System for cell culture in bioreactor

Abstract

To provide a bioreactor cell culture system which can be used for the production of cells of interest, the production of cell assemblies of interest (organoids, tissues) and / or the production of molecules of interest, or the production of complex molecular assemblies (components of extracellular matrices, cell organelles, antibodies, vaccines, exosomes, viroids).SOLUTION: The present invention relates to a bioreactor cell culture system comprising a closed chamber containing a plurality of floating cellular micro-compartments. Each micro-compartment comprises an external hydrogel layer providing a cavity containing a set of self-organized cells and an extracellular matrix or an extracellular matrix substitute. The present invention further relates to the use of such bioreactors for producing cells and / or organoids and / or molecules and / or complex molecular assemblies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a system for cell culture in bioreactor.The system according to the present invention can be used for producing cells of interest, producing cell aggregates of interest (organoids, tissues) and / or molecules, or producing complex molecular aggregates (extracellular matrix, cell organelles, antibodies, vaccines, exosomes, components of viroids), or other materials of interest that are derived from or produced by cells that are grown in such a system. [Background technology]

[0002] Bioreactor cell culture systems are of increasing interest, particularly to the pharmaceutical industry. Indeed, eukaryotic cells are increasingly being used as therapeutic tools, particularly in cell and tissue therapy, and as tools for the biological production of molecules of interest, from protein fractions (insulin, antibodies, etc.) to complexes of proteins, lipids, and sugars derived from cells or cell organelles, via extracellular vesicles and exosomes, to viral derivatives (especially for the production of vaccines). Bioreactor cell culture systems enable the mass cultivation of these cells, thus meeting the needs of cells and / or molecules of interest on an industrial scale.

[0003] Currently, there are three main classes of bioreactor cell culture methods: - Batch culture methods allow cells to be inoculated into a fixed volume of medium. After a sufficient incubation time to allow sufficient growth, the molecules and / or cells are harvested. The main problem with these methods is that the nutrients present in the medium are depleted over time, leading to the accumulation of toxic metabolic products. -Fed-batch methods allow for the addition of medium as needed to feed the cells while maintaining an acceptable cell density. The main problem with these systems is that metabolic waste products are not removed and accumulate in the bioreactor, ultimately affecting yield. - A method that allows for perfusion culture, where the medium is continuously exchanged to feed the cells and remove waste products. Such systems allow for higher yields, but rapid and continuous exchange of the medium is required to preserve the cells without damaging them (mechanical stress generated by the flow).

[0004] In the prior art, these large-scale biological production methods have little or no applicability to fragile cells or fragile cell aggregates. Indeed, cells and cell aggregates in suspension, aggregates, or on microcarriers are directly exposed to mechanical stresses (such as impact, shear stress, and pressure) in the medium. When the volume is large, the mechanical forces used to stir or circulate the medium can destroy cells or cell aggregates, especially due to shear stress applied by the liquid flow or collisions with moving components that stir the medium. Summary of the Invention

[0005] Summary of the Invention By addressing these issues of cell culture in bioreactors, we discovered that it is possible to culture large numbers of cells in a bioreactor by creating a culture space within microcompartments separated by an outer hydrogel layer. Thus, the desired cell niche is surrounded by a hydrogel shell that advantageously allows nutrients to penetrate and proteins and metabolites to escape, while retaining components larger than 150 kDa in size (extracellular matrix, exosomes, viral particles, cells). Furthermore, because the cells are protected by the hydrogel shell from stresses that may be present within the reactor, flow through the bioreactor can be as strong as the hydrogel shell can support. Furthermore, unlike existing culture systems, the hydrogel shell of the cell microcompartment protects cells from mechanical stresses associated with collisions and prevents fusion of multicellular factors (aggregates, microcarriers) present in liquid suspension cultures, which can cause reproducibility issues by altering the local conditions experienced by the cells (diffusion distances in the medium, mechanical stress). The microcompartments are suspended in the bioreactor, allowing uniform access to medium and diffusion into the microcompartments, as well as good convection. Additionally, because the cellular niche is protected by the hydrogel shell, even the most fragile cell types can be cultured under optimal yield conditions with minimal cell death and well-controlled phenotypes. Unlike simple gel-encased spheroids, the cavity within the capsule leaves cells room to proliferate and / or self-organize on the extracellular matrix. Advantageously, each microcompartment contains a unique cellular niche. In other words, a specific hydrogel shell surrounds a single cellular niche. Because the outer layer of the microcompartment is made of hydrogel, it can be easily dissolved at the end of production to recover cells. The 3D nature of these microcompartments advantageously allows for up to 100,000-fold cell expansion within the microcompartment.

[0006] Thus, the present invention has as its object a bioreactor cell culture system comprising an enclosed chamber containing a plurality of cellular microcompartments, each of which comprises an outer hydrogel layer providing a cavity containing a set of self-organizing cells and an extracellular matrix or extracellular matrix substitute.

[0007] According to the present invention, an outer hydrogel layer surrounds the set of cells. The hydrogel layer forms a hollow capsule, providing a cavity that contains the set of cells.

[0008] Advantageously, the hydrogel capsule contains a unique set of cells.

[0009] According to the present invention, a plurality of cell microcompartments are suspended within a bioreactor chamber, more specifically, the microcompartments are suspended in a medium contained within the bioreactor chamber.

[0010] The present invention also has as its object the use of such a bioreactor cell culture system comprising a sealed chamber for the production and / or amplification of cells of interest. The amplification is advantageously between 2 and 100,000 fold during each passage. This amplification rate corresponds to the number of viable cells recovered at the end of the amplification divided by the number of viable cells inoculated.

[0011] The present invention also has as its object the use of such a bioreactor cell culture system for the production of molecules and / or complex molecular assemblies of interest, such as components of the extracellular matrix, cell organelles, antibodies, vaccines, exosomes, viroids, etc., wherein said molecules and / or assemblies are excreted from the microcompartments by the cells of said microcompartments into the culture medium, or conversely, accumulated inside the microcompartments for subsequent recovery.

[0012] The present invention also provides for its object a process for the production of organoids or cells of interest, comprising: - introducing a plurality of cellular microcompartments into a bioreactor comprising a sealed chamber, wherein said microcompartments each comprise an outer hydrogel layer encapsulating cells and an extracellular matrix or extracellular matrix substitute; - culturing the microcompartments under conditions that allow proliferation of the cells within the microcompartments and / or their self-organization into organoids; - Recovering the cell microcompartments and optionally hydrolyzing the hydrogel layer to recover the organoids or cells of interest. The method includes the steps of:

[0013] The present invention also has as its object a process for the production of differentiated cells from multipotent, pluripotent or totipotent cells, comprising: - introducing into a bioreactor a plurality of cellular microcompartments, each of said microcompartments comprising an outer hydrogel layer encapsulating multipotent, pluripotent or totipotent cells and an extracellular matrix or extracellular matrix substitute; - culturing the microcompartments under conditions that allow proliferation and / or differentiation of the cells in the microcompartments into one or more cell types of interest; - Recovering the cell microcompartments and optionally hydrolyzing the hydrogel layer to recover the cell type of interest. The method includes the steps of: DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description The present inventors have found that it is possible and particularly advantageous to culture cells in a reactor comprising a sealed chamber by retaining the cells within an outer capsule of a crosslinked hydrogel. More precisely, the present inventors have developed cellular microcompartments, each comprising an outer hydrogel layer encapsulating a set of self-organizing cells and an extracellular matrix or extracellular matrix substitute. According to the present invention, the cellular microcompartments are suspended in a bioreactor.

[0015] According to the present invention, self-organizing cells refer to a set of cells that are uniquely positioned relative to one another to allow for cellular interaction and communication to form a desired three-dimensional microstructure. Thus, each microcompartment comprises an outer hydrogel layer, or hydrogel capsule, that contains a set of self-organizing cells. The cells can proliferate, organize, and / or differentiate within the hydrogel capsule.

[0016] In some embodiments, the hydrogel capsules contain a unique set of self-organizing cells. By unique, we mean that the capsule contains only one group of cells, which may be more or less cohesive. In particular, a unique set of cells refers to a three-dimensional cellular structure in which each cell of the set is in physical contact with at least one other cell of the set.

[0017] According to the present invention, it is possible to encapsulate all types of eukaryotic cells, more particularly mammalian cells. In particular, the cells are selected from differentiated cells, progenitor cells, stem cells, multipotent cells, pluripotent cells, totipotent cells, genetically modified cells, and mixtures thereof. In one embodiment, the encapsulated cells are pluripotent stem cells, particularly embryonic stem cells and / or induced pluripotent cells (IPS). In one embodiment, the encapsulated cells are embryonic stem cells, particularly pluripotent embryonic stem cells. In one embodiment, the encapsulated cells are embryonic stem cells, excluding human embryonic stem cells that require the destruction of the human embryo. In another embodiment, the encapsulated cells are human embryonic stem cells derived from supernumerary human embryos conceived in the context of medically assisted reproduction that are no longer the subject of a parent project in accordance with the bioethics laws in force at the time and in the country from which the embryonic stem cells were obtained. In another embodiment, the encapsulated cells are induced pluripotent cells (IPS), particularly human induced pluripotent cells (hIPS). In another embodiment, the encapsulated cells are embryonic stem cells and induced pluripotent cells. In one embodiment, the encapsulated cells comprise a mixture of embryonic stem cells and induced pluripotent cells.

[0018] In the context of the present invention, the term "outer hydrogel layer" or "hydrogel shell" refers to a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferably water. Such an outer hydrogel layer is obtained by crosslinking a hydrogel solution. Advantageously, the polymer of the hydrogel solution is a polymer that can be crosslinked when exposed to stimuli such as temperature, pH, ions, etc. Advantageously, the hydrogel solution used is biocompatible in the sense that it is not toxic to cells. The hydrogel layer advantageously allows the diffusion of dissolved gases (and in particular oxygen and / or carbon dioxide), nutrients, and metabolic waste products, allowing cell survival, proliferation, differentiation, maturation, and / or the production of molecules or molecular assemblies of interest and / or the repeated occurrence of cellular behaviors of interest. The polymer of the hydrogel solution may be of natural or synthetic origin. For example, the hydrogel solution may contain one or more polymers selected from the group consisting of sulfonate-based polymers such as sodium polystyrene sulfonate, acrylic acid-based polymers such as sodium polyacrylate, polyethylene glycol diacrylate, methacrylic acid gelatin compounds, polysaccharides, particularly polysaccharides of bacterial origin such as gellan gum, or those of plant origin such as pectin or alginate. In some embodiments, the hydrogel solution contains at least alginate. Preferably, the hydrogel solution contains only alginate. In the context of the present invention, "alginate" refers to a linear polysaccharide formed from β-D-mannuronate (M) and α-L-guluronate (G), their salts and derivatives. Advantageously, the alginate is sodium alginate, composed of more than 80% G and less than 20% M, with an average molecular weight of 100 to 400 kDa (e.g., PRONOVA® SLG100), and a total concentration of 0.5% to 5% (weight / volume) density.

[0019] According to the present invention, the cell microcompartments are sealed. It is the outer hydrogel layer that gives the cell microcompartments their size and shape. The microcompartments can have any shape that is compatible with encapsulating cells.

[0020] Preferably, the extracellular matrix layer forms a gel. The extracellular matrix layer contains a mixture of proteins and extracellular compounds necessary for cell culture, such as pluripotent cells. Preferably, the extracellular matrix contains structural proteins such as laminin 521, 511, or 421, entactin, vitronectin, laminin, collagen, and growth factors such as TGF-beta and / or EGF. In some embodiments, the extracellular matrix layer consists of or comprises Matrigel® and / or Geltrex®.

[0021] According to the present invention, the microcompartments may contain extracellular matrix substitutes instead of extracellular matrix. Extracellular matrix substitutes refer to compounds that can promote cell adhesion and / or survival by interacting with membrane proteins and / or extracellular signal transduction pathways. For example, such substitutes include proteins (laminin, vitronectin, fibronectin, and collagen), biological polymers and their fragments, including non-sulfated glycosaminoglycans (hyaluronic acid) or sulfated glycosaminoglycans (chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate), synthetic polymers containing units derived from or that mimic the properties of biological polymers (RGD units), and small molecules that mimic adhesion to substrates (Rho-A kinase inhibitors, such as Y-27632 or thiazovivin).

[0022] Any method for producing cell microcompartments containing extracellular matrix and cells within hydrogel capsules may be used to carry out the manufacturing process according to the present invention. In particular, the microcompartments can be manufactured by adapting the method and 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, pp. 1593-1604).

[0023] Advantageously, the dimensions of the cell microcompartments are controlled. In one embodiment, the cell microcompartments according to the invention have a spherical shape. Preferably, the diameter of such microcompartments is comprised between 10 μm and 1 mm, more preferably between 50 μm and 500 μm, even more preferably less than 500 μm, preferably less than 400 μm. In another embodiment, the cell microcompartments according to the invention have an elongated shape. In particular, the microcompartments may have an ovoid or tubular shape. Advantageously, the smallest dimension of such ovoid or tubular microcompartments is comprised between 10 μm and 1 mm, more preferably between 50 μm and 500 μm, even more preferably less than 500 μm, preferably less than 400 μm. "Smallest dimension" means twice the smallest distance between a point located on the outer surface of the hydrogel layer and the center of the microcompartment.

[0024] In particular embodiments, the thickness of the outer hydrogel layer represents 5 to 40% of the radius of the microcompartment. The thickness of the extracellular matrix layer represents 5 to 80% of the radius of the microcompartment and is advantageously suspended on the inner surface of the hydrogel shell. This matrix layer is able to fill the space between the cells and the hydrogel shell. In the context of the present invention, the "thickness" of a layer is the dimension of said layer extending radially from the center of the microcompartment.

[0025] In an embodiment of the invention, the bioreactor comprises microcompartments in which cells self-organize into cysts.

[0026] In the context of the present invention, a cyst is defined as at least one layer of pluripotent or totipotent cells organized around a central lumen. Thus, according to the present invention, such microcompartments comprise, around the central lumen, said layer of pluripotent cells, a layer of extracellular matrix or a layer of extracellular matrix substitute, and an outer hydrogel layer in succession. The lumen is produced at the moment of cyst formation by cells growing and developing in the layer above the extracellular matrix layer. Advantageously, the lumen contains a liquid, more specifically a culture medium.

[0027] According to the present invention, the cyst advantageously contains one or more layers of mammalian, human, or non-human pluripotent stem cells. Pluripotent stem cells, or pluripotent cells, refer to cells that are not capable of forming an entire organism but have the ability to form all tissues present in the whole organism of origin. In particular, the cyst may contain embryonic stem cells (ESCs), induced pluripotent stem (IPS) cells, or multilineage-differentiating stress enduring (MUSE) cells found in the skin and bone marrow of adult mammals.

[0028] Advantageously, the thickness of the outer hydrogel layer represents 5 to 40% of the radius of the microcompartment, the thickness of the extracellular matrix layer represents 5 to 80% of the radius of the microcompartment, and the thickness of the pluripotent cell layer represents approximately 10% of the radius of the microcompartment. The pluripotent cell layer is in contact with the extracellular matrix layer at at least one point, and a medium-filled space may exist between the matrix layer and the cyst. The lumen then represents 5 to 30% of the radius of the microcompartment. In a specific example, the cell microcompartment has a spherical shape with a radius equal to 100 μm. The thickness of the hydrogel layer is 5 μm to 40 μm. The thickness of the extracellular matrix layer is 5 μm to approximately 80 μm. The thickness of the pluripotent cell layer is 10 to 30 μm, and the lumen radius is approximately 5 to 30 μm.

[0029] According to an exemplary aspect of the present invention, (a) incubating 600,000 to 2 million mammalian pluripotent stem cells in a medium containing an inhibitor of the RHO / ROCK pathway; (b) mixing these pluripotent stem cells derived from step (a) with an extracellular matrix; (c) encapsulating the mixture from step (b) in a hydrogel layer; (d) culturing the capsules obtained in step (c) in a medium containing an inhibitor of the RHO / ROCK pathway; (e) rinsing the capsules from step (d) to remove inhibitors of the RHO / ROCK pathway; (f) culturing the capsules from step (e) in a fed-batch production mode by daily doubling the volume of pluripotent cell culture medium, such as MTESR1 (Stemcell Technologies), that does not contain an inhibitor of the RHO / ROCK pathway for 3 to 20 days, preferably 5 to 10 days, and optionally harvesting the resulting cell microcompartments; According to the method described above, it is possible to culture such microcompartments in, for example, a 150 mL bioreactor, where the cells form cysts.

[0030] A person skilled in the art knows how to adapt the number of cells and the volume of the bioreactor as needed.

[0031] Thiazovivin (C 15 H 13 N5OS) and / or Y-27632(C 14 H 21 Step (a) of incubation in a medium containing one or more inhibitors of the RHO / ROCK ("Rho-associated protein kinase") pathway, such as NO, and step (d) of culturing promote the survival of pluripotent stem cells and the attachment of cells to the extracellular matrix, once an outer hydrogel layer forms around the extracellular matrix. However, it is desirable that these steps be time-limited so that the inhibitors of the RHO / ROCK pathway do not interfere with cyst formation.

[0032] Thus, preferably, the incubation in step (a) is carried out for a period comprised between a few minutes and a few hours, preferably between 2 minutes and 2 hours, more preferably between 10 minutes and 1 hour.

[0033] Likewise, preferably, the culturing step (d) is carried out for a period comprised between 2 and 48 hours, preferably for a period comprised between 6 and 24 hours, more preferably for a period comprised between 12 and 18 hours.

[0034] Step (e) is necessary to ensure removal of traces of the RHO / ROCK pathway inhibitor, and is carried out, for example, by rinsing, preferably several times, in successive medium that does not contain the RHO / ROCK pathway inhibitor.

[0035] Advantageously, step (f) is carried out for a time sufficient to obtain cellular microcompartments in which the layer of extracellular matrix and pluripotent cells has a cumulative thickness equal to 50 to 100% of the thickness of the outer hydrogel layer. Any medium suitable for the culture of pluripotent stem cells may be used.

[0036] In one embodiment, the process according to the invention comprises an intermediate step (a') between steps (a) and (b), consisting of dissociating the induced pluripotent stem cells by means of a reagent, preferably without enzymes. Advantageously, said reagent is inhibited or rinsed off before the encapsulation step by successive rinses in a medium specific for pluripotent cells. For example, the reagent used is ReLeSR®. Of course, it is also possible to use a reagent containing trypsin or enzymes, but the viability of the pluripotent cells after this step may then be lower compared to the use of a reagent without enzymes.

[0037] Alternatively, such microcompartments may comprise: (A) mixing mammalian differentiated cells with an extracellular matrix and a cell reprogramming agent; (B) encapsulating the mixture from step (A) in a hydrogel layer; (C) culturing the capsules from step (B) for at least 3 days and, optionally, harvesting the resulting cell microcompartments. can be obtained according to

[0038] For example, differentiated cells used are fibroblasts, peripheral blood mononuclear cells, epithelial cells and more generally cells derived from liquid or solid biopsies of human tissue.

[0039] Those skilled in the art know how to reprogram differentiated cells into stem cells by reactivating the expression of genes associated with the embryonic stage by means of specific factors. By way of example, mention may be made of the method 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) and in the international application WO2010 / 105311 entitled "Production of reprogrammed pluripotent cells."

[0040] The reprogramming agent is advantageously co-encapsulated with the differentiated cells to concentrate the product and facilitate contact with the set of cells.

[0041] The reprogramming agent makes it possible to impose on the cells a series of phenotypic changes up to the pluripotent stage. Advantageously, the reprogramming step (A) is carried out using a specific medium to promote these phenotypic changes. For example, the cells are treated with a serine / threonine protein kinase receptor inhibitor (e.g., product SB-431542 (C 22 H 16 N4O3), one or more inhibitors of the RHO / ROCK (“Rho-associated protein kinase”) pathway, e.g., thiazovivin and / or Y-27632, fibroblast growth factors, e.g., FGF-2, ascorbic acid, and antibiotics, e.g., trichostatin A (C 17 H 22 The cells are cultured in a first medium containing 10% human or bovine serum in minimal essential Eagle's medium (DMEM) supplemented with NO. The medium is then replaced with a medium that promotes the growth of pluripotent cells, such as medium mTeSR®1.

[0042] Such cysts can then be forced down a desired differentiation pathway to yield microcompartments containing one or more cell types of interest, particularly for the production of a molecule of interest or for the production of a desired organoid.

[0043] In some embodiments, the bioreactor comprises microcompartments containing cells that self-organize into organoids.

[0044] In the context of the present invention, organoid is defined as a multicellular structure that is organized in three dimensions to reproduce at least part of the microstructure of an organ.Accordingly, according to the present invention, such microcompartment comprises a three-dimensional multicellular structure surrounded by an extracellular matrix, and the whole is encapsulated in an outer hydrogel layer.

[0045] According to the present invention, organoids can be obtained by encapsulating pluripotent or progenitor cells, which then differentiate, within a hydrogel capsule, or by directly encapsulating differentiated or mature cells.

[0046] In one embodiment, the cell microcompartments introduced into the bioreactor contain pluripotent cells, and then a step of cell differentiation into at least one cell type of interest is carried out inside the bioreactor, followed, optionally, by a step of expansion of said differentiated cells within the microcompartments.

[0047] In some embodiments, the cell microcompartments introduced into the bioreactor contain already differentiated cells or progenitor cells, and the process of proliferation and / or maturation of said differentiated cells in the microcompartments is then carried out inside the bioreactor.

[0048] Advantageously, the microcompartments introduced into the bioreactor have an initial cell density of less than 10%, preferably less than 1%, even more preferably less than 0.1% of the internal volume of the microcompartment.

[0049] Advantageously, the microcompartments harvested at the end of the cultivation process in the bioreactor have a cell density occupying more than 10% of the internal volume of the microcompartment.

[0050] According to the present invention, cells contained in hydrogel capsules are contained in a bioreactor and exposed to a flow of culture medium through a layer of hydrogel.

[0051] Advantageously, the ratio of the convective volume outside the microcompartment to the diffusive volume inside the microcompartment is comprised between 1 and 10,000, preferably between 1 and 1000, and more preferably between 1 and 100.

[0052] According to the present invention, the convection volume refers to the volume of medium inside the reactor chamber between the microcompartments. Thus, the microcompartments are suspended in the bioreactor, and the convection volume refers to the medium circulating between the microcompartments. Conversely, the diffusion volume refers to the volume of medium diffusing inside the microcompartments, i.e., in the space / void created around / between / by the self-assembled cells.

[0053] Thus, in the case of microcompartments containing cysts, the diffusion volume is primarily composed of the central lumen and, at the onset of cyst growth, the space between the capsule wall and the cyst. In the case of microcompartments containing organoids, the diffusion volume is primarily composed of the space created within the three-dimensional multicellular structure.

[0054] The microcompartments according to the invention are advantageously characterized by the presence within the hydrogel capsule of one or more lumens or spaces that are free of cells and allow the precise proliferation or self-organization of cells inside the microcompartment. Those skilled in the art know how to recover the cells at the most appropriate moment for the amplification or differentiation process, corresponding in this context to a certain level of saturation of the optimal spaces.

[0055] In some embodiments, the microcompartments occupy between 0.01% and 74% of the volume of the bioreactor chamber.

[0056] The use of cell microcompartments allows cells to be cultured in any type of bioreactor with a sealed chamber, and especially in bioreactors in batch, fed-batch, or continuous feed (perfusion) mode. The use of these microcompartments is particularly advantageous for continuous feed culture. Indeed, the cells are protected by the hydrogel shell and can be exposed to continuous flow without the risk of weakening the cells.

[0057] In some embodiments, the bioreactor comprises a chamber that can be sealed, which allows for controlling the atmosphere inside the bioreactor and for example, culturing the microcompartments under an inert atmosphere.

[0058] The cell culture system according to the present invention may comprise a chamber having a volume comprised between 1 mL and 10,000 L, preferably between 5 mL and 10,000 L, between 10 mL and 10,000 L, between 100 mL and 10,000 L, between 200 mL and 10,000 L, or between 500 mL and 10,000 L. In some embodiments, the chamber has a volume of at least 1 mL. In some embodiments, the chamber has a volume of at least 10 mL. In some embodiments, the chamber has a volume of at least 100 mL. In some embodiments, the chamber has a volume of at least 500 mL. In some embodiments, the chamber has a volume of at least 1 L. In some embodiments, the chamber has a volume of at least 10 L. In some embodiments, the chamber has a volume of 100 L or more. Advantageously, any bioreactor comprising a sealed chamber and capable of industrial-scale production of cells, organoids, molecules and / or complex molecular assemblies can be used.

[0059] In general, the use of a closed chamber allows for fine control of the culture environment without the risk of interference from the external environment, facilitates the production of a sterile product, and allows for better volumetric yields.

[0060] In some embodiments, the microcompartments contain 10% to 98% by volume of cells at harvest, i.e., 100 to 1,000,000 cells, depending on the diameter of the relevant compartment and the size of the cells produced. This can be calculated by the ratio of the total number of cells produced (as measured by those skilled in the art using a Malassez cell or automated cell counter) to the number of capsules obtained (as measured by those skilled in the art by manual counting under a light microscope or by characterizing the capsule volume by automated image analysis). Of course, it is possible to start cell cultures with microcompartments containing a small number of cells at the start, particularly 1 to 1,000 cells, i.e., 0.01% to 10% by volume occupied by cells within the microcompartment, depending on the diameter of the relevant compartment and the size of the cells produced. More typically, microcompartments according to the present invention contain 0.01% to 98% by volume of cells.

[0061] Cells can then grow inside the microcompartments and self-organize into organoids.

[0062] In one embodiment, the cells of a microcompartment are all of the same cell type. According to the present invention, cells of the same microcompartment are considered to be all of the same cell type if at least 50%, preferably 70%, more preferably 90%, and even more preferably 98% or more of the cells of said microcompartment have the same phenotype, according to the knowledge of a person skilled in the art that allows characterizing cell types. In another embodiment, the cells of the microcompartments are of at least two different cell types. Advantageously, 20 to 100% of the cells of the compartments have the same phenotype.

[0063] According to the present invention, it is possible to culture in the same bioreactor microcompartments all containing the same cell type, or conversely with different cell types. For example, a bioreactor may contain two types of microcompartments, each containing a specific cell type.

[0064] The culture system according to the present invention is particularly advantageous for the production and / or expansion of cells of interest. Indeed, the organization of cells within the hydrogel capsule, together with the extracellular matrix, allows for proliferation of 2 to 100,000-fold during each passage.

[0065] Passaging refers to the manipulation of cells to add space or culture surface to continue amplification or to initiate differentiation or self-organization into organoids. In the case of microcarriers, this manipulation may require the bioreactor to be reloaded with new microcarriers. For standard two-dimensional culture of adherent pluripotent stem cells, this manipulation consists of separating cells from old medium to re-inoculate them into new medium with a larger surface area; for those skilled in the art, this manipulation may result in a loss of 50% of cells. For the culture in microcompartments according to the present invention, this corresponds to the dissociation of the microcompartments, the dissociation of self-organized cell sets, or their dispersion into cell sets small enough to be re-encapsulated in new microcompartments.

[0066] The present invention has as its object in particular the use of such a bioreactor cell culture system for the large scale production of pluripotent cells.

[0067] The present invention also has as its object the use of such a bioreactor cell culture system for producing unipotent or multipotent progenitor cells from pluripotent cells.

[0068] The present invention also has as its object the use of such bioreactor cell culture systems for the production of terminally differentiated cells (i.e., corresponding to one or more specific functions) from pluripotent cells and / or unipotent or multipotent progenitor cells and / or combinations of these progenitor cells.

[0069] The present invention has as its object in particular a process for the production of organoids or cells of interest, comprising: - introducing a plurality of cellular microcompartments into a bioreactor comprising a sealed chamber, wherein said microcompartments each comprise an outer hydrogel layer encapsulating cells and an extracellular matrix or extracellular matrix substitute; - culturing the microcompartments under conditions that allow proliferation of the cells within the microcompartments and / or their self-organization into organoids; - Recovering the cell microcompartments and optionally hydrolyzing the hydrogel layer to recover the organoids or cells of interest. The method includes the steps of:

[0070] Those skilled in the art are able to adapt the culture conditions to the cell types in the microcompartments in order to promote their proliferation and / or self-organization.

[0071] In some embodiments, the introduced cell microcompartment contains pluripotent cells, and the process comprises the step of cell differentiation into at least one cell type of interest in the bioreactor, and the step of proliferation of the differentiated cells in the microcompartment.For example, the production of primitive endodermal organoids for the study of differentiation in human endodermal tissue can be carried out according to the following protocol: - After 2-3 days of culture, from step f) the microcompartments described above are obtained: - culturing the cells in STEMdiff™ Pancreatic Stage 1 Medium from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies, in a 150 mL closed bioreactor for 3 to 6 days; -Use of the obtained primitive endoderm for developmental studies.

[0072] In another embodiment, the introduced cell microcompartments contain already differentiated cells or progenitor cells, and said process comprises a step of expansion of said differentiated cells in the microcompartments inside the bioreactor.

[0073] During the proliferation and / or maturation process, cells advantageously self-organize into specific organoids according to the cell type-specific organization.

[0074] In embodiments relating to amplification, the microcompartments introduced into the bioreactor have a cell density that occupies less than 10%, preferably 1%, and even more preferably 0.1% of the internal volume of the microcompartment. Thereafter, during the culturing process, the cells grow inside the microcompartment.

[0075] In the embodiment of differentiation and / or maturation without amplification, the microcompartment introduced into bioreactor has a cell density that occupies more than 1% of the internal volume of the microcompartment.Then, during the culture process, cells differentiate and / or mature and / or self-organize inside the microcompartment.For example, in the context of cell therapy for Parkinson's disease, the first type of neural organoid production for neural transplantation has been carried out according to the following protocol: - thawing of 5 million dopaminergic progenitor cells, such as those sold by Cellular Dynamics International (iCell® DopaNeurons), -Encapsulation of predifferentiated neural progenitor cells according to the protocol described by Alessandri et al. 2016. -Culture in 150 mL closed bioreactors in media provided by Cellular Dynamics. -Maturation and structuring of dopaminergic neural organoids over 2 weeks in a bioreactor. Preparation of the grafts by dissociation of the hydrogel capsules by rinsing twice for 30 seconds in 1 ml of ReLeSR® (Stemcell Technologies), followed by resuspension in an 11% by weight solution of 70 kDa dextran in neuronal medium and dispensing in a homemade glass cannula. -Transplantation into animal models of Parkinson's disease.

[0076] In the embodiment combining amplification and differentiation / maturation, the microcompartments introduced into the bioreactor advantageously have a cell density of occupancy of less than 10%, preferably 1%, and even more preferably less than 0.1% of the internal volume of the microcompartment. Then, the cells grow inside the microcompartment during the culture step, and then during the differentiation step. Then, the cells self-organize inside the microcompartment during the second culture step, which can be triggered by changing the type of nutrient medium or by physical triggers (temperature, illuminance). For example, in the context of cell therapy for Parkinson's disease, a second type of neural organoid production for neuronal transplantation was carried out according to the following protocol: - from step f) to obtain the above microcompartments after 2-3 days of culture: Culture for 1–2 days in a 150 mL sealed bioreactor in neural induction medium containing 10 μ m 24(S),25-epoxycholesterol in a neurobasal / DMEM-F12 base supplemented with inhibitors of the BMP2 (2 μ m dorsomorphin or 0.5 μ m LDN 193189) and TGFbeta (10 μ m + SB 431542) signaling pathways, N2, and B27. -BMP2 (2 μ m dorsomorphin or 0.5 μ m LDN 193189) and TGFbeta (10 μ m + SB 431542) signaling pathway inhibitors, two activators of the SHH pathway (200 ng / mL SHH; 1 μ m purmorphamine) and FGF8 (100 ng / mL), a WNT pathway inhibitor (3 μ m Chir99021), and cultured for 6 days in a 150 mL sealed bioreactor in neurobasal / DMEM-F12 base supplemented with N2 and B27, containing 10 μ m 24(S),25-epoxycholesterol. -cultured for 1 day in a 150 mL sealed bioreactor in a second neuroregionalization medium containing an inhibitor of the BMP2 signaling pathway (2 μ m dorsomorphin or 0.5 μ m LDN 193189), an inhibitor of the WNT pathway (3 μ m Chir99021), and 10 μM 24(S),25-epoxycholesterol in a neurobasal / DMEM-F12 base supplemented with N2 and B27. - Culture for 2 weeks in a 150 mL closed bioreactor in a medium for maturation and structuring of dopaminergic neural organoids in a bioreactor containing cyclic AMP (500 μM) + ascorbic acid (200 μM) + GDNF (20 ng / mL) + BDNF (20 ng / mL) + FGF-20 (5 ng / mL) + TGFbeta (1 ng / mL) + trichostatin (10 nM) + compound E (1 μM). Preparation of the grafts by dissociation of the hydrogel capsules by rinsing twice for 30 seconds in 1 ml of ReLeSR® (Stemcell Technologies), followed by resuspension in an 11% by weight solution of 70 kDa dextran in neuronal medium and distribution in a glass cannula manufactured in-house. -Transplantation into animal models of Parkinson's disease.

[0077] In another embodiment of the combination of amplification and differentiation / maturation, the microcompartment introduced into the bioreactor advantageously has a cell density of occupancy of less than 10%, preferably 1%, and even more preferably less than 0.1% of the internal volume of the microcompartment.Then, cells grow inside the microcompartment.Then, cells are recovered by dissolving the capsule, and then subjected to a second encapsulation step, then a differentiation step, and then, cells self-organize inside the microcompartment during the second culture step, which can be triggered by changing the type of nutrient medium or physical triggers (temperature, illuminance).For example, the production of human pancreatic organoids for human pancreatic tissue transplantation has been carried out according to the following protocol: - from step f) to obtain the above microcompartments after 2-3 days of culture: -Cultured in STEMdiff™ Pancreatic Stage 1 Medium supplemented with Supplement 1A and Supplement 1B from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies, in a 150 mL sealed bioreactor for 1 day. Cultured in a 150 mL sealed bioreactor for 1 day in STEMdiff™ Pancreatic Stage 1 Medium supplemented with Supplement 1B from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies. -Cultured in STEMdiff™ Pancreatic Stage 2-4 Medium supplemented with Supplement 2A and Supplement 2B from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies, in a 150 mL sealed bioreactor for 1 day. -Cultured for 2 days in a 150mL closed bioreactor in STEMdiff™ Pancreatic Stage 2-4 Medium supplemented with Supplement 2A and Supplement 2B from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies. Cultured in a 150mL sealed bioreactor for 3 days in STEMdiff™ Pancreatic Stage 2-4 Medium supplemented with Supplement 3 from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies. Cultured in a 150mL sealed bioreactor for 5 days in STEMdiff™ Pancreatic Stage 2-4 Medium supplemented with Supplement 3 from the STEMdiff™ Pancreatic Progenitor Cell Kit, available from STEMCELL Technologies. Preparation of the grafts by dissociation of the hydrogel capsules by rinsing twice for 30 seconds in 1 ml of ReLeSR® (Stemcell Technologies), followed by resuspension in an 11% by weight solution of 70 kDa dextran in the previous medium, and dispensing in a glass cannula manufactured in-house. -Transplantation into animal models of type 1 diabetes.

[0078] Advantageously, the microcompartments recovered at the end of the culture process in the bioreactor have a cell density of occupancy of more than 10%, preferably more than 50%, of the internal volume of the microcompartment, which can go up to 98% occupancy in the case of organoids.

[0079] The culture system according to the present invention is also particularly attractive for the production of molecules and / or complex molecular assemblies of interest, which are either excreted by the cells in the microcompartments into the medium or, conversely, accumulated inside the microcompartments for subsequent recovery. This production method allows for limiting the filtration process, particularly of cellular components, by concentrating them inside the microcompartments. This method allows for easier separation of the medium containing dissolved components from insoluble components or components larger than the mesh size of the capsule hydrogel (typically 150 to 250 kDa in the case of alginate), due to the separation in the bioreactor of convective and diffusive volumes through the capsule.

[0080] According to the invention, the microcompartments are then advantageously used in a reactor in continuous breeding mode. As explained above, the presence of a protective hydrogel shell allows for perfusion of the medium at a flow rate without risk of damaging the cells. In particular, it is possible to perfuse the interior of the reactor with medium at a flow rate comprised between 0.001 and 100 volumes of cells per day contained in the bioreactor.

Claims

1. A bioreactor cell culture system comprising an enclosed chamber containing a plurality of suspended cell microcompartments, each of the microcompartments comprising an outer hydrogel layer providing a cavity containing a set of self-organizing cells and an extracellular matrix or extracellular matrix substitute.

2. 2. The bioreactor cell culture system of claim 1, wherein the ratio of the convection volume outside the microcompartment to the diffusion volume inside the microcompartment is comprised between 1 and 10,000.

3. 3. A bioreactor cell culture system according to any one of claims 1 to 2, wherein all or some of the microcompartments contain cells self-organised into cysts.

4. 4. The bioreactor cell culture system of claim 1, wherein all or some of the microcompartments contain cells that have self-organized into organoids.

5. 5. The bioreactor cell culture system according to any one of claims 1 to 4, wherein the bioreactor is selected from a batch mode bioreactor, a fed-batch mode bioreactor and a continuous mode bioreactor, preferably a continuous (perfusion) mode bioreactor.

6. 6. The bioreactor cell culture system according to any one of claims 1 to 5, wherein the chamber has a volume comprised between 1 mL and 10,000 L.

7. 7. The bioreactor cell culture system of claim 1, wherein the microcompartments contain between 0.01% and 98% by volume of cells.

8. 8. The system according to claim 1, wherein the cells of the microcompartments are all of the same cell type or, conversely, of at least two different cell types.

9. 9. The system according to claim 1, wherein the microcompartments all contain the same cell type or, conversely, have at least partially different cell types.

10. 10. Use of a bioreactor cell culture system according to any one of claims 1 to 9 for the production and / or amplification of cells of interest, preferably by 2 to 100,000 fold between each passage.

11. 10. Use of a bioreactor cell culture system according to any one of claims 1 to 9 for the production of molecules or complex molecular assemblies of interest, wherein said molecules or assemblies are excreted from the microcompartments by the cells of said microcompartments into the culture medium or, conversely, accumulated inside the microcompartments for subsequent recovery.

12. 1. A process for producing an organoid or cell of interest, comprising: - introducing a plurality of cell microcompartments into a bioreactor, wherein said microcompartments each comprise an outer hydrogel layer encapsulating cells and an extracellular matrix or extracellular matrix substitute; - culturing the microcompartments under conditions that allow proliferation of the cells within the microcompartments and / or their self-organization into organoids; - Recovering the cell microcompartments and optionally hydrolyzing the hydrogel layer to recover the organoids or cells. The process includes:

13. 13. The process according to claim 12, wherein the introduced cell microcompartments contain pluripotent cells, said process comprising a step of cell differentiation into at least one cell type of interest inside a bioreactor and optionally expansion of said differentiated cells in the microcompartments.

14. 13. The process according to claim 12, wherein the introduced cell microcompartments contain already differentiated cells or progenitor cells, and the process comprises a step of proliferation and / or maturation of the differentiated cells in the microcompartments inside the bioreactor.

15. 15. The process according to any one of claims 12 to 14, wherein the microcompartments introduced into the bioreactor have an initial cell density occupying less than 10% of the internal volume of the microcompartment, preferably less than 1%, even more preferably less than 0.1%.

16. 16. The process according to any one of claims 12 to 15, wherein the microcompartments harvested at the end of the culturing step in the bioreactor have a cell density occupying more than 10% of the internal volume of the microcompartments.