the action of the cells with metabolic and / or secreted elements.

[0080] The cyst-shaped conformation allows for reduced pressures on cells compared to 2D or aggregate cultures. This configuration allows for reduced cell mortality and increased culture amplification factor. Consequently, this allows for reduced number of passages and dissociations required; reduced culture time required to reach the final cell number required. Collectively, these improvements also contribute to maintaining the genetic integrity of cells in microcompartments.

[0081] Advantageously, the presence of several cysts in the same microcompartment makes it possible to improve the reproducibility of the encapsulation process and thereby the reproducibility of the cell batches, improves survival during encapsulation and improves amplification per unit of time.

[0082] The microcompartment may also comprise, in addition to the cysts, cells suspended in the microcompartment.

[0083] According to one embodiment, each cyst present in the microcompartment was obtained by encapsulation in the internal part 14 of the microcompartments: from 1 to 30 cells, preferably from 1 to 10, in particular from 2 to 30 or from 3 to 30, in particular from 2 to 10 or from 3 to 10, more preferably from 1 to 5, even more preferably from 2 to 5 or from 3 to 5, preferably at least 4 cells, and the smallest radius of the internal part 14 being at least 100 pm, preferably at least 200 pm. The encapsulation of a controlled concentration of cells in a large capsule (smallest radius of the internal part of the microcompartment of at least 100 pm) contributes to obtaining at least two cysts in the microcompartment. It is difficult to obtain two cysts with a single cell, and it is preferable to encapsulate in the internal part 14 of the microcompartments at least 2 cells, preferably at least 3, even more preferably at least 4.Encapsulation of an initial number of cells greater than 30 is possible but less advantageous because it is difficult or even impossible (the probability decreases) to obtain two cysts with more than 20 / 25% in cell volumes in the internal part at the time of encapsulation, in particular because this limits the amplification capacity to X4 which is disadvantageous for the production of cell batches. According to the invention it is preferable that there are at least two separate sources of cells which do not meet to each form a cyst.

[0084] The cells present in the microcompartment according to the invention were preferentially obtained after at least two cycles of cell division after encapsulation in an external layer of hydrogel of at least one cell.

[0085] Preferably, the cells present in the microcompartment according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division after encapsulation in an external hydrogel layer, preferably of at least 2 cells, preferably at least 3, even more preferably at least 4, in particular between 2 and 30 cells. For example, the cells present in the microcompartment have been obtained after at least six cycles of cell division after encapsulation of the cells in the external hydrogel layer.

[0086] Preferably the number of cell divisions for implementing the method according to the invention is less than 300, even more preferably less than 200.

[0087] Preferably the microcompartment is obtained after at least 2 passages after encapsulation, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage can last for example between 2 and 15 days, in particular between 3 and 10 days.

[0088] Preferably, the microcompartment is obtained after at least one re-encapsulation, more preferably between 1 and 14 re-encapsulations, in particular between 2 and 7 re-encapsulations. Very preferably, one re-encapsulation corresponds to a new passage and each encapsulation cycle corresponds to one passage.

[0089] 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.

[0090] Thus, according to one embodiment, the cells present in the microcompartment according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division, after encapsulation in an external layer of hydrogel of cell(s) representing a volume less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0091] 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.

[0092] In the microcompartment according to the invention, the volume of the internal part 14 preferably represents at least 20% of the total volume of the microcompartment, preferably at least 40%. This makes it possible to accommodate more cells after amplification, therefore more cysts and / or larger cysts.

[0093] The thickness of the layer of cells forming each cyst is preferably between 6 and 200 pm, more preferably between 6 and 60 pm.

[0094] According to a variant shown in [Fig.4a] and in [Fig.4b], at least two cysts can fuse together. In this case, the microcompartment according to the invention comprises at least one cyst resulting from the fusion of two cysts.

[0095] The cellular microcompartment according to the invention is closed or partially closed, that is to say that the external layer is closed or partially closed. Preferably the microcompartment is closed.

[0096] The microcompartment according to the invention can be in any three-dimensional form, that is to say it can have the shape of any object in space. The microcompartment can have any shape compatible with the encapsulation of cells. Preferably, the microcompartment according to the invention is in a spherical or elongated or substantially spherical or elongated shape. It can have the shape of an ovoid, a cylinder, a spheroid or a sphere or substantially this shape.

[0097] It is the outer layer of the microcompartment, i.e. 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 202 pm and 1 mm, preferably between 202 pm and 700 pm, even more preferably 202 pm and 600 pm, in particular between 202 pm and 500 pm.

[0098] Its largest dimension is preferably greater than 202 pm, more preferably between 202 pm and 1 m, even more preferably between 202 pm and 50 cm.

[0099] The invention also relates to several microcompartments together.

[0100] The invention also relates to a set or series of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to the invention.

[0101] Preferably, the series of microcompartments according to the invention is in a culture medium, in particular in an at least partially convective culture medium. Any culture medium suitable for cell culture can be used, and in particular saline phosphate buffer such as, for example, “dulbecco's modified eagle medium” or “Roswell Park Memorial Institute medium” provided that the concentration of dissolved salts is compatible with maintaining the crosslinking of the alginate by the divalent cations.

[0102] According to a particularly suitable embodiment, the invention relates to a series of cellular microcompartments in a closed enclosure, such as a bioreactor, preferably in a culture medium in a closed enclosure, such as a bioreactor. Thus, preferably, the microcompartments are arranged in a culture medium in a closed bioreactor.

[0103] The set or series of microcompartments according to the invention preferably comprises between 2 and 1016 microcompartments.

[0104] The microcompartments according to one of the invention can be used for all applications, in particular as a medicament in cell therapy in humans or animals.

[0105] The microcompartment according to the invention may optionally be frozen for storage. It should then preferably be defrosted before use.

[0106] Method for obtaining micro compartments according to the invention

[0107] The invention also relates to a method for preparing microcompartments according to the invention.

[0108] The method for preparing a microcompartment or a set of microcompartments according to the invention may comprise the following steps: - (a) incubating human or animal cells in a culture medium containing at least one cytoprotective factor, in particular an inhibitor of apoptosis and / or Rho / A kinases, - (b) mixing the cells from step (a) with extracellular matrix elements, in particular a biological or synthetic extracellular matrix, - (c) encapsulating the cell suspension in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an external hydrogel layer defining an internal part, the smallest radius or the average radius of said internal part being at least 100 pm; - (d) culturing the microcompartments obtained in an isotonic rinsing buffer, preferably for less than 30 minutes, then in a culture medium, preferably in a culture medium containing at least one cytoprotective factor, in particular an inhibitor of apoptosis and / or Rho / A kinases; - (e) preferentially rinsing the microcompartments, so as to eliminate the cytoprotective factor (inhibitor of apoptosis and / or Rho / A kinases), preferentially within 48 hours after encapsulation, even more preferentially within 24 hours; - (f) culturing the microcompartments for at least two cell division cycles (amplification), preferably between 1 and 20 days, even more preferably between 2 and 10 days, in particular between 5 and 7 days, in a culture medium devoid of cytoprotective factor (apoptosis inhibitor and / or Rho / A kinases), and - (g) optionally recover the cellular microcompartments obtained.

[0109] The number of cells encapsulated in each microcompartment in step c) is preferably from 1 to 30 cells, in particular from 2 to 30 or from 3 to 30, preferably from 1 to 10, in particular from 2 to 10 or from 3 to 10, more preferably from 1 to 5, even more preferably from 2 to 5 or from 3 to 5, preferably at least 4 cells. The encapsulation of a controlled concentration of cells in a large capsule (smallest radius of the internal part of the microcompartment of at least 100 μm) contributes to obtaining at least two cysts in the microcompartment. It is difficult to obtain two cysts with a single cell, and it is preferable to encapsulate in the internal part 14 of the microcompartments at least 2 cells, preferably at least 3, even more preferably at least 4.Encapsulation of an initial number of cells greater than 30 is possible but less advantageous because it is difficult or even impossible (the probability decreases) to obtain two cysts with more than 20 / 25% in cell volumes in the internal part at the time of encapsulation, in particular because this limits the amplification capacity to X4 which is disadvantageous for the production of cell batches. According to the invention it is preferable that there are at least two separate sources of cells which do not meet to each form a cyst.

[0110] Any culture medium suitable for the culture of cells, in particular pluripotent stem cells, can be used, and in particular saline phosphate buffer such as the medium “Roswell Park Memorial Institute medium” or Mtesrl or E8 Essential for example.

[0111] The cytoprotective factor may for example be one or more inhibitor(s) of the RHO / ROCK (“Rho-associated protein kinase”) pathways and / or an apoptosis inhibitor known to those skilled in the art or any other cytoprotective factor known to those skilled in the art. The cytoprotective factor must make it possible to promote the survival of the cells, and in the case of the presence of an extracellular matrix, their adhesion of the cells to the extracellular matrix at the time of the formation of the external layer of hydrogel around said extracellular matrix.

[0112] In the method according to the invention, all of the cells initially encapsulated in step (c) preferably 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.

[0113] The method according to the invention may comprise a step prior to step a) or b) of dissociating the cells by chemical, enzymatic or mechanical dissociation. This step is very preferably necessary because epithelial type cells, in particular stem cells, are adherent cells.

[0114] The encapsulated cells are suspended in the form of single cells and / or clusters or group(s) of at least two cells ("cluster(s)"). Preferably, when there are at least 3 cells, the single cell(s) represent less than 50% in number of all the cells initially encapsulated in step (b). Indeed, it is preferable to encapsulate clusters of cells because this reduces distortions of chromosomal segregation and bad chromosomal segregations and consequently reduces the occurrence of new mutagenesis and contributes to maintaining the genomic integrity of the cells, because the isolated cells risk dying due to a lack of cell-cell interaction and the complete dissociation of cells leads to increased genetic abnormalities.

[0115] Preferably, each cluster of cells initially encapsulated in step (c) has a largest dimension less than 20% of the largest dimension of a microcompartment in which it is encapsulated, even more preferably less than 10%. Indeed, the clusters of cells must not be too large in size compared to the size of the microcompartment because too large a dimension of these initial cell clusters could lead, during cell divisions, to earlier cell confluence in the capsule; this too early confluence of all or part of the capsules could lead to an increase in pressures. intracellular and cause cellular stress, impacting cell growth but also chromosome segregation.

[0116] The method according to the invention comprises a step b) of mixing the cells with an extracellular matrix between step a) and step c). According to a variant, this step b) can optionally be implemented before step (a) or simultaneously with the encapsulation in step (c).

[0117] Step c) of encapsulation is carried out according to techniques known to those skilled in the art. Indeed, any method for producing cellular microcompartments containing extracellular matrix and cells inside a hydrogel capsule can be used for implementing the preparation method according to the invention. In particular, it is possible to prepare microcompartments by adapting the method and the microfluidic device described in Alessandri et al., 2016 (“A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)”, Lab on a Chip, 2016, vol. 16, no. 9, p. 1593-1604), in accordance with the steps described below.

[0118] Preferably, step c) is implemented in a device capable of generating hydrogel capsules using a microfluidic chip. For example, the device may comprise syringe pumps for several solutions injected concentrically using a microfluidic injector which makes it possible to form a jet which splits into drops then collected in a calcium bath. According to a particularly suitable embodiment, two or three solutions are loaded onto two or three syringe pumps: - a hydrogel solution, for example alginate, - optionally an isotonic intermediate solution, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ to avoid crosslinking of the hydrogel too early in the injector, such as for example a sorbitol solution,

[0119] - the solution resulting from step b) comprising cells, culture medium and the extracellular matrix. The three solutions are co-injected (injected simultaneously) concentrically using a microfluidic injector or microfluidic chip which allows the formation of a jet which splits into drops whose outer layer is the hydrogel solution and the core the solution from step b) comprising cells; These drops are collected in a calcium bath which crosslinks and / or gels the alginate solution to form the shell.

[0120] To improve the mono-dispersity of the cellular microcompartments, the hydrogel solution is preferentially charged with a direct current at (between 1 and lOkV). A mass ring can optionally be placed at a distance from the tip of between 1mm and 20cm, preferably 3mm to 10cm, even more preferably 1cm to 5cm, from the tip in the plane perpendicular to the axis of the jet leaving the microfluidic injector (coextrusion chip) to generate the electric field.

[0121] Preferably, the solution is maintained at a temperature below 4°C before being injected, to prevent the solution from gelling due to the presence of extracellular matrix elements.

[0122] According to the invention, it is necessary to generate capsules whose internal part has a mean radius or smaller radius of at least 100 pm. To generate capsules with such dimensions with a coextrusion chip (microfluidic injector or microfluidic chip) the invention proposes in particular to modify the flow rate of the coextruded solutions and the final opening of the coextrusion chip. By flow rate is meant the flow rate of each solution that arrives at the injector. By final opening of the coextrusion chip is meant the internal opening of the outlet channel of the chip.- for the flow rate: we preferentially go from a value between 20 and 40 ml / h for each coextruded solution for the standard sizes of capsules known from the prior art, to a value between 45 and 150 mL / h, preferentially between 45 and 110 mL per hour for a variant of the invention, - for the diameter of the final opening of the coextrusion chip (microfluidic injector) which goes from a value preferentially between 50 and 120 pm for the standard sizes of capsules known from the prior art, to a diameter value between 150 and 300 pm, preferentially between 180 and 240 pm.

[0123] Thus, according to a particular embodiment, the encapsulation step (c) is carried out using a microfluidic injector whose final opening diameter is between 150 and 300 μm, preferably between 180 and 240 μm, and with the flow rate of each of the 3 solutions between 45 and 150 mL / h, preferably between 45 and 110 mL / h.According to one embodiment, this encapsulation is carried out by co-injection of three solutions: - a hydrogel solution, - an isotonic intermediate solution such as for example a sorbitol solution, - the solution resulting from step b) comprising cells, culture medium and the extracellular matrix, concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of the three solutions, said jet breaking up into drops, said drops being collected in a calcium bath which stiffens the hydrogel solution to form the external layer of each microcompartment, . the internal part of each drop being constituted by the solution resulting from step (b) comprising cells, culture medium and the extracellular matrix.

[0124] Very preferably, steps (d), (e) and (f) are carried out with permanent or sequential stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusive gradient. For example, it allows homogeneous control of the level of cellular oxygenation; thus avoiding the phenomena of necrosis linked to hypoxia, or oxidative stress linked to hyperoxia. By avoiding an increase in cell mortality and / or oxidative stress, stirring helps to maintain the genetic integrity of the cells.

[0125] The method according to the invention is preferably implemented in a closed enclosure such as a closed bioreactor.

[0126] The number of cell division cycles in step (f) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cell division cycles.

[0127] Preferably the microcompartment is obtained after at least 2 passages (one passage corresponding here to a complete cycle of steps (a), (b), and (e), optionally (c) and (d)), 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.

[0128] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the cells after step (f), i.e. at least two encapsulation cycles. Preferably, each encapsulation cycle corresponds to one passage. In this variant of the method (at least one re-encapsulation of the cells after step (e)) the number of cell divisions of the entire method (for all the passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0129] In a method according to the invention there may be several re-encapsulations, preferably between 1 and 100, in particular between 1 and 10 re-encapsulation(s).

[0130] Each re-encapsulation may comprise: - a step of dissociating the microcompartment or series of microcompartments to obtain a cell suspension or a cell cluster suspension; removal of the external hydrogel layer may be achieved in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, removal may be achieved using a saline phosphate buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection, and - a step of re-encapsulation of all or part of the cells or clusters of cells in a hydrogel capsule. Re-encapsulation is a suitable means for a increase in cellular amplification obtained from the pluripotent stage, and reduce the risks of mutation.

[0131] Re-encapsulation consists of removing the external layer of hydrogel, preferably resuspending in a partially or totally dissociated manner the cells which were in the form of cysts in the microcompartments and implementing the steps of the process again.

[0132] According to one embodiment, the re-encapsulation comprises the following steps: - (i) removing the external hydrogel layer, - (ii) resuspending the cells which were contained in the microcompartment so as to obtain single cells and / or at least one set or cluster of cells in a culture medium containing at least one cytoprotective factor, - (iii) mixing the cells from step (a) with extracellular matrix elements, - (iv) encapsulating the cell solution in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an outer hydrogel layer defining an inner portion, the smallest radius or the average radius of said inner portion being at least 100 pm; - (v) culturing the microcompartments obtained in a culture medium containing at least one cytoprotective factor, in particular an inhibitor of apoptosis and / or Rho / A kinases, - (vi) preferentially rinse the microcompartments, so as to remove the cytoprotective factor; - (vii) culturing the microcompartments in a culture medium devoid of cytoprotective factor, for at least one cell division cycle, and - (viii) optionally recover the cellular microcompartments obtained.

[0133] Compartmentalization in microcompartments makes it possible to eliminate microcompartments containing more mutated cells than other capsules. Even if the mutated cells have rapid growth, they will reach the capsular confluence which will contain their multiplication. Compartmentalization also makes it possible not to contaminate the entire cell population, and also to eliminate capsules containing mutant cells, at any time, in particular before a re-encapsulation step. This sorting can be done either by online analysis, or by eliminating capsules filled more quickly than the others, for example.

[0134] According to a variant of the invention, the encapsulated cells are differentiated cells which are reprogrammed into pluripotent cells inside the hydrogel capsule during the formation of the microcompartments. According to a variant, the cell reprogramming agents may be added in step (a) and / or (b) and / or (c) and / or (d) and / or (ii) and / or (iii) and / or (iv) and / or (v). Preferably, these are cell reprogramming agents that are non-permeant with respect to the hydrogel layer. The addition of reprogramming agents is particularly relevant when the initially encapsulated cells are differentiated cells that it is desired to dedifferentiate, in particular to the pluripotent stage. A person skilled in the art knows how to reprogram a differentiated cell into a stem cell by reactivating the expression of genes associated with the embryonic stage using specific factors, referred to in the present invention as "reprogramming agents". Examples include the methods described in Takahashi et al., 2006 (“Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” Cell, 2006 Vol 126, pages 663-676), Ban et al., 2009 (“Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome” Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85(8):348-62) and in international application WO2010 / 105311 entitled “Production of reprogrammed pluripotent cells”. The reprogramming agents are advantageously co-encapsulated with the differentiated cells, so as to concentrate the product and promote contact with all the cells. In the case of reprogramming agents permeable to the hydrogel layer, it is possible to add said agents to the culture medium after the encapsulation step. The reprogramming agents make it possible to impose on the cells a succession of phenotypic changes up to the pluripotent stage. Advantageously, the reprogramming step is carried out using specific culture media, promoting these phenotypic changes.For example, the cells are cultured in a first medium comprising 10% human or bovine serum in a minimum essential medium of Eagle (DMEM) supplemented with a serine / threonine protein kinase receptor inhibitor (such as the product SB-431542 (C22Hi6N4O3)), one or more inhibitors of the RHO / ROCK pathways (Rho-associated protein kinase), such as thiazovivin and / or Y-27632, fibroblast growth factors, such as FGF-2, ascorbic acid and antibiotics, such as Trichostatin A (C17H22N2O3). Then the culture medium is replaced with a medium promoting the multiplication of pluripotent cells, such as the mTeSR® 1 medium.

[0135] The incubation of step (a) and / or (ii) is preferably carried out for a time between a few minutes and a few hours, preferably between 2 minutes and 2 hours, more preferably between 10 minutes and 1 hour.

[0136] Step (d) and / or (v) of culture with a cytoprotective factor is carried out for a time of between 2 and 48 hours, preferably for a time of between between 6 and 24 hours, more preferably for a period of between 12 and 18 hours.

[0137] The rinsing step can be carried out by one or more rinses, in successive culture media free of RHO / ROCK pathway inhibitors, less than 48 hours, preferably less than 24 hours, more preferably between 12 and 18 hours after the start of step (d) and / or (v).

[0138] In one embodiment, at least one of the steps (preferably all steps) is carried out at a temperature suitable for cell survival, between 4 and 42°C. The temperature during cell proliferation should preferably be between 32 and 37°C to avoid triggering mutations by lowering the performance of the repair enzymes. Similarly, preferably, the temperature should be low (ideally around 4°C) to manage cell stress in step (c).

[0139] At any time, the method according to the invention may comprise a step consisting of verifying the phenotype of the cells contained in the microcompartment. This verification may be carried out by identifying the expression by at least a portion of the cells contained in the microcompartment, of at least one gene specific to the desired phenotype.

[0140] The cellular microcompartments obtained according to the methods of the invention can then be frozen before any use. Freezing is preferably carried out at a temperature between -190°C and -80°C. Thawing can be carried out in a warm water bath (preferably 37 degrees) so that the cells thaw fairly quickly. The microcompartments according to the invention, before their use, can be kept at more than 4°C for a limited period before their use, preferably between 4°C and 38°C.

[0141] The implementation of the method according to the invention makes it possible to obtain microcompartments comprising at least 80, preferably at least 800, at least 1000, at least 5000, in particular at least 8000 cells, these cells being organized in the form of at least two cysts.

[0142] Advantageously, the 3-dimensional structure of the cells in the microcompartment and the low or zero percentage of cells isolated during encapsulation (the majority of cells being encapsulated in the form of clusters of cells), reduces chromosomal desegregation and consequently reduces the occurrence of new mutagenesis.

[0143] The invention especially promotes amplification with a high amplification factor, which consequently reduces the culture time and the number of divisions to obtain a very large number of cells, and therefore limits new mutagenesis.

[0144] The protection of cells thanks to the outer layer and the presence of extracellular matrix elements reduces chromosomal desegregation and limits the mechanical stress of cells, and therefore reduces the occurrence of new mutagenesis.

[0145] Advantageously, the presence of several cysts in each microcompartment also makes it possible to promote initial cell survival and smooth out growth asynchronies.

[0146] The invention is now illustrated by an example and results

[0147] Figures 1a and 1b show hollow hydrogel capsules allowing the growth of multiple human pluripotent stem cell cysts in the central microcompartment.

[0148] Figures 2a and 2b show a microscopy image, at D6.5 post encapsulation showing a plurality of hollow alginate capsules containing human pluripotent stem cells. The scale bar is 500um. The microcompartments according to the invention comprise several 3D stem cell colonies. These colonies all have a homogeneous cystic configuration with an average diameter of 170um. On average, 2.6 colonies (cysts) are observed per capsule at this stage of the culture.

[0149] Figures 3a and 3b show a plurality of multi-cyst capsules cultured in suspension in a bioreactor.

[0150] The key parameters and results of an example of encapsulation according to the invention in a 10-liter bioreactor are given in Table 1 below.

[0151] [Tables 1] 10L Bioreactor - Trial 10L Bioreactor - Trial 2 Target Oxygen Threshold 20% DO 20% DO Maximum Volume 10.2L 9.7L Min % Capsule Volume 4.5-15% 3.9-14% Max cells / ml Capsule 33.92m / ml 33.80m / ml hPSCs Seeded 56 Million 51 Million hPSCs Produced 15.2 Billion 14.52 Billions Amplification Factor (AF) X271 / 6.59 Days X282 / 6.69 Days Doubling Time (DTT) 19.4 Hours 19.73 Hours Number of Cycles (NCC) 8.14 8.08 Final Viability 99.5% 99.7% Final OCT4+ 92.0% 96.7% Final SOX2+ 99.6% 99.7% NANOG+ final 97.0% 98.4%

[0152] Encapsulation of hiPSCs and suspension culture were performed for 2 separate cycles. The target oxygen level of 20% dissolved oxygen is described as a hypoxic condition (in contrast to bioreactors with a dissolved oxygen level of 100% which are described as normoxic). A fed batch strategy, i.e., renewal of the culture medium by subtraction / addition of medium at regular intervals, was applied, resulting in an increase in the working volume and a decrease in the concentration of capsules relative to the total volume. Cell density can be expressed in millions of cells per milliliter of capsules. The harvested capsule volume is measured in a graduated glass cylinder. The measured cell number can be related to this previously measured capsule volume to deduce a cell concentration per capsule volume.The amplification factor (AF) is defined as AF=N(tO+Dt) / N(tO), where N(tO) and N(tO+Dt) are the initial cell numbers at tO and tO+Dt respectively. The population doubling level (PDL) is defined as PDL(t)= ln(AF) / ln(2). The population doubling time (PDT) is defined as PDT(t)=Dt / PDL where Dt is the time in culture.

[0153] Viability was assessed by the NC-3000 nucleocounter (Chemometech). Decapsulated and dissociated cells were fixed and stained for OCT4, SOX2 and NANOG and analyzed by flow cytometry, BD Accuri C6 plus. The results regarding the amplification factor are also shown in [Fig.5].

[0154] [Fig.4b] shows a series of photos from a video microscopy of a capsule comprising several cysts according to the invention.

[0155] The observation is carried out in transmission with a Nikon IM bio-station with a lOx objective. The average external diameter of the quasi-spherical capsule is 288um as indicated by the lOOum scale bar. The initial snapshot was taken 4 days after encapsulation. The sequence illustrates the growth of several colonies of pluripotent stem cells in a hollow hydrogel capsule. These colonies are cultured in a closed 3D microcompartment, defined externally by a layer of cross-linked alginate. The internal compartment is described as hollow, in the sense that it does not contain cross-linked alginate. The internal compartment containing the cells also contains extracellular matrix (here Matrigel®) which is visible under microscopy by a granulosity different from that of the alginate.

[0156] During encapsulation, the volume injected into the internal microcompartments is 50% from the cell / matrix mix and 50% from an isomolar sorbitol solution (see method). Thus, the internal space is at least 50% liquid.

[0157] Initially the 3 colonies have a homogeneous cystic configuration with respective diameters of 76um, 100um and 78um (cyst at the bottom right of the image). At this initial stage the epithelial thickness of these cysts is respectively 10um, 13um and 12um. We observe that the 2 cysts at the top left of the capsule gradually come into contact and fuse their internal lumens as well as their epithelia. This results in a larger stem cell colony which also has a circular and symmetrical cystic structure.

[0158] 54 hours after the start of the observation (right images before the last line of the [Fig.4b]), the 2 cystic colonies present in the capsule come into contact. At this stage the diameters of these cysts are 232 and 175um. The respective thicknesses of the cell layer of these cysts are 36 and 24um respectively. It is observed that the contact between these colonies is not followed by fusion of the lumens or epithelia. The boundary between these 2 structures remains clearly identifiable on the last and 12th image (75th hour).

[0159] A shift of 20 um to the left of the largest colony is observed, which appears to be pushed back by the small colony. Despite a slight flattening of the contact zone between the 2 colonies, their symmetrical cystic structures remain, however, maintained for each of the 2 colonies, with homogeneous cell layer thicknesses of 50 and 40 um respectively. This growth of the encapsulated colonies and their likely ability to push back into the internal capsular space is facilitated by the at least partially liquid nature of this internal hollow space. Furthermore, the use of progressively degradable extracellular matrices such as Matrigel, further allows cells to grow in a more permissive 3D environment than during encapsulation in a full hydrogel bead; and thus allows the maintenance of an epithelial cystic structure despite the forces generated by cell multiplication.

[0160] Overall, the partially liquid hollow intracapsular space of this large capsule allows 3D stem cell colonies to grow while preserving a stable cystic epithelial phenotype. The stability of this phenotype allows good phenotypic homogeneity of the produced cells and a robust / reliable bioproduction process, illustrating the persistence of the epithelial phenotype during the growth of several cysts.

[0161] [Fig.4a] illustrates a “multi-cyst” fusion sequence into capsules: - A: Self-organization and luminogenesis of 3D stem cell colonies inside the microcompartment. Formation of encapsulated stem cell cysts - B: Growth of stem cell cysts: progressive increase in cyst diameter, lumen diameter and cyst thickness. - C: The cysts come into contact - D: The cysts fuse their cell layers - E: The cysts also fuse their internal lumens and this results in a single cyst of larger size.

Claims

Claims

1. A three-dimensional microcompartment (10) of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an outer layer (12) of hydrogel defining an inner portion (14), said inner portion (14) comprising at least: - extracellular matrix elements (16), and - at least two cysts, each cyst being formed by at least one layer of human or animal cells (18), excluding human embryonic stem cells, organized in three dimensions around a lumen (20), the cells of each layer (18) being epithelial cells or having an epithelial-like morphology and capable of forming a cyst, the smallest radius of the inner portion (14) being at least 100 pm.

2. Microcompartment (10) according to the preceding claim, characterized in that the cells of each layer (18) are chosen from induced pluripotent stem cells (iPSC) and the following cells: glandular epithelium cells, renal epithelium cells, intestinal epithelium cells, skin epithelium cells, retinal pigment epithelium cells, epicardium cells, endocardium cells.

3. Microcompartment (10) according to one of the preceding claims, characterized in that the internal part (14) also comprises liquid zones without extracellular matrix elements.

4. Microcompartment according to one of the preceding claims, characterized in that the smallest radius of the internal part (14) is at least 200 pm.

5. Microcompartment according to one of the preceding claims, characterized in that the volume of the internal part (14) represents at least 20% of the total volume of the microcompartment, preferably at least 40%.

6. Microcompartment according to one of the preceding claims, characterized in that it is closed.

7. Microcompartment according to one of the preceding claims, characterized in that the external layer comprises alginate.

8. Cellular microcompartment according to one of the preceding claims, characterized in that at least one cyst results from the fusion of two cysts.

9. Microcompartment according to one of the preceding claims, characterized in that the cells present in the microcompartment have been obtained by encapsulation in the internal part of an external layer of hydrogel, from 2 to 30 cells.

10. Microcompartment according to one of the preceding claims, for use as a medicament.

11. Set of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to one of claims 1 to 9.

12. Set of microcompartments according to the preceding claim, characterized in that the microcompartments are arranged in a culture medium in a bioreactor.

13. A method for preparing a cellular microcompartment according to one of claims 1 to 9 or a set of cellular microcompartments according to one of claims 11 to 12, comprising the following steps: - (a) incubating at least two human or animal cells in a culture medium containing at least one cytoprotective factor, - (b) mixing the cells from step (a) with extracellular matrix elements, preferably a biological or synthetic extracellular matrix, - (c) encapsulating the cell suspension in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an external hydrogel layer defining an internal part, the smallest radius or the average radius of said internal part being at least 100 μm;- (d) culturing the microcompartments obtained in an isotonic rinsing buffer, then in a culture medium, preferably in a culture medium containing at least one cytoprotective factor, - (e) preferentially rinsing the microcompartments, so as to eliminate the cytoprotective factor; - (f) culturing the microcompartments for at least two cell division cycles (amplification), preferably between 1 and 20 days, even more preferably between 2 and 10 days, in particular between 5 and 7 days, in a culture medium devoid of cytoprotective factor, and - (g) optionally recovering the cell microcompartments obtained.

14. Method according to claim 13, characterized in that step c) is carried out by co-injection of two or three solutions: - a hydrogel solution, - optionally an isotonic intermediate solution, - the solution from step b) comprising cells, culture medium and the extracellular matrix, concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of solutions, said jet breaking up into drops, said drops being collected in a calcium bath which stiffens the hydrogel solution to form the external layer of each microcompartment, the internal part of each drop being constituted by the solution from step (b) comprising cells, culture medium and the extracellular matrix.

15. Method according to claim 14, characterized in that the final opening diameter of the microfluidic injector is between 150 and 300 pm, preferably between 180 and 240 pm, and the flow rate of each of the solutions is between 45 and 150 mL / h, preferably between 45 and 110 mL / h.

16. Method according to one of claims 13 to 15, characterized in that all of the cells initially encapsulated in step (c) represent a volume less than 50% of the volume of the microcompartment in which they are encapsulated.

17. Method according to one of claims 13 to 16, characterized in that step b) of mixing the cells with an extracellular matrix is carried out either between step (a) and step (c), or simultaneously with the encapsulation in step (c).

18. Method according to one of claims 13 to 17, characterized in that steps (d), (e) and (f) are carried out with permanent or sequential stirring.

19. Method according to one of claims 13 to 18, characterized in that it is implemented in a bioreactor.

20. Method according to one of claims 13 to 19, characterized in that prior to or simultaneously with step (a), the method comprises a step of dissociating the cells by chemical, enzymatic or mechanical dissociation.

21. Method according to one of claims 13 to 20, characterized in that the method comprises at least one re-encapsulation of the cells after step (f).

22. Method according to claim 21, characterized in that each reencapsulation corresponds to a passage.

23. Method according to one of claims 21 or 22, characterized in that each re-encapsulation consists of eliminating the external layer of hydrogel, preferably of resuspending in a partially or totally dissociated manner, the cells which were in the form of cysts in the microcompartments and of implementing the steps of the method again.

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