EP3797150A1

EP3797150A1 - System for cell culture in a bioreactor

Info

Publication number: EP3797150A1
Application number: EP19733852.8A
Authority: EP
Inventors: Maxime FEYEUX; Kevin ALESSANDRI; Pierre Nassoy
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut d'Optique Theorique et Appliquee; Universite de Bordeaux
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut d'Optique Theorique et Appliquee; Universite de Bordeaux
Priority date: 2018-05-21
Filing date: 2019-05-20
Publication date: 2021-03-31
Also published as: US20210123013A1; BR112020023707A2; AU2019273006A1; CN112424333A; JP2021524253A; WO2019224467A1; SG11202011501XA; FR3081168B1; KR20210011975A; CA3101003A1; MX2020012412A; CL2020003003A1; FR3081168A1; IL278775A

Abstract

The invention relates to a system for cell culture in a bioreactor comprising a closed chamber containing a plurality of cellular micro-compartments, wherein the micro-compartments each comprise an external hydrogel layer providing a cavity containing a set of self-organised cells and an extra-cellular matrix or an extra-cellular matrix substitute. The invention also relates to the use of such bioreactors for producing cells and/or organoids and/or molecules and/or complex molecular assemblies.

Description

Bioreactor cell culture system

The invention relates to cell culture systems in a bioreactor. The system according to the invention can be used for the production of cells of interest, cell sets (organoids, tissues) and / or the production of molecules of interest, or complex molecular assemblies (components of extracellular matrices, cell organelles, antibodies, vaccines, exosomes, viroids), or other material of interest derived from cells or produced by cells cultured in such systems.

Bioreactor cell culture systems are of growing interest to the pharmaceutical industry, among others. Indeed, eukaryotic cells are increasingly used as a therapeutic tool, particularly in cell and tissue therapy, and as a bioproduction tool of molecules of interest, since protein fractions (insulin, antibodies, etc. .), through protein complexes, lipids and sugars from cells or cell organelles, extracellular vesicles and exosomes, to viral derivatives (for the production of vaccines in particular). Bioreactor cell culture systems make it possible to mass culture these cells and thus meet the needs of cells and / or molecules of interest on an industrial scale.

Currently, there are three major classes of bioreactor cell culture methods:

- Methods allowing batch culture, in which the cells are seeded in a fixed volume of culture medium. After a suitable culture time to allow sufficient growth, the molecules and / or cells are harvested. The major problem with these methods is that nutrients in the environment become depleted over time, and toxic metabolites accumulate;

- The methods allowing the culture in fed lots ("fed batch"), in which the culture medium is added as and when to feed the cells while keeping an acceptable cell density. The main problem with these systems is that the metabolic wastes are not removed and accumulate in the bioreactor, which ultimately affects the yield;

- Methods for culture in infusion, in which the culture medium is continuously changed, to feed the cells and eliminate waste. Such systems allow a higher yield but the rapid and continuous change of the culture medium requires retaining the cells without damaging them (mechanical stress generated by the flow). In the state of the art, these methods of mass bioproduction are not or hardly applicable to fragile cells or fragile cell assemblies. In fact, in suspension, in aggregate or on micro-carriers, cells and cell assemblies are directly exposed in the culture medium to mechanical constraints (shock, shear stress, pressure, etc.). When the volumes become large, the mechanical forces used to stir or circulate the medium can destroy cells or cell assemblies including the shear stress applied by the liquid flow or the impact with the moving elements that stir the medium.

Summary of the invention

By working on these cell culture problems in a bioreactor, the inventors have discovered that it is possible to provide a culture space within microcompartments delimited by an outer layer of hydrogel for culturing a large number of cells within a bioreactor. . The cell niche of interest is thus surrounded by a hydrogel shell, which advantageously infiltrates the nutrients and exfiltrates the proteins and metabolites but retains elements whose size exceeds 150 KDa (extracellular matrix, exosomes, viral particles, cells). In addition, the cells being protected from the stresses that may exist within the reactor by the hydrogel shell, the flow through the bioreactor can be as strong as the hydrogel shell can support it. In addition, the hydrogel shell of cellular microcompartments, contrary to existing culture systems, preserves the cells from mechanical constraints related to collisions and prevents fusions of multicellular elements (aggregates, micro-carriers) that exist during suspension culture. liquid, and which cause problems of reproducibility by varying the local conditions felt by the cells (diffusion distance in the middle, mechanical stresses). The microcompartments are suspended in the bioreactor, which allows access to the culture medium and diffusion in homogeneous microcompartments, as well as good convection. In addition, since the cell niche is protected by the hydrogel shell, it is possible to cultivate the most fragile cell types under optimal yield conditions with low cell death and a well-controlled phenotype. Unlike the simple spheroid embedded in a gel, the cavity in the capsule gives room for cells to multiply and / or self-organize on extracellular matrix. Advantageously, each microcompartment comprises a single cellular niche. In other words, a given hydrogel shell surrounds a single cell niche. The outer layer of microcompartments being hydrogel, it can easily be dissolved to recover the cells after production. These microcompartments being in 3D, they advantageously allow cell amplification in the microcompartment by a factor of up to 100,000. The subject of the invention is therefore a bioreactor cell culture system comprising a closed chamber containing a plurality of cellular microcompartments, in which the microcompartments each comprise an outer layer of hydrogel forming a cavity containing a set of self-organized cells and the extracellular matrix. or an extracellular matrix substitute.

According to the invention, an outer layer of hydrogel surrounds a set of cells. The hydrogel layer forms a hollow capsule, providing a cavity containing the set of cells.

Advantageously, the hydrogel capsule contains a single set of cells.

According to the invention, the plurality of cellular microcompartments is suspended in the enclosure of the bioreactor. More particularly, microcompartments float in the culture medium contained in the enclosure of the bioreactor.

The subject of the invention is also the use of such a bioreactor cell culture system, comprising a closed chamber, for the production and / or amplification of cells of interest. The amplification is advantageously a factor of 2 to 100,000 between each pass. This amplification factor corresponds to the number of living cells harvested after amplification, divided by the number of living cells seeded.

The subject of the invention is also the use of such a bioreactor cell culture system for the production of molecules of interest and / or complex molecular assemblies, such as components of extracellular matrices, cellular organelles, antibodies, vaccines, exosomes, viroids, etc., said molecules and / or assemblages being excreted by the microcompartment cells out of said microcompartment into the culture medium, or conversely accumulated inside the microcompartment for a subsequent harvest.

The subject of the invention is also a process for producing organoids or cells of interest comprising the steps according to which:

introducing a plurality of cellular microcompartments into a bioreactor, comprising a closed chamber, said microcompartments each comprising an outer layer of hydrogel encapsulating cells and extracellular matrix or an extracellular matrix substitute;

the microcompartment is cultured under conditions allowing the multiplication of the cells inside the microcompartments, and / or the self organization of the cells into organoids; the cellular microcompartments are recovered;

and optionally, the hydrogel layer is hydrolyzed to recover the organoids or the cells of interest.

The invention also relates to a method for producing differentiated cells from multipotent, pluripotent or totipotent cells comprising the steps according to which:

introducing a plurality of cellular microcompartments into a bioreactor, said microcompartments each comprising an outer layer of hydrogel encapsulating multipotent, pluripotent or totipotent cells and extracellular matrix or an extracellular matrix substitute;

the microcompartment is cultivated under conditions allowing the multiplication of the cells inside the microcompartment, and / or the differentiation in one or more cell types of interest;

the cellular microcompartments are recovered;

and optionally, the hydrogel layer is hydrolyzed to recover the cell type (s) of interest.

detailed description

The inventors have discovered that it is possible and particularly advantageous to cultivate cells within a reactor comprising a closed enclosure, by keeping the cells inside an outer crosslinked hydrogel capsule. More specifically, the inventors have developed cell microcompartments each comprising an outer layer of hydrogel encapsulating a set of self-organized cells and the extracellular matrix or an extracellular matrix substitute. According to the invention, the cellular microcompartments are suspended in the bioreactor.

According to the invention, the term "self-organized cells" is understood to mean a set of cells positioned in a particular manner with respect to one another in order to create cellular interactions and communications and to form a three-dimensional microstructure of interest. Each microcompartment thus comprises an outer layer of hydrogel, or hydrogel capsule, enclosing a set of self-organized cells. The cells can multiply, organize and / or differentiate within the hydrogel capsule.

In one embodiment, the hydrogel capsule contains a single set of self-organized cells. By single means that the capsule contains only a group of cells, which can be more or less cohesive. In particular, a single set of cells means a three-dimensional cellular structure in which each cell of said set is in physical contact with at least one other cell of said set.

According to the invention, it is possible to encapsulate all kinds of eukaryotic cells, and more particularly mammalian cells. In particular, the cells are chosen from differentiated cells, progenitors, stem cells, multipotent cells, pluripotent cells, totipotent cells, genetically modified cells, and mixtures thereof, etc. In one embodiment, the encapsulated cells are pluripotent stem cells, chosen in particular from embryonic stem cells and / or pluripotency-induced cells (IPS). In one embodiment, the encapsulated cells are embryonic stem cells, including pluripotent embryonic stem cells. In one embodiment, the encapsulated cells are embryonic stem cells, excluding human embryonic stem cells that required the destruction of a human embryo. In another embodiment, the encapsulated cells are human embryonic stem cells derived from supernumerary human embryos designed in the context of a medically assisted procreation no longer the subject of a parental project, in accordance with the bioethical laws. in force at the time and in the country of collection of the said embryonic stem cells. In another embodiment, the encapsulated cells are pluripotency-induced cells (IPS), including pluripotency-induced human cells (hIPS). In another embodiment, the encapsulated cells are embryonic stem cells and pluripotency-induced cells. In one embodiment, the encapsulated cells comprise a mixture of embryonic stem cells and pluripotency-induced cells.

In the context of the invention, the "outer layer of hydrogel", or "hydrogel shell", designates a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, and preferably of water. Such an outer layer of hydrogel is obtained by crosslinking a hydrogel solution. Advantageously, the polymer or polymers of the hydrogel solution are crosslinkable polymers when subjected to a stimulus, such as a temperature, a pH, ions, etc. Advantageously, the hydrogel solution used is biocompatible, in that it is not toxic to the cells. The hydrogel layer advantageously allows the diffusion of dissolved gases (and in particular oxygen and / or carbon dioxide), nutrients, and metabolic waste to allow the survival, proliferation, differentiation, maturation of the cells and or the production of molecules or molecular assemblies of interest and / or the recapitulation of cellular behaviors of interest. The polymers of the hydrogel solution may be of natural or synthetic origin. For example, the hydrogel solution contains one or more polymers among sulfonate-based polymers, such as sodium polystyrene sulfonate, acrylate-based polymers, such as sodium polyacrylate, polyethylene glycol diacrylate, gelatin methacrylate compound, polysaccharides, and especially polysaccharides of bacterial origin, such as gellan gum, or of plant origin, such as pectin or alginate. In one embodiment, the hydrogel solution comprises at least alginate. Preferably, the hydrogel solution comprises only alginate. In the context of the invention, "alginate" is understood to mean linear polysaccharides formed from bD-mannuronate (M) and α-L-gluluronate (G), salts and derivatives thereof. Advantageously, the alginate is a sodium alginate, composed of more than 80% of G and less than 20% of M, with an average molecular mass of 100 to 400 kDa (for example: PRONOVA® SLG100) and a total concentration of between 0.5% and 5% by weight (weight / volume).

According to the invention, the microcompartment cell is closed. It is the outer layer of hydrogel that gives its size and shape to the cellular microcompartment. The microcompartment can have any form compatible with cell encapsulation.

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

According to the invention, the microcompartment can contain, in place of the extracellular matrix, an extracellular matrix substitute. An extracellular matrix substitute is a compound capable of promoting attachment and / or survival of cells by interacting with membrane proteins and / or extracellular signal transduction pathways. For example, such a substitute comprises biological polymers and their fragments, in particular proteins (laminins, vitronectins, fibronectins and collagens), non-sulfated (hyaluronic acid) or sulphated glycosaminoglycans (chondroitin sulfate, dermatan sulphate, keratan sulphate, heparan sulphate), and synthetic polymers containing units derived from biological polymers or reproducing their properties (RGD pattern) and small molecules mimicking attachment to a substrate (Rho-A kinase inhibitors such as Y-27632 or thiazovivin).

Any method of producing cellular microcompartments containing within a hydrogel capsule of the extracellular matrix and cells can be used for carrying out the preparation method according to the invention. In particular, it is possible to prepare microcompartments by adapting the method and the micro fluidic device described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of fimctionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC), "Lab on a Chip, 2016, vol. 16, no. 9, p. 1593-1604).

Advantageously, the dimensions of the cellular microcompartment are controlled. In one embodiment, the cellular microcompartment according to the invention has a spherical shape. Preferably, the diameter of such a microcompartment is between 10 mh and 1 mm, more preferably between 50 mhi and 500 mh, more preferably less than 500 mhi, preferably less than 400 mhi. In another embodiment, the cellular microcompartment according to the invention has an elongate shape. In particular, the microcompartment can have an ovoid or tubular shape. Advantageously, the smallest dimension of such an ovoid or tubular microcompartment is between 10 mh and 1 mm, more preferably between 50 mhi and 500 mhi, more preferably less than 500 mhi, preferably less than 400 mhi. "Smallest dimension" means twice the minimum distance between a point on the outer surface of the hydrogel layer and the center of the microcompartment.

In a particular embodiment, the thickness of the hydrogel outer layer is 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 attached to the inner face of the hydrogel shell. This matrix layer can fill the space between the cells and the hydrogel shell. In the context of the invention, the "thickness" of a layer is the dimension of said layer extending radially from the center of the microcompartment.

In one embodiment of the invention, the bioreactor comprises microcompartments in which the cells are self-organized in a cyst.

In the context of the invention, at least one layer of pluripotent or totipotent cells organized around a central lumen is designated by cyst. According to the invention, such a microcompartment therefore comprises successively, around a central lumen, said layer of pluripotent cells, an extracellular matrix layer, or an extracellular matrix substitute, and the outer layer of hydrogel. Light is generated at the time of cyst formation by the cells that multiply and grow in layers on the extracellular matrix layer. Advantageously, the light contains a liquid and more particularly the culture medium.

According to the invention, a cyst advantageously contains one or more layers of pluripotent stem cells of a mammal, human or non-human. A pluripotent stem cell, or pluripotent cell, is a cell that has the capacity to form all the tissues present in the whole organism, without being able to form an organism. whole as such. In particular, a cyst may contain embryonic stem cells (ESC), pluripotency-induced stem cells (IPS), or MUSE cells ("Multilineage-differentiating stress enduring") found in the skin and bone marrow. adult mammals. Advantageously, the thickness of the hydrogel outer layer represents 5 to 40% of the radius of the microcompartment, the thickness of the extracellular matrix layer represents 5 to 80% of the microcompartment radius and the thickness of the pluripotent cell layer represents about 10% of the radius of the microcompartment. The pluripotent cell layer is in contact at least at one point with the extracellular matrix layer, a space filled with culture medium may be present between the matrix layer and the cyst. The light then represents from 5 to 30% of the radius of the microcompartment. In a particular example, the cellular microcompartment has a spherical shape of radius equal to 1 OOmhi. The hydrogel layer has a thickness of 5mhi to 40mhi. The extracellular matrix layer has a thickness of 5 μm to about 80 μm. The pluripotent cell layer has a thickness of 10 to 30 mHi, the light having a radius of about 5 to 30 mHi.

According to an exemplary embodiment of the invention, it is possible to cultivate in a bioreactor, for example, 150 ml of such microcompartments, in which the cells form cysts, according to the steps below:

(a) incubate from 600,000 to 2 million mammalian pluripotent stem cells in culture medium containing RHO / ROCK inhibitor;

(b) mixing said pluripotent stem cells from step (a) with an extracellular matrix;

(c) encapsulating the mixture of step (b) in a hydrogel layer;

(d) culturing the capsules obtained in step (c) in a culture medium containing a RHO / ROCK inhibitor;

(e) rinsing the capsules from step (d) so as to remove the RHO / ROCK inhibitor;

(f) cultivating in a fed-batch type of production the capsules resulting from step (e) for 3 to 20 days, preferably 5 to 10 days, by diluting the volume of medium by a factor of two each day with a pluripotent cell culture medium such as MTESR1 (Stemcell technologies) lacking RHO / ROCK pathway inhibitor, and optionally recovering the cell microcompartments obtained. Those skilled in the art will be able to adapt the number of cells and the volume of the bioreactor according to the needs.

The step (a) of incubation and the step (d) of culture in a medium containing one or more inhibitors of RhO / ROCK ("Rho-associated protein kinase") pathways, such as thiazovivin (C. , N: OS) and / or Y-27632 (C14H21N3O) promote the survival of pluripotent stem cells, and the adhesion of cells to the extracellular matrix at the time of formation of the outer layer of hydrogel around said matrix extracellular. It is desirable, however, that these steps be time-limited so that RHO / ROCK inhibitors do not prevent cyst formation.

Thus, preferably, the incubation of step (a) is conducted for a time of between a few minutes and a few hours, preferably between 2 minutes and 2 hours, more preferably between 10 minutes and 1 hour.

Likewise, preferably, step (d) of culture is conducted for a time of between 2 and 48 hours, preferably for a time of between 6 and 24 hours, more preferably for a time of between 12 and 18 hours.

Step (e) is necessary to ensure removal of any trace of RHO / ROCK pathway inhibitors. Step (e) is for example carried out by rinsing, and preferably by several rinses, in successive culture media free of RHO / ROCK channel inhibitors.

Advantageously, step (f) is conducted for a time sufficient to obtain a cellular microcompartment in which the extracellular matrix and pluripotent cell layers have a cumulative thickness equal to 50 to 100% of the thickness of the hydrogel outer layer. . Any culture medium suitable for pluripotent stem cell culture can be used.

In one embodiment, the method according to the invention comprises an intermediate step (a ') of dissociating the pluripotent stem cells resulting from step (a) before step (b), preferably by means of an enzyme-free reagent. Advantageously, said reagent is inhibited or rinsed before the encapsulation step, in particular by successive rinsing in a specific medium for pluripotent cells. For example, the reagent used is ReLeSR®. Of course, it is also possible to use trypsin or an enzyme-containing reagent, but the survival rate of the pluripotent cells at the end of this step may then be lower compared to the use of a reagent free of 'enzyme.

Alternatively, such microcompartments can be obtained according to the steps below: (A) mixing differentiated mammalian cells with an extracellular matrix and cell reprogramming agents;

(B) encapsulating the mixture of step (A) in a hydrogel layer;

(C) culturing the capsules from step (B) for at least 3 days, and optionally recovering the obtained cell microcompartments.

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

Those skilled in the art can reprogram a differentiated cell into a stem cell by reactivating the expression of genes associated with the embryonic stage by means of specific factors. By way of examples, the methods described in Takahashi et al., 2006 ("Induction of pluripotent stem cells from embryonic and adult fibroblast cultures by defiined factors" Cell, 2006 Vol 126, pages 663-676) and in the international application W02010 / 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 to promote contact with all the cells.

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 (A) is carried out using specific culture media, promoting these phenotypic changes. For example, the cells are cultured in a first medium comprising 10% human serum, or bovine, in Eagle's Minimal Essential Medium (DMEM) supplemented with a serine / threonine protein kinase receptor inhibitor (such as the SB product). -431542 (C ₂₂ H ₁₆ N ₄ O ₃ )), one or more RHO / ROCK ("Rho-associated protein kinase") pathway inhibitors, such as thiazovivin and / or Y-27632, fibroblast growth factors such as FGF-2, ascorbic acid and antibiotics, such as Trichostatin A (C ₁₇ H ₂₂ N ₂ O ₃ ). Then the culture medium is replaced by a medium promoting the multiplication of pluripotent cells, such as mTeSR®l medium. Such cysts can then be forced into a differentiation path of interest, so as to obtain microcompartments containing one or more cell types of interest, in particular for the production of molecules of interest, or the production of organoids. interest. In one embodiment, the bioreactor comprises microcompartments including organo-organ-organized cells.

In the context of the invention, the term "organoid" denotes a multicellular structure organized in three dimensions so as to reproduce the microstructure of at least a part of an organ. According to the invention, such a microcompartment therefore comprises a multicellular structure in 3 dimensions, surrounded by extracellular matrix, the whole being encapsulated in the outer layer of hydrogel.

According to the invention, the organoids can be obtained by encapsulating pluripotent or progenitor cells which are then differentiated inside the hydrogel capsule, or by directly encapsulating differentiated cells or mature cells.

In one embodiment, the cellular microcompartments introduced into the bioreactor contain pluripotent cells. A cell differentiation step in at least one cell type of interest is then carried out inside the bioreactor, and optionally a step of multiplication of said differentiated cells in the microcompartments.

In one embodiment, the cellular micro-compartments introduced into the bioreactor contain already differentiated cells or progenitors. A step of multiplication and / or maturation of said differentiated cells in the microcompartments is then carried out inside the bioreactor.

Advantageously, the micro-compartments introduced into the bioreactor have an initial cell density of less than 10% of occupancy of the internal volume of the microcompartment, preferably less than 1%, more preferably less than 0.1%.

Advantageously, the microcompartment recovered at the end of the culture step in the bioreactor have a cell density greater than 10% occupancy of the internal volume of microcompartments.

According to the invention, the cells contained in the hydrogel capsules are subjected to the flow of medium contained in the bioreactor and which passes through the hydrogel layer.

Advantageously, the convective volume ratio outside the microcompartment on diffusive volume inside the microcompartment is between 1 and 10,000, preferably between 1 and 1000, more preferably between 1 and 100.

According to the invention, the convective volume designates the volume of culture medium inside the chamber of the reactor, between the microcompartments. The microcompartments being in suspension in the bioreactor, the convective volume thus represents the medium flowing between the microcompartments. Conversely, the diffusive volume refers to the volume of culture medium diffusing inside the microcompartment, that is to say in the space / spaces / voids formed around / between / by the cells once self-organized.

Thus, in the case of a microcompartment containing a cyst, the diffusive volume is mainly constituted by the central lumen and at the beginning of the growth of said cyst, the space between the wall of the capsule and the cyst. In the case of a microcompartment containing an organoid, the diffusive volume mainly consists of the spaces formed within the multicellular structure in 3 dimensions.

The microcompartments according to the invention are advantageously characterized by the presence within the hydrogel capsule of one or more lumens, or one or more spaces, devoid of cells and allowing precisely the multiplication or self-organization. cells inside the microcompartment. Those skilled in the art will be able to harvest the cells at the most appropriate moment for its amplification or differentiation process corresponding to a certain level of optimal space saturation in this context.

In one embodiment, the microcompartment occupies between 0.01% and 74% of the volume of the enclosure of the bioreactor.

The use of cellular micro-compartments makes it possible to cultivate the cells in any type of bioreactor, provided with a closed enclosure, and in particular in a bioreactor in "batch" feed mode, in feed mode with "fed" batch "or in continuous feeding mode (infusion). The use of these microcompartments is particularly advantageous in the case of culture in continuous feed mode. Indeed, the cells being protected by the hydrogel shell, it is possible to subject them to continuous flows, without the risk of weakening them.

In one embodiment, the bioreactor comprises a hermetically sealable enclosure. This makes it possible to control the atmosphere inside the bioreactor, and for example to cultivate microcompartments in an inert atmosphere.

The cell culture system according to the invention may comprise an enclosure having a volume of 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, between 500 mL and 10,000 L. In one embodiment, the enclosure has a volume of at least 1 mL. In one embodiment, the enclosure has a volume of at least 10 mL. In one embodiment, the enclosure has a volume of at least 100 mL. In one embodiment, the enclosure has a volume of at least 500 mL. In one embodiment, the enclosure has a volume of at least 1 L. In a mode of In one embodiment, the enclosure has a volume of at least 10 L. In one embodiment, the enclosure has a volume of 100 L, or more. Advantageously, any bioreactor comprising a closed chamber, and capable of producing, on an industrial scale, cells, organoids, molecules and / or complex molecule assemblies may be used.

In general, the use of a closed enclosure allows a fine control of the culture environment, without risk of disturbance by the external environment. It is also easy to obtain sterile products. This also allows better volumetric efficiency.

In one embodiment, the microcompartments comprise between 10% and 98% by volume of cells at harvest, ie between 100 and 1,000,000 cells depending on the diameter of the compartment concerned and the size of the cells produced, which can be calculated by realizing the ratio between the total number of cells produced (as measured by those skilled in the art with a Malassez cell or an automated cell counter) and the number of capsules obtained (as measured by the human the art by characterizing the volume of capsules by manual counting under an optical microscope or by an automated image analysis). Of course, it is possible to start the cell culture with microcompartments having a smaller number of cells initially, and in particular between 1 and 1,000 cells, ie 0.01% and 10% by volume occupied by the cells within the microcompartment following the diameter of the compartment concerned and the size of the cells produced. More generally, the microcompartments according to the invention comprise between 0.01% and 98% by volume of cells.

The cells can then multiply inside the microcompartment and self-organize, including organoids.

In one embodiment, the cells of a microcompartment are all of the same cell type. According to the invention, it is considered that the cells of the same microcompartment are all of the same cell type if at least 50%, preferably 70%, more preferably 90%, even more preferentially 98% or more of the cells of said microcompartment have the same phenotype, following the knowledge of those skilled in the art to characterize this cell type. In another embodiment, the cells of a microcompartment are at least two different cell types. Advantageously, between 20 and 100% of the cells of a compartment have the same phenotype.

According to the invention, it is possible to cultivate micro-compartments within the same bioreactor comprising all the same cell types, or conversely having different cell types. For example, the bioreactor may contain two types of microcompartments, each containing a particular cell type. The culture system according to the invention is particularly advantageous for the production and / or amplification of cells of interest. Indeed, the organization of the cells within the hydrogel capsule, with the extracellular matrix, allows their multiplication by a factor of 2 to 100,000 between each passage.

By passage is meant the manipulation of cells to add space or culture surface in order to continue amplification or to initiate differentiation or self-organization into organoids. This operation may require in the example of micro-carriers to reload the bioreactor with new micro-carriers. For the standard two-dimensional culture of pluripotent, adherent stem cells, this operation consists in detaching the cells from the old culture support in order to reseed a new culture medium with more surface, for those skilled in the art. operation can result in the loss of 50% of the cells. For the microcompartment culture according to the invention, this corresponds to the dissociation of the microcompartment, the dissociation of self-organized cell sets or their dispersion in cell sets sufficiently small to be encapsulated again in new microcompartments.

The invention particularly relates to the use of such a bioreactor cell culture system for the mass production of pluripotent cells.

The invention also relates to the use of such a bioreactor cell culture system for the production of unipotent or multipotent progenitors from pluripotent cells.

The invention also relates to the use of such a bioreactor cell culture system for the production of end-differentiated cells (that is to say corresponding to one or more specific functions) from pluripotent cells and / or or unipotent or multipotent progenitors and / or combinatorics of these progenitors.

The subject of the invention is in particular a method for producing organoids or cells of interest comprising the steps according to which:

introducing a plurality of cellular microcompartments into a bioreactor comprising a closed chamber, said microcompartments each comprising an outer layer of hydrogel encapsulating cells and extracellular matrix or an extracellular matrix substitute;

the microcompartment is cultivated under conditions allowing the multiplication of the cells inside the microcompartments, and / or the self-organization of the cells into organoids; cellular microcompartments are recovered

Those skilled in the art are able to adapt the culture conditions to the cell type of microcompartments, in order to promote their multiplication and / or self-organization.

In one embodiment, the cellular microcompartments introduced contain pluripotent cells, said method comprising, within the bioreactor, a cell differentiation step in at least one cell type of interest and a step of multiplication of said differentiated cells in the cells. microcompartments. For example, the production of primitive endoderm organoids for the study of differentiation in human endodermal tissues can be carried out according to the following protocol:

- From step f) for obtaining microcompartments, described above, at 2-3 days of culture:

Culture in a closed bioreactor of 150mL in STEMdiff ™ Pancreatic stage 1 medium of STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL technologies for 3 to 6 days.

- Use of the primitive endoderm obtained for developmental studies.

In another embodiment, the introduced cell microcompartments contain already differentiated cells or progenitors, said method comprising, within the bioreactor, a step of multiplying said differentiated cells in microcompartments.

During the multiplication and / or maturation step, the cells will advantageously self-organize into a specific organoid, according to an organization specific to said cell type.

In one embodiment concerning the amplification, the microcompartments introduced into the bioreactor have a cell density of less than 10% of occupancy of the internal volume of the microcompartment, preferably 1% even more preferably 0.1%. The cells will then multiply inside the microcompartments during the culturing step.

In one embodiment relating to differentiation and / or maturation without amplification, microcompartments introduced into the bioreactor have a cell density greater than 1% occupancy of the internal volume of microcompartments. The cells will then differentiate and / or mature and / or self-organize inside the microcompartments, during the culture stage. For example, a first type of production of neural organoids for neuronal transplantation as part of the cellular therapy of Parkinson's disease has been carried out according to the following protocol:

- Thawing of 5 million dopaminergic progenitors such as those marketed by Cellular Dynamics International (iCell® DopaNeurons),

Encapsulation of pre-differentiated neural progenitors according to the protocol described in Alessandri et al. 2016.

Culture in a closed bioreactor of 150mL in the culture medium provided by Cellular Dynamics.

- Maturation and structuration of neural dopaminergic organoids for two weeks in the bioreactor.

- Preparation of the graft by dissociation of the hydrogel capsule with two rinses of thirty seconds in 1 mL of ReLeSR® (Stemcell technologies) and resuspension in a solution of 11% by mass of dextran 70KDa in the medium of neuron culture, distribution in a glass cannula manufactured by us.

Graft in a model animal of Parkinson's disease.

In an embodiment combining amplification and differentiation / maturation, the microcompartments introduced into the bioreactor advantageously have a cell density of less than 10% of occupancy of the internal volume of the microcompartment, preferably 1%, even more preferably 0.1%. The cells will then multiply inside the microcompartments, during the culture step and then during the differentiation step. The cells will then self-organize within the microcompartments, during a second culture step that can be triggered by a change in the nature of the nutrient medium or a physical trigger (temperature, illumination). For example, a second type of production of neural organoids for neuronal transplantation in the context of Parkinson's disease cell therapy has been carried out according to the following protocol:

- From step f) for obtaining microcompartment described above at 2-3 days of culture:

Culture in a closed bioreactor of 150mL in a neural induction medium containing inhibitors of BMP2 signaling pathways (2mM Dorsomorphin or 0.5mM LDNA 193189) and TGLbeta (+ 10mM SB 431542), 24 (S), 25- 10 mM epoxycholesterol on a neurobasal basis / DMEM-Ll2 supplemented with N2 and B27 for 1 to 2 days.

Culture in a closed bioreactor of 150mL in a neural regionalization medium containing inhibitors of signaling pathways BMP2 (2mM Dorsomorphin or 0.5mM LDNA 193189) and TGLbeta (+ 10mM SB 431542), two activators of the SHH pathway (SHH 200 ng / mL, 1 mM Purmorphamine) and LGL8 (100 ng / mL), an inhibitor of the WNT pathway 3 mM Chir9902l, 24 (S), 25 mM epoxycholesterol on a neurobasal basis / DMEM-Fl2 supplemented with N2 and B27 for 6 days.

- Culture in a closed bioreactor of 150mL in a second neural regionalization medium containing an inhibitor of the BMP2 signaling pathway (2mM Dorsomorphin or 0.5mM LDNA 193189), an inhibitor of the WNT Chir9902I 3 mM pathway, 24 (S ), 25-epoxycholesterol 10mM on a neurobasal basis / DMEM-Fl2 supplemented with N2 and B27 for 1 day.

Culture in a closed bioreactor of 150mL in a medium of maturation and structuration of the neural dopaminergic organoids for two weeks in the bioreactor containing cyclic AMP (500mM) + ascorbic acid (200mM) + GDNF (20ng / mL) + BDNF (20ng / mL) + FGF-20 (5ng / mL) + TGFbeta (lng / mL) + trichostatin (10hM) + Compound E (ImM).

Graft in a model animal of Parkinson's disease.

In another embodiment combining amplification and differentiation / maturation, the micro-compartments introduced into the bioreactor advantageously have a cell density of less than 10% occupancy of the internal volume of the microcompartment, preferably 1% even more preferably 0.1%. The cells will then multiply inside the microcompartments. The cells are then recovered by dissolution of the capsule, then subjected to a second encapsulation step followed by the differentiation step, the cells will then self-organize inside the microcompartments, during a second culture step. which can be triggered by a change in the nature of the nutrient medium or a physical trigger (temperature, illumination). For example, the production of human pancreas organoids for the transplantation of human pancreatic tissues has been carried out according to the following protocol:

From step f) of obtaining microcompartment described above at 2-3 days of culture:

Culture in a closed bioreactor of 150mL in a STEMdiff ™ Pancreatic stage 1 medium supplemented with complement 1A and complement 1B of STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL technologies for 1 day.

Culture in a closed bioreactor of 150mL in a STEMdiff ™ Pancreatic stage 1 medium supplemented by the supplement 1B of the STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL Technologies for 1 day. Culture in a closed bioreactor of 150mL in STEMdiff ™ Pancreatic stage 2-4 supplemented by complement 2A and complement 2B of STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL technologies for 1 day.

Culture in a closed bioreactor of 150mL in a STEMdiff ™ Pancreatic stage 2-4 medium complemented by complement 2A and complement 2B of STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL technologies for 2 days.

Culture in a closed bioreactor of 150mL in a medium STEMdiff ™ Pancreatic stage 2-4 supplemented by complement 3 of the STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL technologies for 3 days.

Culture in a closed bioreactor of 150mL in a medium STEMdiff ™ Pancreatic stage 2-4 complemented by the supplement 3 of the STEMdiff ™ Pancreatic Progenitor Kit marketed by STEMCELL technologies for 5 days.

- Preparation of the graft by dissociation of the hydrogel capsule with two rinses of thirty seconds in lmL ReLeSR® (Stemcell technologies) and resuspension in a solution of 11% by mass of dextran 70KDa in the previous medium, distribution in a glass cannula manufactured by us.

Graft in a model animal type 1 diabetes.

Advantageously, the microcompartment recovered at the end of the step of culture in the bioreactor have a cell density greater than 10% occupancy of the internal volume of the microcompartment, preferably greater than 50%, and can go in the case of organoids up to at 98% occupancy.

The culture system according to the invention is also particularly advantageous for the production of molecules of interest and / or complex molecular assemblies, said molecules and / or complex molecular assemblies being excreted by the cells of the microcompartments out of said microcompartment. in the culture medium, or conversely accumulated inside the microcompartment for a subsequent harvest. This production method notably makes it possible to limit the filtration steps of the cellular elements by concentrating them inside the microcompartments. This method makes it possible, thanks to the separation in the bioreactor of the convective volume and the diffusive volume by the capsule, a facilitated segregation of the medium containing the dissolved elements of the insoluble elements or of a size greater than the size of the hydrogel of the capsule ( typically 150 to 250 KDa for alginate).

According to the invention, the microcompartments are then advantageously used in a reactor in continuous feed mode. As explained above, the presence of the protective hydrogel shell makes it possible to infuse the culture medium with a flow rate without risk of damaging the cells. In particular, it is possible to perfuse the interior of the reactor in a culture medium with a flow rate of between 0.001 and 100 volumes of cells contained in the bioreactor per day.

5

Claims

A bioreactor cell culture system comprising a closed chamber containing a plurality of cell microcompartments in suspension, wherein the microcompartments each comprise an outer hydrogel layer providing a cavity containing a set of self-organized cells and extracellular matrix or a substitute for extracellular matrix.

2- bioreactor cell culture system according to claim 1, wherein the convective volume ratio outside the microcompartment on diffusive volume within the microcompartment is between 1 and 10,000.

3- bioreactor cell culture system according to one of the preceding claims, wherein all or part of the microcompartment comprises self-organized cyst cells.

4- bioreactor cell culture system according to one of the preceding claims, wherein all or part of the microcompartments include cells self-organoed organoids

5- bioreactor cell culture system according to one of the preceding claims, wherein the bioreactor is selected from bioreactors in batch feed mode, fed batch mode bioreactors and bioreactors in the bioreactor mode. continuous feeding, preferably among the bioreactors in continuous feeding mode (infusion). Bioreactor cell culture system according to one of the preceding claims, wherein the enclosure has a volume between 1 mL and 10,000L.

Bioreactor cell culture system according to one of the preceding claims, wherein the microcompartment comprises between 0.01% and 98% by volume of cells.

8. The system according to one of the preceding claims, wherein the cells of a microcompartment are all of the same cell type, or conversely are at least two different cell types.

9. System according to one of the preceding claims, wherein the microcompartments all comprise the same cell types, or conversely present at least partially different cell types. 10- Use of the bioreactor cell culture system according to one of claims 1 to 9, for the production and / or amplification of cells of interest, preferably a factor of 2 to 100,000 between each pass.

11- The use of the bioreactor cell culture system according to one of claims 1 to 9 for the production of molecules of interest or complex molecular assemblies, said molecules or assemblies being excreted by the cells of the microcompartments out of said microcompartments up to the culture medium or conversely accumulated inside the microcompartment for a subsequent harvest.

A process for producing organoids or cells of interest comprising the steps of:

introducing a plurality of cellular microcompartments into a bioreactor, said microcompartments each comprising an outer layer of hydrogel encapsulating cells and extracellular matrix or an extracellular matrix substitute;

the microcompartment is cultured under conditions allowing the multiplication of the cells inside the microcompartments, and / or the self organization of the cells into organoids;

cellular microcompartments are recovered

and optionally, the hydrogel layer is hydrolyzed to recover the organoids or the cells.

13- Method according to claim 12, wherein the introduced microcompartmental cell contain pluripotent cells, said method comprising, within the bioreactor, a cell differentiation step in at least one cell type of interest and optionally a multiplication step said differentiated cells in microcompartments.

14. The method of claim 12, wherein the introduced microcompartment cell contain already differentiated cells or progenitors, said method comprising, within the bioreactor, a step of multiplication and / or maturation of said differentiated cells in microcompartments.

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

16- Method according to one of claims 12 to 15, wherein the microcompartment recovered at the end of the step of culture in the bioreactor have a cell density greater than 10% occupancy of the internal volume of the microcompartment.