Transduction and / or transfection in a three-dimensional microcompartment

Abstract

The present invention relates to the fields of cell transduction and transfection in a three-dimensional cell culture model and concerns, in particular, a specific cell microcompartment enabling transduction and / or transfection to be carried out within the internal part of said microcompartment.

Description

Title of the invention: Transduction and / or transfection in a three-dimensional microcompartment technical field

[0001] The present invention relates to the field of transient, constitutive, or conditional genetic and proteomic modification of cells in a three-dimensional cell culture model. The invention particularly relates to a specific cell microcompartment for performing transduction and / or transfection within the internal portion of said microcompartment. State of the art

[0002] In recent years, cell culture methods have evolved in terms of both complexity and performance. Traditional cell culture methods, which consist of culturing cells on flat surfaces in two dimensions, are being replaced by three-dimensional (3D) culture techniques, offering better reproduction of the cellular environment in vivo.

[0003] 3D cell culture offers an alternative closer to biological reality by reproducing cell-extracellular matrix and cell-cell interactions more accurately, leading to more reliable and relevant results for cell biology studies, medical research and cell therapy.

[0004] The cells cultured in these 3D systems can be of any type. They can be differentiated cells with different phenotypes, progenitor cells, or stem cells.

[0005] However, certain constraints associated with 3D cell culture may limit its use, particularly for specific applications such as transgene delivery and expression. Indeed, common transduction or transfection techniques that work well in 2D cell culture may not be effective for 3D cultures, where cells are encapsulated in denser extracellular matrices, thus limiting the access of transduction vectors or chemical or physical transfection agents to the cells.

[0006] For example, the document "Neumann AJ, Schroeder J, Alini M, Archer CW, Stoddart MJ. Enhanced adenovirus transduction of hMSCs using 3D hydrogel cell carriers. Mol Biotechnol. 2013;53(2):207-216. doi:10.1007 / sl2033-012-9522-y" presents a method for transducing cells in a 3D culture via the encapsulation of cells and viral vectors in alginate. The capsules thus formed are filled with alginate of the same density, limiting transduction within the capsule. Encapsulation in alginate forming "full" capsules limits or even blocks the movement of cells and viral vectors and consequently reduces the efficiency of transduction.

[0007] Thus, existing 3D cell culture systems do not exploit their performance potential, particularly for the implementation of genetic and / or proteomic modification of cells, more specifically by transfection or transduction.

[0008] There is therefore an important need for a new solution enabling the transfection and / or transduction of 3D cultured cells so that said cells can express or receive at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner. Summary of the invention

[0009] To meet this need, the invention proposes a new cellular microcompartment comprising an outer hydrogel layer defining an inner part, said inner part comprising: - at least one human, animal or plant eukaryotic cell; - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the outer layer.

[0010] Preferably, the hydrogel of the inner part is different from the hydrogel constituting the outer layer, in that it has at least one different chemical property and / or at least one different physical property.

[0011] Advantageously, the hydrogel of the outer layer retains the hydrogel of the inner part so as to form a three-dimensional cellular microcompartment. The hydrogel and / or aqueous solution of the inner part are particularly well-suited to maintaining the viability of human, animal, or plant eukaryotic cells while allowing the diffusion of said cells and transduction and / or transfection agents within the inner part.

[0012] Preferably, the microcompartment according to the invention is characterized by a diffusive conductance of the outer layer to at least one transduction agent and / or transfection agent that is strictly lower than the diffusive conductance of the inner part to the same transduction agent and / or transfection agent.

[0013] Diffusive conductance corresponds to the capacity of a material, such as a hydrogel layer, to allow the diffusion of an object, such as a transduction and / or transfection agent in solutions. Thus, the higher the diffusive conductance of a material to an object, the more the object will be able to diffuse into said material.

[0014] According to one embodiment, the outer layer of the microcompartment according to the invention is characterized by the presence of pores whose largest dimension is less than the smallest dimension d of said at least one transduction agent and / or transfection agent and, when the inner part comprises a hydrogel, the latter comprises pores whose smallest dimension is greater than this dimension d.

[0015] Advantageously, the pore size of the outer layer is smaller than the smallest dimension d of said at least one transduction and / or transfection agent, preventing the diffusion of at least one transduction and / or transfection agent through the outer layer. In other words, the pores of the outer layer, by virtue of their adapted dimensions, allow at least one transfection and / or transduction agent to be retained within the inner part of the microcompartment. Conversely, when the inner part comprises a hydrogel, the latter includes pores whose adapted dimensions allow at least one transduction and / or transfection agent to diffuse into the inner part.

[0016] In the context of the invention, the pore size can be measured by techniques well known to those skilled in the art, namely: - By transmission electron microscopy; - By super-resolution optical microscopy; - By X-ray diffraction imaging; - By neutron diffraction imaging; and - By excluding molecules of known size and conformation.

[0017] Preferably, the outer hydrogel layer is characterized by the presence of pores whose largest dimension is less than 25nm, preferably less than 20nm, in particular less than 1nm, even more preferably less than 5nm.

[0018] According to a preferred object of the invention, the hydrogel of the inner part is characterized by the presence of pores whose smallest dimension is greater than 10 nm, preferably greater than 25 nm, even more preferably between 25 and 500 nm.

[0019] When the internal part comprises at least one aqueous solution, this does not limit the diffusion of soluble particles such as transfection and / or transduction agents according to their size through said solution, in particular soluble particles whose size would be greater than 10 nm, preferably 25 nm.

[0020] According to one embodiment, the internal part of the microcompartment according to the invention comprises at least one solution and / or at least one hydrogel comprising pores whose smallest dimension is greater than 10 nm, in particular greater than 25 nm, preferably greater than 100 nm, even more preferably greater than 500 nm.

[0021] The invention thus relates to a microcompartment comprising two distinct structural elements allowing transfection and / or transduction within the microcompartment, namely: - an outer layer or envelope, formed by a hydrogel layer, preventing cells and transduction and / or transfection agents from the inner part from passing through it. Preferably, said outer layer has a diffusive conductance to at least one transduction and / or transfection agent strictly lower than the diffusive conductance of the inner part to that same transduction and / or transfection agent, and / or comprises pores whose largest dimension is smaller than the smallest dimension d of said transduction and / or transfection agent, so that the transduction agent cannot diffuse through this outer layer; and - an inner portion, comprising in particular at least one aqueous solution and / or at least one hydrogel different from that of the outer layer, intended to promote interaction between cells and transduction and / or transfection agents comprising at least one molecule of interest, it being understood that said inner portion is delimited by the outer hydrogel layer. Preferably, said inner portion has a diffusive conductance to at least one transduction and / or transfection agent strictly greater than the diffusive conductance of the outer layer to that same transduction and / or transfection agent, and / or when the inner portion comprises at least one hydrogel, the latter comprises pores whose smallest dimension is greater than the smallest dimension d of said transduction and / or transfection agent, so that the transduction agent can diffuse through this inner portion.

[0022] The outer hydrogel layer allows, on the one hand, the formation of the protective outer envelope, and thus the formation of the capsule or microcompartment, on the other hand, the solution and / or hydrogel of the less rigid, looser inner part allows cell growth and promotes the movement of cells and transfection and / or transduction agents within the inner part, given the particularly suitable conductance and / or pore size of the solution and / or hydrogel.

[0023] Conversely, the prior art, composed of a single layer of hydrogel, is either too rigid, preventing the movement of cells and transduction and / or transfection agents and resulting in low transfection / transduction efficiency, or, on the contrary, too loose and therefore incompatible with bioreactor culture. Indeed, the mechanical stresses of bioreactor culture, particularly due to the significant shear forces generated during culture, necessitate a suitable cell microcompartment.

[0024] The outer hydrogel layer, different from the inner part comprising a A solution and / or a different hydrogel is used to form the outer shell of the microcompartment or capsule according to the invention, which protects the capsule's contents from the external environment, particularly when the capsules are cultured in a bioreactor. Furthermore, the outer hydrogel layer ensures the retention of cells and transduction / transfection agents within it. This outer layer thus allows for the separation of the concentration of transduction / transfection agents near the cells, also known as the "local concentration," from the overall concentration of transduction / transfection agents in the culture system, also known as the "global concentration."

[0025] Thus, the structure of the microcompartment optimizes cell-transfection and / or transduction agent encounters and thus makes it possible to reduce the amount of transfection and / or transduction agent needed to obtain the desired transfection and / or transduction efficiency, in particular compared to 2D cultures or 3D cultures described in the prior art.

[0026] According to another preferred embodiment of the invention, the microcompartment comprises at least one transfection agent and / or at least one transduction agent capable of diffusing into the inner part. Preferably, the majority of the transfection and / or transduction agents present in the inner part of the microcompartment are capable of diffusing. According to one embodiment, all the transfection and / or transduction agents present in the inner part of the microcompartment are capable of diffusing.

[0027] Advantageously, the properties of the aqueous solution and / or hydrogel in the internal part allow the movement of cells and transfection and / or transduction agents, thus promoting their encounter and consequently promoting transfection / transduction within the internal part of the microcompartment.

[0028] Also, the microcompartment according to the invention comprises four major constituents, namely: - at least one outer layer of hydrogel forming the envelope of said microcompartment; - at least one solution and / or hydrogel, allowing the diffusion of transduction and / or transfection agents as well as, optionally, of cells present in the internal part of the microcompartment; -at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest; - at least one human, animal or plant eukaryotic cell.

[0029] The cells present in the internal part can be of any cell type. More preferably, the cells are chosen from human, animal and plant eukaryotic cells, and even more preferably from stem cells Pluripotent cells include progenitors, cells undergoing differentiation, and differentiated cells. Preferably, the cells are not derived from a human embryo or require the destruction of a human embryo.

[0030] The cells present in the internal part can be isolated and / or in the form of at least one layer and / or in the form of at least one three-dimensional aggregate and / or in the form of at least one three-dimensional cellular micro-tissue, possibly with at least one lumen.

[0031] According to one embodiment, the microcompartment comprises at least one cell layer and at least one lumen. When the microcompartment comprises at least one lumen, at least one cell layer, the transduction agent(s) and / or transfection agent(s), the aqueous solution layer or the hydrogel of the inner part and the outer layer are preferably, successively organized around said lumen.

[0032] According to another aspect, the invention also relates to a set of microcompartments, in which said set comprises at least one microcompartment according to any of the preceding embodiments.

[0033] The microcompartment according to the invention or the set of microcompartments according to the invention, is also particularly suitable for use in cell culture, in particular in three-dimensional cell culture, allowing the production of cells of interest in large quantities, in particular cells, micro-tissue or organoid comprising and / or expressing at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner, capable of being used, for example in the context of cell therapy.

[0034] Advantageously, the conformation of the microcompartment according to the invention makes it possible to carry out in capsulo transduction and / or transfection and consequently produce cells comprising and / or expressing at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner.

[0035] Also, the invention also relates to a microcompartment according to the invention or a set of microcompartments according to the invention, for its use as a drug.

[0036] According to one variant, the invention also relates to a use of the microcompartment according to the invention or of a set of microcompartments according to the invention to insert at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner into a eukaryotic cell.

[0037] According to another variant, the invention also relates to the use of the microcom A compartment according to the invention for manufacturing microtissues comprising and / or expressing at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive, or conditional manner. It is understood that in this particular embodiment, the microtissues are preferably not intended for implantation in a human or animal. By way of example, they can be used as an ex vivo model.

[0038] The invention also relates to a transduction method and a transfection method implemented in at least one microcompartment according to the invention.

[0039] On the other hand, the microcompartment according to the invention can be produced in different ways; however, in a particular aspect, the invention relates to a method for preparing the cell microcompartment according to the invention, comprising the following steps: (a) prepare a solution containing cells, (b) prepare at least one aqueous solution and / or at least one hydrogel comprising at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, (c) encapsulate the solutions and / or hydrogels from steps (a) and (b) by collinear flow in an outer layer of hydrogel different from that of step (b); (d) cultivate the microcompartments obtained in step (c) in a culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably from 3 to 50 days, and (e) optionally recover the resulting cellular microcompartments.

[0040] According to a particular object of the invention, step (c) comprises the following substeps: - bring the solution from step (a) and the solution from step (b) into contact with a layer of hydrogel to form at least one drop, and - collect the drop obtained in a calcium bath suitable for stiffening said hydrogel solution to form the outer layer of each microcompartment.

[0041] In a particularly preferred manner, step (c) is carried out by simultaneous co-injection of the solution from step (a), the solution from step (b) and an outer layer of hydrogel, said co-injection is carried out concentrically via a microfluidic or millifluidic injector forming a jet at the injector outlet consisting of the mixture of said solutions, said jet breaking into drops.

[0042] Advantageously, the final opening diameter of the microfluidic injector is between 50 and 800 pm, preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 0.1 and 2000 mL / h, preferably between 10 and 2000 mL / h, more preferably between 10 and 150 mL / h, even more preferably between 11 and 100 mL / h.

[0043] According to a variant of the process according to the invention, step (c) is carried out by co-injection of the solution of step (a) and the solution of step (b) into an outer layer of hydrogel having pores whose largest dimension is less than the smallest dimension d of said transduction agent and / or transfection agent of step (a); said co-injection is carried out concentrically via a microfluidic or millifluidic injector, said injector comprising a tip, said tip being in contact with a calcium solution, forming a jet at the injector outlet consisting of the mixture of said solutions, said jet forming a tube.

[0044] When said injector includes a tip, said tip being in contact with the calcium solution, the final opening diameter of the microfluidic injector is preferably between 50 and 1000 pm, more preferably between 80 and 300 pm, and the flow rate of each of the solutions is between 1 and 100 mL / h.

[0045] Other features and advantages will become apparent from the detailed description of the invention, the examples and figures that follow. Brief description of the drawings

[0046] [Fig-1] Fig. 1 represents the experimental setup of Example 1 relating to the transduction of primary human CD3+ T lymphocytes.

[0047] [Fig.2A] Fig.2A is a graphical representation of a comparative study of the transduction efficiency of a 2D T lymphocyte culture system vs a 3D system as a function of the MOI of a Lentiviral vector.

[0048] [Fig.2B] Fig.2B is a graphical representation of a comparative study showing the fluorescence intensity of a transgene in cells transduced by a Lentiviral vector in a 2D T lymphocyte culture system vs a 3D system.

[0049] [Fig.2C] Fig.2C represents the average number of GFP transgenes integrated into the genome of T lymphocytes by a lentiviral vector, as measured by qPCR, in a 2D culture system versus a 3D system.

[0050] [Fig.2D] Fig.2D is a graphical representation of a comparative study of transduction efficiency in a 2D T lymphocyte culture system vs a 3D system as a function of the MOI of a Sendai viral vector.

[0051] [Fig.2E] Fig.2E is a graphical representation of a comparative study showing the fluorescence intensity of a transgene in T lymphocytes transduced by a Sendai viral vector in a 2D cell system vs a 3D system.

[0052] [Fig.3A] Fig.3A is a graphical representation of a comparative study of the cell viability of a 2D T lymphocyte culture system vs a 3D culture system in the presence of different MOI of a Sendai viral vector.

[0053] [Fig.3B] The [Fig.3A] is a graphical representation of a comparative study of the cell viability of a 2D T lymphocyte culture system vs a 3D culture system in the presence of different MOI of a Sendai viral vector.

[0054] [Fig.4] Fig.4 represents the experimental scheme of Example 2 relating to the transduction of human induced pluripotent stem cells (hIPS).

[0055] [Fig.5] The [Fig.5] is a graphical representation of a comparative study of the transduction efficiency of a 2D human induced pluripotent stem cell culture system vs a 3D system as a function of the MOI of several different transduction agents.

[0056] [Fig.6] The [Fig.6] is a graphical representation showing the cotransduction efficiency of 1, 2 or 3 lentiviral vectors in T lymphocytes.

[0057] [Fig.7A] The [Fig.7A] is a graphical representation of a comparative study of transduction efficiency in microcompartments according to the invention vs microcompartments outside the invention.

[0058] [Fig.7B] Fig.7B is a graphical representation of a comparative study of the fluorescence intensity of transgenes within microcompartments according to the invention vs microcompartments outside the invention after transduction.

[0059] [Fig.7C] The [Fig.7C] is a graphical representation of a comparative study of the cell viability of microcompartments according to the invention vs of microcompartments outside the invention after transduction.

[0060] [Fig.7D] The [Fig.7D] is a graphical representation of a comparative study showing the amplification factor of microcompartments according to the invention vs of microcompartments outside the invention after transduction. Detailed description of the invention

[0061] Definitions

[0062] For the purposes of this invention, "differentiated cells" means cells that exhibit a particular phenotype, as opposed to pluripotent stem cells that are not differentiated or progenitor cells that are undergoing differentiation.

[0063] For the purposes of this invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even if not explicitly stated, the cells, stem cells, progenitor cells, and tissues according to this invention are constituted or obtained from human cells or from immunologically humanized non-human mammalian cells. For the purposes of this invention, "mutant cell" means a cell carrying at least one mutation.

[0064] For the purposes of this invention, "progenitor cell" means a stem cell already engaged in cell differentiation but not yet differentiated.

[0065] For the purposes of this invention, "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG, and SOX2, and surface markers such as SSEA4 / 5, Tra-1-60, and Tra-1-81. The embryonic stem cells used in the invention are obtained without destroying the embryo from which they are derived, for example, using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, human embryonic stem cells may be excluded.

[0066] For the purposes of this invention, "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire organism of origin, without, however, being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. These may include, in particular, induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells, or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0067] For the purposes of this invention, "induced pluripotent stem cell" refers to a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are notably positive for pluripotency markers, such as alkaline phosphatase staining and the expression of the proteins NANOG, SOX2, OCT4, and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 2007, 131(5): 861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1): 101-106).

[0068] For the purposes of this invention, "diffusive conductance" refers to the ability of a material to allow the diffusion of an object in solutions. Where the diffusive conductance of the object is: [Math] Pobjrt = JJ Where D is the effective diffusion coefficient of objects in the material, L is the length of the material through which the objects diffuse, and JJdS corresponds to the integration over the entire surface of the material through which the objects diffuse.

[0069] In the context of the invention, the diffusive conductance of the hydrogel of the part External diffusivity can be measured using two compartments separated by the hydrogel of the outer layer whose diffusive conductance is to be measured. To measure its diffusive conductance, simply place the solution containing the transfection or transduction agent in question in the first compartment and the solution without the agent in the other compartment. Then, using a suitable method such as counting, fluorescence, or titration, measure the change in the amount of agent in each compartment over time to deduce the diffusive conductance. For example, the two compartments could be two reservoirs separated by a membrane made of the hydrogel of the outer layer whose diffusive conductance is to be measured.

[0070] Alternatively, the diffusive conductance of the hydrogel's outer layer can be measured locally using the Fluorescence Recovery After Photobleaching (FRAP) technique for fluorescent agents of interest. This technique involves irradiating an area of ​​the material with a laser, thereby photobleaching the fluorescent agents in the irradiated area, and observing the return to a normal fluorescence level. The time to return to normal is directly related to the diffusion coefficient, which then allows the diffusive conductance to be calculated.

[0071] In the context of the invention, the diffusive conductance of the hydrogel in the inner part of a transfection or transduction agent is defined as follows: [Math2] IL = Dint*ir(R)2 / r Where D is the diffusion coefficient, R is the radius of the largest sphere inscribed in the inner part and r is the diameter of the smallest sphere circumscribed about the inner part.

[0072] For the purposes of this invention, a "cell layer" or "cell layer" refers to several cells forming a layer or layer that can be structured around a lumen; for example, a cellular tissue or micro-tissue, or a three-dimensionally grouped culture. The thickness of the cell layer can be variable. This layer is organized in three dimensions within the microcompartment.

[0073] By "Feret diameter" in the sense of the invention, we mean the distance, in particular "d" or "D", between two tangents, these two tangents being parallel, such that the whole of the projection is contained between these two parallel tangents.

[0074] For the purposes of this invention, "drop" also means a three-dimensional structure formed from at least one liquid solution comprising the constituents of an unrigidified hydrogel (polymerization precursors, chains of non- or partially cross-linked polymers), of hydrogel precursor elements. Also, the droplet constitutes a transitional state between the co-injection of the different constituents and the microcompartment according to the invention.

[0075] By "hydrogel of plant or synthetic origin" is meant a hydrogel that is not of animal origin and / or derived from cancer cell lines such as Matrigel®. It may be, for example and without limitation, alginate.

[0076] By "the smallest dimension" of X in the sense of the invention, we mean the value of the smallest diameter of Feret of X.

[0077] By "the largest dimension" of X in the sense of the invention, we mean the value of the largest diameter of Feret of X.

[0078] For the purposes of this invention, "light" or "lumen" refers to a volume of aqueous solution topologically surrounded by cells. Preferably, its contents are not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment.

[0079] For the purposes of this invention, "microcompartment" or "capsule" means a three-dimensional structure that is partially or totally closed and contains several cells.

[0080] By "MOI or multiplicity of infection" in the sense of the invention, we mean the ratio between the number of functional particles of each transduction agent and the number of cells within the microcompartment.

[0081] By "pore" in the sense of the invention, we mean a sub-volume within the general volume of hydrogel in which monomers and / or polymer of the hydrogel do not have covalent or ionic chemical bonds in continuity with the three-dimensional network of the general volume of the hydrogel.

[0082] For the purposes of this invention, "tissue" or "biological tissue" refers to the common biological meaning of tissue, that is, the intermediate level of organization between the cell and the organ. A tissue is a group of similar cells of the same origin (most often derived from a common cell lineage, although they may originate from the association of distinct cell lineages), grouped into clusters, networks, or bundles (fibers). A tissue forms a functional unit, meaning that its cells work together to perform the same function. Biological tissues regenerate regularly and are assembled to form organs.

[0083] Microcompartment according to the invention

[0084] The present invention relates to a three-dimensional cellular microcompartment comprising an outer hydrogel layer defining an inner part, said inner part comprising: - at least one human, animal or plant eukaryotic cell; - at least one transduction agent and / or at least one transfection agent including at least one molecule of interest, and - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the outer layer.

[0085] The microcompartment according to the invention is a three-dimensional microcompartment, delimited by the outer hydrogel layer and inside said outer layer, an inner part comprises one or more cells, one or more transduction and / or transfection agents, and one or more aqueous solutions and / or one or more hydrogels.

[0086] Advantageously, the outer layer delimits an inner part and allows the elements present within the inner part to be contained, in particular the cells and the transfection and / or transduction agents.

[0087] Preferably, the hydrogel of the inner part is different from the hydrogel constituting the outer layer, in that it has at least one different characteristic, in particular at least one different chemical property and / or at least one different physical property.

[0088] Advantageously, the hydrogel of the inner part, different from that of the outer layer, allows the transfection and / or transduction agents to diffuse into the inner part and consequently improves the efficiency of transduction / transfection, in particular compared to capsules or microcompartment made up of a single hydrogel.

[0089] According to one embodiment, the hydrogel of the outer layer has a density and / or viscosity strictly greater than the density and / or viscosity of the solution and / or hydrogel present within the inner part.

[0090] According to another embodiment, the hydrogel of the outer layer has a different molecular weight than the hydrogel in the inner part. Preferably, in this embodiment, the cell microcompartment according to the invention comprises in its inner part at least one cell, at least one transfection and / or transduction agent, at least one solution, and / or at least one hydrogel with a molecular weight lower than that of the hydrogel in the outer layer. Thus, in this embodiment, the molecular weight of the hydrogel in the outer layer is greater than the molecular weight of the hydrogel in the inner part.

[0091] Advantageously, the hydrogel and / or solution of the inner part allows cells and transfection and / or transduction agents to diffuse within the inner part. Furthermore, the outer layer exhibits greater diffusion resistance than the solution and / or hydrogel of the inner part, thereby improving transfection and / or transduction within the microcompartment. In addition, the outer hydrogel layer also protects cells from the external environment, limits uncontrolled cell proliferation, and restricts their differentiation should differentiation occur.

[0092] Preferably, the microcompartment according to the invention is characterized by a diffusive conductance of the outer layer to at least one transduction agent and / or transfection agent that is strictly lower than the diffusive conductance of the inner part to the same transduction agent and / or transfection agent.

[0093] According to one embodiment, the microcompartment according to the invention has a maximum diffusive conductance of the hydrogel of the outer layer to at least one transduction agent and / or transfection agent of less than 0.1 / h, preferably less than 0.05 / h, even more preferably less than 0.01 / h.

[0094] According to one embodiment, the microcompartment according to the invention has a maximum diffusive conductance of the hydrogel of the inner part to at least one transduction agent and / or transfection agent greater than 0.1 / h, preferably greater than 1 / h, even more preferably greater than 10 / h, in particular greater than 50 / h.

[0095] According to another embodiment, the microcompartment according to the invention has a ratio of the diffusive conductance of the hydrogel of the inner part to the diffusive conductance of the hydrogel of the outer layer with respect to a transfection and / or transduction agent greater than 5, preferably greater than 10, even more preferably greater than 100, in particular greater than 1000.

[0096] In the context of the invention, diffusive conductance is preferably measured at 25°C and at a pH of 7.

[0097] According to one embodiment of the invention, the three-dimensional cellular microcompartment comprises an outer hydrogel layer defining an inner part, said inner part comprising: - at least one human, animal or plant eukaryotic cell; - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and - at least one aqueous solution and / or at least one hydrogel, in which the porosity of the outer layer is characterized by the presence of pores whose largest dimension is less than the smallest dimension d of said at least one transduction agent and / or transfection agent and, where the inner part comprises a hydrogel, the latter comprises pores whose smallest dimension is greater than this dimension d.

[0098] In the context of the invention, the pore size can be measured by techniques well known to those skilled in the art, namely: - By transmission electron microscopy; - By super-resolution optical microscopy; - By X-ray diffraction imaging; - By neutron diffraction imaging; - By excluding molecules of known size and conformation.

[0099] Preferably the pore size of the hydrogel of the outer layer and of the hydrogel of the inner part is measured by neutron scattering or small angle X-ray scattering (SANS / SAXS) and by polymer scattering test of known molecular mass.

[0100] Preferably, the hydrogel of the outer layer is characterized by the presence of pores whose largest dimension is smaller than the smallest dimension d of at least one transduction and / or transfection agent present in the inner part. Even more preferably, the hydrogel constituting the outer layer is characterized by the presence of pores whose largest dimension is smaller than the smallest dimension d of the majority of the transduction and / or transfection agents present in the inner part. According to one embodiment, the hydrogel of the outer layer is characterized by the presence of pores whose largest dimension is smaller than the smallest dimension d of all the transduction and / or transfection agents present in the inner part.

[0101] According to a particularly suitable embodiment, the hydrogel constituting the outer layer is characterized by the presence of pores whose largest dimension is less than the smallest dimension d of the transduction agent or transfection agent having the smallest dimension d within the inner part.

[0102] Preferably, the hydrogel of the outer layer is characterized by the presence of pores whose largest dimension is less than 25 nm, preferably less than 20 nm, in particular less than 1 nm, and even more preferably less than 5 nm. Advantageously, the particular pore size of the outer hydrogel layer allows for the retention of cells and transduction and / or transfection agents in the inner part.

[0103] According to one embodiment, the hydrogel of the outer layer is characterized by the presence of at least one continuous diffusion path between the outer face and the inner face of the outer hydrogel layer for which the smallest dimension is less than 25 nm, preferably less than 10 nm. In other words, the hydrogel of the outer layer is characterized by the absence of a continuous diffusion path between the outer face and the inner face of the outer hydrogel layer for which the smallest dimension is less than 25 nm, preferably less than 10 nm.

[0104] According to one embodiment, the hydrogel of the inner part comprises pores whose smallest dimension is greater than the smallest dimension d of at least one transduction and / or transfection agent present in the inner part. Preferably, the hydrogel constituting the inner part comprises pores whose smallest dimension is greater than the smallest dimension d of the majority of the transduction and / or transfection agents present in the inner part. According to a In this embodiment, the hydrogel of the inner part comprises pores whose smallest dimension is greater than the smallest dimension d of all the transduction agents and / or all the transfection agents present in the inner part.

[0105] Preferably, the hydrogel of the inner part of the microcompartment comprises pores whose smallest dimension is greater than the smallest dimension d of the transduction agent and / or the transfection agent having the smallest dimension d within the inner part.

[0106] According to a particularly suitable embodiment, the smallest dimension of the pores of the hydrogel of the inner part is greater than 10nm, preferably greater than 25nm, even more preferably between 25 and 500nm.

[0107] Preferably, the microcompartment according to the invention is characterized in that it comprises at least one continuous diffusion path within the hydrogel of the inner part between a cell and a transfection and / or transduction agent for which the smallest dimension is greater than 1nm, preferably between 25 and 500nm.

[0108] According to a preferred embodiment, the microcompartment according to the invention comprises at least one aqueous solution and / or at least one hydrogel, in which the hydrogel of the inner part is characterized by the presence of pores whose smallest dimension is greater than the largest dimension D of said transduction agent and / or transfection agent present in the inner part, and in that the hydrogel of the outer layer is characterized by the presence of pores whose largest dimension is less than that of dimension D so that the transduction agent cannot diffuse through this outer layer.

[0109] Preferably, the hydrogel of the inner part of the microcompartment comprises pores whose smallest dimension is greater than the largest dimension D of the transduction agent and / or the transfection agent having the largest dimension D within the inner part.

[0110] According to a particular embodiment, the microcompartment according to the invention comprises an outer hydrogel layer defining an inner portion, said inner portion comprising at least one hydrogel, in which the Young's modulus of the hydrogel in the inner portion is strictly lower than the Young's modulus of the hydrogel in the outer layer. Thanks to this Young's modulus of the hydrogel in the inner portion, the outer layer will be more rigid than the hydrogel in the inner portion, the latter being looser, more flexible, particularly due to the presence of shorter chains, thus facilitating cell growth and the movement of transduction and / or transfection agents.

[0111] According to one aspect of the invention, the advantages mentioned above can be improved when the Young's modulus of the hydrogel in the inner part is included between 0.01 and 200 kPa, more preferably between 0.1 and 60 kPa, even more preferably between 0.1 and 5 kPa. Preferably, the Young's modulus of the outer layer hydrogel is greater than 10 kPa, more preferably greater than 60 kPa, even more preferably greater than 100 kPa.

[0112] According to one embodiment, the hydrogel of the inner part is entangled with the hydrogel of the outer layer, particularly on the inner face of the outer layer. Therefore, the boundary between the two hydrogels, despite having different Young's moduli, may not be perfectly sharp. Consequently, at least a portion of the hydrogel of the inner layer or mesh may be entangled with the inner face of the outer layer.

[0113] The hydrogel of the inner part with a low Young's modulus then exhibits viscoelastic and viscoplastic properties that are particularly advantageous for obtaining an extracellular matrix substitute, in which cells can lodge, grow, move and be transfected and / or transduced satisfactorily.

[0114] Advantageously, a hydrogel, for example an alginate hydrogel, with a low Young's modulus, exhibits faster-relaxing properties, also known as "fast-relaxing", which allows cells to move, change shape, extend, proliferate and mechanically remodel the hydrogel-based matrix and also to allow the diffusion of transfection and / or transduction agents, making the microcompartment according to the invention particularly suitable for in-capsulo transfection and / or transduction.

[0115] Preferably, the hydrogel(s) used in the microcompartments according to the invention are biocompatible, that is to say, they are not toxic to cells. They must allow the diffusion of oxygen and nutrients to nourish the cells contained in the microcompartment and allow their survival.

[0116] The hydrogel of the outer layer and / or the inner part may comprise or be made of alginate.

[0117] Alginate hydrogel is shear-thinning, that is to say that its viscosity decreases when the shear rate increases; this is particularly advantageous when passing through a micro-fluidic injector to form the microcompartments according to the invention.

[0118] According to a preferred embodiment, the outer hydrogel layer comprises at least alginate. It may consist exclusively of alginate.

[0119] The alginate constituting the outer layer may in particular be a sodium alginate, composed of 80% a-L-guluronate and 20% [3-D-mannuronate, having a Young's modulus greater than 100 kPa, preferably greater than 60 kPa, more preferably greater than 100 kPa.

[0120] According to one embodiment, the alginate of the outer layer has an average molecular weight of 100 to 400 kDa, more preferably between 150 and 250 kDa. When the hydrogel of the outer layer is alginate, the concentration of the alginate solution used to form said outer layer of the microcompartment is preferably between 0.5 and 5% by mass, more preferably the concentration is equal to 2% (plus or minus 0.5%) by mass.

[0121] When the concentration of the alginate solution intended to form the outer layer of the microcompartment is equal to 2%, the viscosity of the alginate forming the outer layer is preferably between 100 and 200 mPa / s.

[0122] Preferably, the outer hydrogel layer is cell-free.

[0123] The hydrogel of the inner part can be in the form of a layer or a mesh. According to one embodiment, the hydrogel of the inner part is juxtaposed at least partially to the inner face of the outer layer.

[0124] According to one embodiment, the microcompartment according to the invention comprises in its internal part: - a layer of aqueous solution; and / or a layer and / or mesh of hydrogel, or - an aqueous solution; and / or a layer and / or mesh of hydrogel, or - a layer of aqueous solution; and / or a hydrogel.

[0125] Preferably, the microcompartment according to the invention comprises in its internal part: - at least one layer of aqueous solution; and / or - at least one layer and / or mesh made of hydrogel.

[0126] Preferably, the hydrogel(s) used in the internal part of the microcompartments according to the invention are biocompatible, that is, they are not toxic to cells. They must allow the diffusion of oxygen and nutrients to nourish the cells contained in the microcompartment and enable their survival. These hydrogels can be chosen from any biocompatible hydrogels exhibiting the characteristics of the invention. They may include, for example, alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid, and / or collagen. They may also include an extracellular matrix or an extracellular matrix substitute such as Matrigel®.

[0127] According to one embodiment, the hydrogel of the inner part comprises alginate or is an alginate but different from the alginate of the outer layer if the outer layer is alginate. This difference may be characterized, in particular, by a difference in diffusive conductance, viscosity, density, molecular weight, Young's modulus, and / or pore size. According to another variant, the inner part comprises a layer and / or a mesh of hydrogel comprising at least alginate. The layer and / or mesh may consist exclusively or partially of alginate. The alginate present in the inner part may in particular be a sodium alginate, composed of 80% a-L-guluronate and 20% [3-D-mannuronate. The alginate of the inner part may be an alginate comprising pores whose smallest dimension is greater than the smallest dimension d of at least one transfection and / or transduction agent, and / or a Young's modulus between 0.01kPa and 200kPa, preferably between 0.1kPa and 60kPa, more preferably between 0.1kPa and 5kPa.

[0128] Preferably, the alginate present in the inner part has an average molecular weight of at most 75 kDa. When the hydrogel of the inner part is alginate, the concentration of the alginate solution intended to form the inner part of the microcompartment is preferably between 0.25 and 2%, more preferably between 0.25 and 1%, and even more preferably 0.5% (mass percentage). When the concentration of the alginate solution intended to form the inner part of the microcompartment is 0.5%, the viscosity of the alginate is preferably 3 mPa / s.

[0129] Advantageously, although mammalian cells are unable to interact with alginate, notably because it allows minimal protein adsorption, alginate is well characterized, easy to sterilize and store, can potentially be chemically modified, and offers interesting mechanical properties. Furthermore, alginate allows for good growth, low cell death, good amplification, and a good proportion of transfected and / or transduced cells.

[0130] According to one embodiment, the hydrogel of the inner part comprises or is fibrin, the fibrin being preferably obtained from the polymerization of fibrinogen by a fibrinogen polymerizing agent, such as thrombin. This polymerizing agent may be added during and / or after encapsulation. Furthermore, the polymerization of the fibrinogen solution by the thrombin solution takes place during and / or after encapsulation. When it occurs after encapsulation, the polymerization takes place within the newly formed droplet or capsule, that is, after the rigidification of the outer layer.

[0131] The hydrogel of the inner part may comprise other constituents such as, for example, at least one peptide sequence, more preferably a peptide sequence of interest capable of interacting with the cells present in the microcompartment according to the invention. By way of example, the peptide sequence may be a peptide or a protein. The peptide sequence may, for example, be a YIGSR motif and / or an RGD motif. The YIGSR motif is a peptide derived from the

[31] chain of laminin with the sequence Tyrosine-Isoleucine-Glycine-Serine-Arginine, facilitating cell adhesion to the matrix. The RGD motif is a peptide with the sequence Arginine-Glycine-Asparagine, also facilitating cell adhesion to the matrix.

[0132] According to a particular embodiment of the invention, the inner part comprises at least two different hydrogels, each being different from the hydrogel of the outer layer. These at least two hydrogels present in the inner part are preferably chosen from alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid, and collagen.

[0133] When the internal part comprises at least two hydrogels, the second hydrogel may be in the form of particles.

[0134] Advantageously, this form of the second hydrogel does not prevent the diffusion of transfection and / or transduction agents within the inner part while providing a beneficial effect on cells, preferentially by improving cell survival.

[0135] When the internal part comprises at least one aqueous solution, this does not limit the diffusion of soluble particles, in particular transfection agents and / or transduction agents depending on their size through said solution, in particular soluble particles whose size would be greater than 10 nm, preferably 25 nm.

[0136] According to another embodiment, when the internal part comprises at least one aqueous solution, the latter has a viscosity between 0.1 and 10mPa / s.

[0137] Preferably, when the inner part comprises an aqueous solution and a hydrogel, the inner part has a viscosity between 0.1 and 50 mPa / s.

[0138] Preferably, the viscosity of the internal part is between 0.1 and 50mPa / s.

[0139] Advantageously, the particular viscosity of the aqueous solution and / or hydrogel facilitates the meeting of transfection agents and / or transduction agents and cells within the internal part of the microcompartment and consequently increases the proportion of transduced / transfected cells compared to 2D or 3D cultures described in the prior art.

[0140] In the context of the invention, the viscosity of the aqueous solution and / or the hydrogel can be measured using a viscometer at 20°C.

[0141] Advantageously, the hydrogel and / or aqueous solution of the inner part promotes encounter with cells and consequently improves transduction / transfection efficiency.

[0142] Preferably, when the inner part comprises an aqueous solution, this solution is preferably a culture medium suitable for the cells of the microcompartment. Typically, the culture medium is chosen from among those commonly compatible with the culture of the cells present within the microcompartment, for example, from those used in two-dimensional culture or spheroid culture.

[0143] Preferably the hydrogel of the outer layer and / or in the inner part is chosen from fibrin, collagen, fibronectin, entactin, hyaluronic acid, alginate, laminin, an extracellular matrix substitute and mixtures thereof.

[0144] In addition to the hydrogel and / or aqueous solution, the internal part of the microcompartment comprises at least one transduction agent and / or a transfection agent comprising at least one molecule of interest.

[0145] Preferably, the microcompartment according to the invention comprising at least one transduction agent and / or at least one transfection agent in the aqueous solution and / or in the hydrogel of the inner part.

[0146] When the microcompartment according to the invention comprises at least one transduction agent, said agent may be chosen from a viral particle, a viral pseudoparticle or their combination.

[0147] Preferably, the transduction agent is chosen from an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a baculovirus and their combinations.

[0148] Preferably, when the microcompartment comprises at least one transduction agent, the MOI (“multiplicity of infection”) of said microcompartment is between 0.01 and 100, in particular between 0.1 and 50, even more preferably between 0.1 and 10.

[0149] According to one variant, when the microcompartment includes at least one transduction agent, the MOI of said microcompartment is less than 10.

[0150] When the microcompartment according to the invention comprises at least one transfection agent, said agent may be selected from an extracellular vesicle of endosomal origin such as an exosome, a plasma membrane derivative, a liposome, a nanoparticle, a peptide complex, calcium phosphate and combinations thereof.

[0151] According to a particular embodiment, the microcompartment comprises at least one transfection agent, said agent being a plasma membrane derivative comprising proteins of viral origin

[0152] Advantageously, the microcompartment according to the invention is suitable for all types of transduction and transfection agents capable of modifying the genome, transcriptome, or proteome of a eukaryotic cell, preferably capable of understanding and / or expressing at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive, or conditional manner. Furthermore, the microcompartment according to the invention requires a lower concentration of the transduction and / or transfection agent than the solutions proposed by the prior art while achieving similar transfection / transduction efficiency.

[0153] According to one embodiment, the smallest dimension d of at least one transfection agent and / or at least one transduction agent present in the internal part of the The microcompartment is larger than lnm, preferably between 15 and 25 nm. According to one variant, the majority of transfection agents and / or transduction agents present in the inner part of the microcompartment, preferably all of them, have a smaller dimension d of less than 25 nm, preferably between 15 and 25 nm.

[0154] According to another embodiment, the largest dimension D of at least one transfection agent and / or at least one transduction agent present in the inner part of the microcompartment is greater than 25 nm, preferably greater than 100 nm, in particular greater than 500 nm, and even more preferably greater than 1 pm. According to a variant, the majority of the transfection agents and / or transduction agents present in the inner part of the microcompartment, preferably all of them, have a largest dimension D greater than 50 nm, in particular greater than 100 nm, and even more preferably between 300 and 500 nm.

[0155] According to one embodiment, the microcompartment according to the invention comprises at least one transduction agent and / or at least one transfection agent in the aqueous solution and / or the hydrogel of the inner part.

[0156] Advantageously, said transduction agent and / or transfection agent can diffuse into the aqueous solution and / or into the hydrogel of the inner part so as to increase its chances of encountering a cell and inserting at least one molecule of interest.

[0157] Preferably, the microcompartment according to the invention comprises at least one transfection agent and / or at least one transduction agent comprising at least one molecule of interest selected from a nucleic acid, a ribonucleic acid, a protein, a peptide and their combinations.

[0158] Advantageously, the transfection and / or transduction agent makes it possible to modify the behavior of the cells present in the internal part by inserting at least one molecule of interest.

[0159] In addition to the hydrogel and / or aqueous solution and at least one transfection agent and / or at least one transduction agent, the internal part of the microcompartment comprises at least one cell.

[0160] In the context of the invention, the cells present in the microcompartment can be any type of cell, in particular eukaryotic cells. More preferably, the cells are human, plant, or animal cells.

[0161] Preferably, the cells present in the internal part are chosen from pluripotent cells, progenitors, cells undergoing differentiation, differentiated cells and mixtures thereof.

[0162] In a particular embodiment, the microcompartment comprises cells Pluripotent stem cells. A pluripotent stem cell, or pluripotent cell, is defined as a cell that has the capacity to form all the tissues present in the entire organism of origin, without, however, being able to form an entire organism as such. Pluripotent stem cells can be, in particular, induced pluripotent stem cells (iPSCs), MUSE cells (Multilineage-Differentiating Stress Enduring), which are found in the skin and bone marrow of adult mammals, or embryonic stem cells (ESCs). In one embodiment, the microcompartment according to the invention does not comprise embryonic stem cells (ESCs).

[0163] According to a particularly adapted variant of the invention, the microcompartment according to the invention comprises human or animal induced pluripotent stem cells.

[0164] In another particular embodiment, the microcompartment according to the invention comprises human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells and / or cells undergoing differentiation. The multipotent cells and / or progenitor cells were preferably obtained from pluripotent stem cells, in particular human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile was artificially modified to match that of particular multipotent cells and / or progenitors, typically by forced expression of transcription factors specific to the target cell phenotype.Preferably, multipotent and / or progenitor cells were obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells.

[0165] According to another embodiment, the microcompartment according to the invention comprises differentiated human or animal cells. The differentiated cells were preferably obtained from pluripotent stem cells or progenitor cells, in particular from human pluripotent stem cells or human progenitor cells, or possibly from non-pluripotent human cells whose transcriptional profile has been artificially modified to match that of particular differentiated cells, typically by forced expression of transcription factors specific to the target cell phenotype. Preferably, the differentiated cells were obtained from pluripotent or multipotent stem cells or progenitor cells after contact with a solution capable of initiating the differentiation of said stem cells.According to one variant, the cellular content of the microcompartment comprises homogeneous or mixed cellular identities.

[0166] Differentiated cells may in particular be in the form of at least one layer of cells or in the form of a tissue, a cell aggregate or a micro three-dimensional fabric or multiple fabrics or micro-fabrics within the micro-compartment. It may be a compacted or uncompacted fabric or micro-fabric, with or without light.

[0167] The microcompartment according to the invention can therefore comprise several types of cells. Thus, according to one embodiment, the microcompartment according to the invention has at least two different cell types in its internal part. In particular, the microcompartment according to the invention can comprise, for example, induced pluripotent stem cells and / or multipotent cells and / or progenitor cells and / or cells undergoing differentiation and / or differentiated cells.

[0168] When the microcompartment according to the invention comprises at least one cell layer or cell layer, the aqueous solution and / or hydrogel are preferably arranged between the outer hydrogel layer and said cell layer or cell layer. It is understood that the microcompartment may also include, in its inner part, cells suspended in the aqueous solution or hydrogel of the inner part.

[0169] When the microcompartment according to the invention comprises at least one cell aggregate and / or tissue and / or microtissue, the aqueous solution and / or hydrogel are preferably arranged between the outer hydrogel layer and said cell aggregate and / or tissue and / or microtissue. It is understood that the microcompartment may also include, in its inner part, cells suspended in the aqueous solution or hydrogel of the inner part.

[0170] According to another preferred object of the invention, the aggregate and / or cell layer and / or cell base and / or tissue and / or microtissue comprises at least one lumen. Said lumen may contain a liquid, in particular culture medium and / or a liquid secreted by the cells.

[0171] Advantageously, the presence of this hollow part allows the cells to have a small diffusive volume whose composition they can control, promoting cell communication.

[0172] According to one embodiment, the cell layer and the hydrogel layer and / or mesh and / or aqueous solution layer of the inner part are organized around at least one lumen, more preferably they are organized successively around at least one lumen.

[0173] According to one embodiment, the cell layer and the hydrogel layer and / or mesh and / or aqueous solution layer of the inner part are successively arranged around a lumen. This is referred to as a cyst-shaped conformation.

[0174] According to an embodiment in which the microcompartment comprises only one cyst, the cell layer, the hydrogel layer and / or mesh and / or the aqueous solution layer of the inner part, and the outer layer are organized successively around a light.

[0175] This cyst-like conformation reduces the pressure exerted on the cells. This configuration also decreases cell mortality and increases the culture amplification factor. Consequently, this reduces the number of passages and dissociations required, and the time in culture needed to reach the desired final cell count.

[0176] According to one embodiment, the microcompartment may comprise one or more cysts, and / or one or more tissues and / or micro-tissues and / or cell aggregates with or without lumen(s).

[0177] Advantageously, the microcompartment according to the invention is suitable for the transduction and / or transfection of any type of eukaryotic cell.

[0178] Such a microcompartment is thus particularly suitable for carrying out genetic and / or transcriptomic and / or protein modification of cells of interest, preferably so that said cells can understand and / or express at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner.

[0179] According to a particular aspect of the invention, the microcompartment according to the invention is obtained after several cycles of cell division. Indeed, the cells included in the microcompartment according to the invention can be cells obtained by amplification, starting from at least one cell.

[0180] In particular, the cells present in the microcompartment according to the invention can be obtained after at least two cycles of cell division after encapsulation in an outer layer of hydrogel of at least one cell.

[0181] Preferably, the cells present in the microcompartment according to the invention were obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28 or 30 cell division cycles after encapsulation in an outer layer of hydrogel of at least 1 cell, preferably between 1 and 5, between 1 and 10, between 1 and 15, between 1 and 20, between 1 and 30, between 1 and 40, between 1 and 50, between 1 and 60, between 1 and 100 cells. For example, the cells present in the microcompartment were obtained after at least six cycles of cell division after encapsulation in an outer layer of hydrogel of at least 1 cell, preferably between 1 and 50 cells.

[0182] Preferably the microcompartment is obtained after at least 2 passages after encapsulation, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage can last, for example, at least 1 day, or between 2 and 50 days, in particular between 3 and 10 days.

[0183] Preferably, the total volume of cells initially encapsulated in the microcompartment before the first cell division cycle is less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0184] Thus, according to one embodiment, the cells present in the microcompartment according to the invention were obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division, after encapsulation in an outer layer of hydrogel of cell(s) representing a volume less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0185] Preferably, in the microcompartment according to the invention, the cells represent more than 50% by volume relative to the volume of the microcompartment, even more preferably more than 60%, 70%, 75%, 80%, 85%, 90% by volume relative to the volume of the microcompartment.

[0186] The microcompartment according to the invention comprises several cells, preferably at least 20 cells, even more preferably at least 100, at least 500, at least 1000, at least 10000.

[0187] The microcompartment according to the invention can be obtained by encapsulation carried out by means of a concentric co-injection via a microfluidic injector forming a jet at the injector outlet consisting of a mixture of the different useful solutions, said jet being fractionated into droplets. The droplets are then collected in a bath, in particular a calcium bath, suitable for rigidifying the hydrogel solution to form the outer layer of each microcompartment.

[0188] According to an embodiment enabling the formation of a tube, the co-injection is also carried out concentrically via a microfluidic injector, said injector comprising a tip, said tip being in contact with a calcium solution, forming a jet at the injector outlet consisting of the mixture of said solutions, said jet forming the tube in the calcium solution.

[0189] The cellular microcompartment according to the invention is preferably closed or partially closed, that is to say that the outer layer is closed or partially closed. More preferably the microcompartment is closed.

[0190] The microcompartment according to the invention can be any three-dimensional shape, that is, it can be any three-dimensional object. The microcompartment can have any shape compatible with cell encapsulation. Preferably, the microcompartment according to the invention is spherical or elongated. It can be ovoid, teardrop, cylindrical, spheroidal, or spherical. In particular, it can be hollow spheroidal, ovoidal, cylindrical, or sphere-shaped. hollow.

[0191] It is the outer layer of the microcompartment, that is to say the hydrogel layer, which gives the microcompartment according to the invention its size and shape. Preferably, the smallest dimension of the microcompartment according to the invention is between 10 pm and 1 mm, preferably between 100 pm and 700 pm. It may be between 200 pm and 600 pm, in particular between 300 pm and 500 pm.

[0192] Its largest dimension is preferably greater than 1m, more preferably between 1m and 1m, even more preferably between 1m and 50cm.

[0193] The microcompartment according to the invention can optionally be frozen for storage. It must then be thawed before use.

[0194] The invention also relates to several microcompartments together. Therefore, the invention also relates to an assembly or series of cellular microcompartments as described above, comprising at least one cellular microcompartment according to the invention.

[0195] The invention also relates to an assembly or series of microcompartments of at least two three-dimensional cellular microcompartments, wherein at least one microcompartment is a microcompartment according to the invention, preferably the majority of the microcompartments in the assembly are microcompartments according to the invention. According to a particular embodiment, all the microcompartments in the assembly are microcompartments according to the invention.

[0196] The microcompartment assembly according to the invention preferably comprises between 2 and 1016 microcompartments, preferably between 1000 and 109 microcompartments according to the invention.

[0197] Preferably the series of microcompartments according to the invention is in a culture medium, in particular in a culture medium that is at least partially convective.

[0198] According to a particularly suitable embodiment, the invention relates to a set of cellular microcompartments in a closed enclosure, such as a bioreactor, preferably in a culture medium in a closed enclosure, such as a bioreactor.

[0199] The presence of an outer hydrogel layer and the aqueous solution and / or hydrogel of the inner part allows for a uniform distribution of cells between the microcompartments. Furthermore, the outer hydrogel layer prevents microcompartment fusion, which is a major source of variability detrimental to the phenotypic homogeneity of the cells.

[0200] According to one embodiment, the microcompartment according to the invention can be obtained by a process comprising the implementation of the following steps: (a) prepare a solution containing cells, (b) prepare at least one aqueous solution and / or at least one hydrogel comprising at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, said hydrogel having pores whose smallest dimension is greater than the smallest dimension d of said at least one transduction agent and / or transfection agent, (c) encapsulate the solutions and / or hydrogels of steps (a) and (b) by collinear flow in an outer hydrogel layer having pores whose largest dimension is less than the smallest dimension d of said transduction agent and / or transfection agent of step (a); (d) cultivate the microcompartments obtained in step (c) in a culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably from 3 to 50 days, and (e) optionally recover the resulting cellular microcompartments.

[0201] Thus, the microcompartment according to any one of the embodiments described above, or the assembly of microcompartments according to any one of the embodiments described above, is also particularly suitable for use in cell culture, especially in three-dimensional cell culture. These allow for the production of large quantities of cells of interest, in particular cells, microtissues, or organoids expressing at least one peptide sequence, a coding or non-coding RNA sequence, or a transgene in a transient, constitutive, or conditional manner, which can be used, for example, in cell therapy. The invention also relates to a microcompartment according to the invention, or an assembly of microcompartments according to the invention, for use as a medicinal product.

[0202] Thus, the microcompartment is particularly suitable for use in clinical settings.

[0203] According to another object, the invention relates to the use of the microcompartment according to any of the preceding purposes, to insert at least one peptide sequence, coding or non-coding RNA sequence, or transgene in a transient, constitutive or conditional manner into at least one eukaryotic cell.

[0204] The invention also relates to the use of the microcompartment according to the invention for the implementation of transfection and / or transduction process(es).

[0205] According to another object, the invention relates to the use of the microcompartment according to any one of the preceding objects, for the production, preferably on a large scale, of cells, micro tissues or tissues expressing at least one transgene in a transient, constitutive or conditional manner.

[0206] Preparation process

[0207] The microcompartment can be obtained by various means known to Man of the trade to prepare microcompartments or capsules.

[0208] The invention also relates to a particular method for preparing microcompartments.

[0209] According to one embodiment, the invention relates to a method for preparing a microcompartment comprising the following steps: (a) prepare a solution containing cells, (b) prepare at least one aqueous solution and / or at least one hydrogel, said solution and / or hydrogel comprising at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, (c) encapsulate the solutions and / or hydrogel(s) of steps (a) and (b) by collinear flow in an outer layer of hydrogel, said hydrogel being different from the possible hydrogel of step (b).

[0210] According to one embodiment, the preparation process according to the invention also includes a step (d) consisting of culturing the microcompartments obtained in step (c) in a culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably from 3 to 50 days.

[0211] According to another embodiment, step (c) consists of encapsulating the solutions and / or hydrogel(s) of steps (a) and (b) by collinear flow in an outer layer of hydrogel, said hydrogel having a diffusive conductance to at least one transduction agent and / or transfection agent less than strictly the diffusive conductance of the inner part to that same transduction agent and / or transfection agent.

[0212] According to one embodiment, the process for preparing a microcompartment according to the invention comprises carrying out the following steps: (a) prepare a solution containing cells, (b) prepare at least one aqueous solution and / or at least one hydrogel comprising at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, said hydrogel having pores whose smallest dimension is greater than the smallest dimension d of said at least one transduction agent and / or transfection agent. (c) encapsulate the solutions and / or hydrogels of steps (a) and (b) by collinear flow in an outer hydrogel layer having pores whose largest dimension is less than the smallest dimension d of said transduction agent and / or transfection agent of step (a).

[0213] According to one embodiment, the solution from step (a) is prepared in at least one aqueous solution, preferably a suitable culture medium. Said culture medium preferably comprises at least one cytoprotective factor, more preferably at least one apoptosis inhibitor.

[0214] The apoptosis inhibitor may, for example, be one or more inhibitors of the RHO / ROCK (Rho-associated protein kinase) pathways, or any other apoptosis inhibitor known to those skilled in the art. The apoptosis inhibitor must promote cell survival during the formation of the outer hydrogel layer.

[0215] The process according to the invention may include a cell dissociation step by chemical, enzymatic, or mechanical dissociation, carried out prior to or simultaneously with the cell incubation step, itself performed prior to step a) of mixing. This step is particularly important in the case of adherent cells.

[0216] The encapsulated cells are suspended as single cells and / or cell clusters. Preferably, single cells represent less than 50% by number of all encapsulated cells, and more preferably, the single cells are hPSCs. Indeed, it is preferable to encapsulate cell clusters because this reduces apoptosis.

[0217] Preferably, the cell density of the solution in step (a) is between 0.1 and 40 million cells per mL, preferably between 0.8 and 20 million.

[0218] The solution of step (a) preferentially comprises at least one cell selected from pluripotent cells, progenitors, cells undergoing differentiation, differentiated cells and mixtures thereof.

[0219] According to a preferred embodiment, the solution of step (a) comprises at least 2 different cell types.

[0220] Preferably, the transduction agent is chosen from an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a baculovirus and their combinations.

[0221] According to another embodiment, the solution of step (b) comprises at least one transfection agent comprising at least one molecule of interest, said agent may be selected from an extracellular vesicle of endosomal origin such as an exosome, a plasma membrane derivative, a liposome, a nanoparticle, a peptide complex, calcium phosphate and combinations thereof.

[0222] Preferably, encapsulation step (c) comprises the following substeps: - bring the solution from step (a), the solution from step (b) and a hydrogel solution intended to form the outer layer into contact to form at least one droplet, and - collect the drop obtained in a calcium bath suitable for stiffening said hydrogel solution to form the outer layer of each microcompartment.

[0223] Once the outer hydrogel layer has been hardened by the calcium bath, the microcompartment is formed. This can then be rinsed. Advantageously, if the Young's modulus of the hydrogel in the inner part is lower than that of the hydrogel in the The outer layer prevents / limits the rigidification of the hydrogel in the inner part by the calcium bath. Specifically, given that the Young's modulus of the hydrogel in the inner part is strictly lower than that of the hydrogel in the outer layer, the rigidification process by the calcium bath prevents the hydrogel in the inner part from stiffening during the capsules' lifespan, thus hindering cell proliferation.

[0224] Preferably, step (c) is carried out by a simultaneous co-injection of the hydrogel solution intended to form the outer layer, the solution of step (a) and the solution of step (b); said co-injection is carried out concentrically via a microfluidic or millifluidic injector forming a jet at the injector outlet consisting of the mixture of said solutions, said jet breaking into drops.

[0225] According to one object of the invention, the final opening diameter of the microfluidic injector is between 50 and 800 pm, preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 0.1 and 2000 mL / h, preferably between 10 and 2000 mL / h, more preferably between 11 and 100 mL / h.

[0226] According to one variant, step (c) is carried out by a simultaneous co-injection of the hydrogel solution intended to form the outer layer, the solution of step (a) and the solution of step (b); said co-injection is carried out concentrically via a microfluidic or millifluidic injector, said injector comprising a tip, said tip being in contact with a calcium solution, forming a jet at the injector outlet consisting of the mixture of said solutions, said jet forming a tube.

[0227] When the tip of the injector is in contact with the calcium solution, the final opening diameter of the microfluidic injector is preferably between 50 and 1000 pm, more preferably between 80 and 300 pm, and the flow rate of each of the solutions is between 1 and 100 mL / h.

[0228] According to another embodiment of the invention, the inner part comprising at least one hydrogel comprising pores whose smallest dimension is greater than the smallest dimension d of said transduction agent and / or transfection agent also comprises at least one second hydrogel distinct from the first hydrogel constituting the layer or mesh of the inner part.

[0229] When at least one hydrogel of the inner part is fibrin, it is preferably obtained from the polymerization of fibrinogen by a fibrinogen polymerizing agent, advantageously said agent is thrombin, said agent may be added during encapsulation and / or after encapsulation.

[0230] Also, according to one variant the thrombin solution is co-injected with the other solutions, the latter is preferentially mixed with the isotonic intermediate solution and the fibrinogen solution is mixed with the solution from step (b).

[0231] Preferably, the fibrinogen concentration is between 10 and 25 mg / mL, preferably 14-20 mg / mL, more preferably 20 mg / mL.

[0232] According to another aspect of the invention, the concentration of thrombin is preferably between 0.01 U / mL and 2 U / mL, more preferably between 0.01 U / mL and 1 U / mL, between 0.01 U / mL and 0.05 U / mL, and even more preferably 0.04 U / mL. "U" is understood to mean a unit of enzymatic activity (i.e., the concentration for an enzyme) which represents the quantity of enzyme necessary to process one micromole of substrate in 1 minute. It is understood that the concentration indicated is that in the mixture. Advantageously, the thrombin is mixed with the other constituents in a 1:1 ratio. Therefore, within the capsule, when the concentration of thrombin before mixing is 0.01 U / mL, the concentration in the capsule is on the order of 0.01 U / mL.

[0233] The post-encapsulation steps can be carried out without agitation or with agitation. Preferably, the post-encapsulation steps are carried out with continuous or sequential agitation. This agitation is important because it maintains the homogeneity of the culture environment and prevents the formation of any diffusive gradient. For example, it allows for homogeneous control of cellular oxygenation levels, thus preventing hypoxia-related necrosis or hyperoxia-related oxidative stress. Consequently, it prevents an increase in cell death and / or oxidative stress.

[0234] Preferably, after the step of culturing the capsules obtained, the process includes a step which consists of rinsing the capsules from step (d), advantageously so as to remove the cytoprotective factor, such as the apoptosis inhibitor.

[0235] When the process according to the invention includes a step of rinsing the capsules obtained, the solution constituting the calcium bath is removed and replaced by a medium suitable for culturing the microcompartments according to the invention, preferably an isotonic solution, more preferably a culture medium containing an apoptosis inhibitor. The step of rinsing the capsules obtained is carried out after step (d).

[0236] In a preferred embodiment, the process according to the invention includes at least one reencapsulation of the cells after step (d), preferably after the rinsing step if such a step is present after step (d). By "at least one reencapsulation of the cells," we mean at least two encapsulation cycles. Preferably, each encapsulation cycle corresponds to one pass. In this embodiment of the process (at least one reencapsulation of the cells after step (d), the number of cell divisions in the entire process (for all passes) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 cell division cycles.

[0237] In a process according to the invention there may be several re-encapsulations, preferably- normally between 1 and 100, especially between 1 and 10 re-encapsulation.

[0238] Each re-encapsulation may include: - a step that consists of dissociating the microcompartment or series of microcompartments to obtain a cell suspension or a suspension of cell clusters; the removal of the outer hydrogel layer can be carried out, in particular, by hydrolysis, dissolution, drilling and / or rupture by any biocompatible means, i.e., non-toxic to cells. For example, removal can be carried out using a phosphate saline buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel contains alginate and / or laser microdissection, and - a step of re-encapsulating all or part of the cells or clusters of cells in a hydrogel capsule.

[0239] Re-encapsulation is a suitable means for increasing cell amplification obtained from the pluripotent stage, and reducing the risks of mutation.

[0240] According to a particular embodiment, re-encapsulation comprises the following steps: - remove the outer hydrogel layer, - resuspend the cells that were contained in the microcompartment so as to obtain single cells and / or at least a set or cluster of cells in an isotonic medium, preferably a culture medium containing an apoptosis inhibitor, - encapsulate the cell suspension in a hydrogel layer; - preferably, cultivate the microcompartments obtained in an isotonic solution containing an apoptosis inhibitor, preferably a culture medium containing an apoptosis inhibitor; - preferably, rinse the microcompartments, advantageously, so as to eliminate the apoptosis inhibitor; - cultivate the microcompartments in an isotonic solution, preferably a culture medium, for at least one cell division cycle, and - optionally collect the resulting cellular microcompartments.

[0241] The invention also relates to a transduction method implemented in at least one cellular microcompartment defined in any of the previously described embodiments.

[0242] Finally, the invention also relates to a transfection process implemented in at least one cellular microcompartment defined in any of the previously described embodiments.

[0243] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results. Examples

[0244] Example 1: Transduction of primary human CD3+ T lymphocytes

[0245] The experimental setup of Example 1 is described in [Fig.1].

[0246] Primary human CD3+ T lymphocytes are thawed, counted and seeded at a rate of 1 million cells per ml in TexMACS medium (Miltenyi Biotec).

[0247] After seeding, the cells are activated by adding "T Cell TransAct human" to the cell culture medium (dilution to l / 100e) and IL-2 (final concentration of 300 U / mL).

[0248] The cells are cultured in a T75 cell culture flask (maintained in an upright position) at 37°C, 5% CO2.

[0249] 48 hours after activation, the cells are collected and concentrated to 10 million of cells per ml in TexMACS medium supplemented with IL-2 (300 U / mL).

[0250] In parallel, the viral vectors are diluted in a volume of TexMACS medium (supplemented with IL-2 at 300 U / mL) to the desired final MOI (see Table 1 below) and added to the cells to obtain a final density of 5 million cells per mL. [Table 1] Titer: Null Low Medium High Lentivirus (LV) 1.00E+09 TU / ml 0 0.1 1 10 Sendai virus (SeV) 1.10E+08 CI / ml 0 0.3 1 5

[0251] The resulting cell population (containing viral particles) is divided into 2 samples, one for 2D cell culture in a 12-well plate (final volume of medium from ImL to 0.5 million cells per mL) and the other for 3D cell encapsulation.

[0252] For encapsulation, 400 µl of the sample of cells and viral vectors (at a rate of 5 million cells per ml) are injected into the encapsulation system programmed with the following parameters: flow rate of the alginate syringe (100 ml / h); flow rate of cells and intermediate solutions (50 ml / h); electric field (2000 V).

[0253] The cells pass through the encapsulation device and the capsules are collected in a calcium bath (25 mM HEPES, 100 mM CaC12, Tween80) located below the encapsulation chip's outlet nozzle (large capsule configuration). The capsules are then collected and rinsed with DMEM / F12 medium. The rinsed capsules are resuspended in 4 volumes (using the volume of the capsules as a reference) of TexMACS medium supplemented with CaC12 (3mM final).

[0254] Once the capsules have settled (~30 minutes to 1 hour), they are collected on a 100 µm filter and resuspended in 13 ml of TexMACS medium supplemented with IL-2 (300 U / mL), transferred into a T75 bottle and incubated at 37°C, 5% CO2.

[0255] The capsules thus formed comprise an outer hydrogel layer containing 2% alginate with a molecular weight of 150-250 kDa XXXXXXX. The inner part comprises an aqueous solution composed of TexMACS culture medium.

[0256] 2 days (for SeV vectors) or 5 days (for LV vectors) after the transduction / encapsulation, the cells are uncapped using 10 volumes of ReLeSR, pelleted and resuspended in TexMACS medium to be counted and further processed for flow cytometry.

[0257] Cells to be analyzed by flow cytometry are incubated with LIVE / DEAD™ Fixable Near IR (780) (Thermo Fisher Scientific, catalog # L34994) for 30 minutes at room temperature (RT) according to the manufacturer's instructions. The cells are then incubated with various fluorescent antibodies (see Table 2), fixed in PBS / 4% PFA, and analyzed by flow cytometry (LSR Fortessa 5L). Flow cytometry results for GFP expression are shown in [Fig. 2A] and [Fig. 2B] for the Lentiviral vector and in [Fig. 2D] and [Fig. 2E] for the Sendai viral vector. Additionally, cell viability results comparing the 2D and 3D systems are shown in [Fig. 3A] for the Lentiviral vector and [Fig. 3B] for the Sendai viral vector. [Table 2] Target Reference Dilution Mouse Anti-Human CD4 BUV737 612748 1 / 200 Mouse Anti-Human CD25 PE 341009 20 / 100 Mouse Anti-Human CD8 APC 340584 1 / 100 Mouse Anti-Human CD3 BUV395 563548 1 / 100

[0258] The transduction efficiency (% of GFP+ cells) of T lymphocytes by lentiviruses is better under 3D cell encapsulation conditions than under 2D conditions, particularly for the lowest MOIs tested (0.1 and 1), without affecting cell viability ([Fig. 3A] and 3B). Furthermore, in addition to transducing more cells, the 3D cell encapsulation condition allows for better Transgene expression in transduced cells, as demonstrated by higher fluorescence intensities ([Fig. 2B] and 2E). This enhanced transgene expression is associated with a greater number of transgenes inserted into the T lymphocyte genome under 3D conditions than under 2D conditions at equivalent MOI ([Fig. 2C]).

[0259] Similarly, the transduction efficiency (% of GFP+ cells) of T lymphocytes by Sendai viral vectors is better under 3D cell encapsulation conditions than under 2D conditions for all MOIs tested (0.3, 1, and 5) ([Fig. 2A] and 2D), without affecting cell viability. Furthermore, in addition to transducing more cells, 3D cell encapsulation allows for better transgene expression in transduced cells, as demonstrated by the higher fluorescence intensities.

[0260] Example 2: Transduction of human induced pluripotent stem cells (hIPS)

[0261] For hiPSCs, the cell culture conditions for the 2D and 3D configurations (before cell encapsulation) are different from Example 1.

[0262] The experimental setup of Example 2 is described in [Fig.4].

[0263] The day before encapsulation, the cells for the 2D transduction experiments are seeded at 25,000 cells / cm2 in a 12-well plate in mTeSRl supplemented with Rock inhibitor (10 pM final).

[0264] For 3D transduction experiments, on the day of encapsulation, cells previously cultured in 2D are washed twice in PBS and detached from the plate using Accutase for 3 minutes at 37°C. Once in suspension, the cells are collected in 2 volumes of mTeSRl, concentrated to 10 million cells / mL in mTeSRl medium supplemented with Rock inhibitor (10 pM final) and incubated with viral vectors at different MOIs (described in Table 3) to a final density of 5 million cells / mL. 400 µL of the cell and viral particle suspension are then injected into the encapsulation system programmed with the following parameters: Alginate syringe flow rate (100 mL / h); Cell and intermediate solution flow rate (50 mL / h); Electric field (2000 V).The cells pass through the encapsulation device and the capsules are collected in a calcium bath (25 mM HEPES, 100 mM CaC12, Tween80) located below the encapsulation chip outlet nozzle (large capsule configuration).

[0265] The capsules are then collected and rinsed with DMEM / F12 medium supplemented with CaC12 (3 mM final concentration) on a 100 µm filter. The rinsed capsules are resuspended in 4 volumes (using the capsule volume as a reference) of DMEM / F12 medium supplemented with CaC12 (3 mM final concentration). Once the capsules have settled (~30 minutes to 1 hour), they are collected on a 100 µm filter and resuspended in 13 mL of mTeSRl medium supplemented with Rock inhibitor (10 pM final concentration), transferred to a T75 flask, and incubated at 37°C, 5% CO2.

[0266] The capsules thus formed comprise an outer hydrogel layer containing 2% alginate with a molecular weight of 150-250 kDa. The inner part comprises an aqueous solution of mTeSRL culture medium

[0267] On the same day, the medium of the cells cultured for 2D cell transduction (seeded the previous day in a 12-well plate) is replaced with mTeSRl medium supplemented with Rock inhibitor (10 pM final) and transduced with viral vectors at the same MOIs as those used in the 3D configuration (see Table 3). [Tables 3] Null Titer Low Medium High Lentivirus (LV) 1.00E+09 TU / ml 0 1 3 10 Sendai virus (SeV) 1.10E+08 CI / ml 0 0.3 1 3 Adeno-associated virus (AAV) 5.00E+12 GC / cell 0 1E2 1E3 1E4

[0268] The day after transduction, the medium for 2D cultured cells is replaced with mTeSRl without Rock inhibitor, and the medium for 3D cultured cells is replaced with mTeSRl supplemented with Rock inhibitor (10 pM). Two days after transduction, the medium is again replaced with mTeSRl without Rock inhibitor for both 2D and 3D cultured cells.

[0269] Three days after transduction, the 3D-cultured cells are decapsulated by removing the growth medium and adding 10 volumes of ReLeSR (for each volume of capsules). After decapsulation, the cells are dissociated using 0.5X TrypLE, resuspended in mTeSRl medium, and subjected to flow cytometry. The cells to be analyzed by flow cytometry are incubated with fixable lime for 30 minutes at room temperature (RT) according to the manufacturer's instructions. The cells are then incubated with various fluorescent antibodies (see Table 4), fixed in PBS / 4% PFA, and analyzed by flow cytometry. The flow cytometry results obtained for GFP expression are shown in [Fig. 5]. [Table 4] Target Dilution Oct3 / 4 antibody, anti-human / mouse PE 1 / 50 Nanog antibody, anti-human APC 1 / 50

[0270] The transduction efficiency (% of GFP+ cells) of iPSCs is better for 3D encapsulation conditions than for 2D conditions, regardless of the MOI used for lentiviral vectors, AAVs or Sendai viral vectors.

[0271] Example 3: Transduction of an encapsulated cell according to the invention with several transduction agents.

[0272] The experimental conditions of this example are identical to those of example 1.

[0273] However, the transduction is performed with three lentilvectors instead of just one in Example 1. The MOIs indicated correspond to the total MOI used for the combination of the three lentilvectors. The results are presented in [Fig. 6]. These results show that it is possible to co-transduce cells using multiple vectors in a 3D microcompartment.

[0274] Example 4: Comparative study of transduction efficiency within a microcompartment comprising a single hydrogel vs. a microcompartment according to the invention

[0275] To carry out this comparative study, this example includes 3 specific conditions:

[0276] *Condition 1: a microcompartment according to the invention comprising an outer hydrogel layer composed of 2% high molecular weight alginate (150-250 kDa) and an inner part comprising an aqueous solution (mTeSRl medium). *Condition 2: A microcompartment according to the invention comprising an outer hydrogel layer composed of 2% high molecular weight alginate (150-250 kDa) and an inner part comprising a 0.5% low molecular weight alginate hydrogel (< 75 kDa). Therefore, the hydrogel of the outer layer and the hydrogel of the inner part are different. *Condition 3: a "full" microcompartment, outside the scope of the invention, comprising a 1.2% high molecular weight (150-250 kDa) alginate hydrogel.

[0277] To obtain condition 3, the protocol described by Neumann et al., 2013 (DOI 10.1007 / sl2033-012-9522-y) was reproduced.

[0278] Results:

[0279] Three days after transduction, [Fig. 7A] shows that transduction is more efficient in condition 1 (82.5% GFP+ cells with a fluorescence intensity of 32338) than in condition 3 (37.1% GFP+ cells with a fluorescence intensity of 11317). Condition 2 shows an intermediate transduction rate (73.3% GFP+ cells with a fluorescence intensity of 20909). In addition to improving transduction efficiency, condition 2 demonstrates better cell viability and amplification compared to condition 3 ([Fig. 7C]).

[0280] Transduction within the microcompartments generated under conditions 1 and 2 is therefore more efficient and less toxic to cells than transduction carried out in Condition 3 ([Fig. 7A] and [Fig. 7B]). Furthermore, the fact that condition 2 performs better than condition 3 ([Fig. 7D]) demonstrates that the physical properties necessary for microcompartment formation can be decoupled from those promoting optimal cell transduction and culture, particularly in a bioreactor. Indeed, it would be impossible to generate / maintain the physical integrity of capsules filled with 0.5% alginate.

Claims

Demands

1. Three-dimensional cellular microcompartment comprising an outer hydrogel layer defining an inner part, said inner part comprising: - at least one human, animal or plant eukaryotic cell, said microcompartment not comprising human embryonic stem cells; - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the outer layer.

2. Cell microcompartment according to claim 1, characterized in that the hydrogel of the inner part is different from the hydrogel constituting the outer layer, in that it has at least one different chemical property and / or at least one different physical property.

3. Cell microcompartment according to any one of the preceding claims, wherein the diffusive conductance of the outer layer to at least one transduction agent and / or transfection agent is strictly less than the diffusive conductance of the inner part to that same transduction agent and / or transfection agent.

4. Cell microcompartment according to any one of the preceding claims, wherein the maximum diffusive conductance of the hydrogel of the outer layer to at least one transduction agent and / or transfection agent is less than 0.1 / h, preferably less than 0.05 / h, even more preferably less than 0.01 / h.

5. Cell microcompartment according to any one of the preceding claims, wherein the maximum diffusive conductance of the hydrogel of the inner part to at least one transduction agent and / or transfection agent greater than 0.1 / h, is preferably greater than 1 / h, even more preferably greater than 10 / h, in particular greater than 50 / h.

6. Cell microcompartment according to any one of the preceding claims, wherein the ratio of the diffusive conductance of the hydrogel of the inner part to the diffusive conductance of the hydrogel of the outer layer relative to a transfection and / or transduction agent is greater than 5, preferably greater than 10, even more preferably greater than 100, in particular greater than 1000.

7. Cell microcompartment according to any one of the preceding claims, wherein the outer layer comprises pores whose largest dimension is less than the smallest dimension d of said at least one transduction and / or transfection agent and, where the inner part comprises a hydrogel, the latter comprises pores whose smallest dimension is greater than this dimension d.

8. Cell microcompartment according to claim 7, characterized in that the smallest dimension of the pores of the hydrogel of the inner part is greater than 1nm, preferably between 25 and 500nm.

9. Cell microcompartment according to claim 7 or 8, characterized in that the outer layer comprises pores whose largest dimension is less than 25nm, preferably less than 1nm.

10. Cell microcompartment according to any one of the preceding claims, characterized in that the hydrogel of the outer layer has a density and / or viscosity strictly greater than the density and / or viscosity of the solution and / or hydrogel present within the inner part.

11. Cell microcompartment according to any one of the preceding claims, characterized in that the hydrogel of the outer layer has a different molecular weight from the hydrogel present within the inner part.

12. Cell microcompartment according to any one of the preceding claims, characterized in that the outer layer has greater diffusion resistance than the solution and / or hydrogel of the inner part.

13. Cell microcompartment according to any one of the preceding claims, characterized in that the viscosity of the inner part is between 0.1 and 50 mPa / s.

14. Cell microcompartment according to any one of the preceding claims, characterized in that the internal part comprises: - at least one layer of aqueous solution; and / or - at least one layer and / or mesh of hydrogel.

15. Cell microcompartment according to the preceding claim, characterized in that at least one transduction agent and / or at least one transfection agent is found in the aqueous solution and / or in the hydrogel of the inner part.

16. Cell microcompartment according to any one of the preceding claims, characterized in that at least one transduction agent is chosen from a viral particle, a pseudo-viral particle and their combination.

17. Cell microcompartment according to any one of the preceding claims, characterized in that at least one transfection agent is selected from an extracellular vesicle of endosomal origin, a plasma membrane derivative, a liposome, a nanoparticle, a peptide complex, calcium phosphate and combinations thereof.

18. Cell microcompartment according to any one of the preceding claims, characterized in that at least one transduction agent and / or at least one transfection agent comprises at least one molecule of interest selected from a nucleic acid, a ribonucleic acid, a protein, a peptide and their combinations.

19. Cell microcompartment according to any one of the preceding claims, characterized in that the internal part comprises at least two different cell types.

20. Cell microcompartment according to any one of the preceding claims, characterized in that the eukaryotic cells present in the internal part are selected from pluripotent cells, progenitors, cells undergoing differentiation, differentiated cells and mixtures thereof.

21. Cell microcompartment according to any one of the preceding claims, characterized in that the inner part comprises at least an aqueous solution and / or a hydrogel whose Young's modulus is strictly less than the Young's modulus of the hydrogel of the outer layer.

22. Cell microcompartment according to any one of the preceding claims, characterized in that the Young's modulus of the aqueous solution or hydrogel in the inner part is between 0.01 and 200kPa, preferably between 0.1 and 60 kPa.

23. Cell microcompartment according to any one of the preceding claims, characterized in that the Young's modulus of the hydrogel in the inner part is between 0.1 and 5kPa.

24. Cell microcompartment according to the preceding claim, characterized in that the Young's modulus of the hydrogel of the outer layer is greater than 10kPa, preferably greater than 60kPa.

25. Cell microcompartment according to any one of the preceding claims, characterized in that the hydrogel of the outer layer and / or in the inner part is selected from fibrin, collagen, fi- bronectin, entactin, hyaluronic acid, alginate, laminin, an extracellular matrix substitute and mixtures thereof.

26. Cell microcompartment according to any one of the preceding claims, characterized in that the aqueous solution of the inner part is a culture medium suitable for culturing the cells present in the inner part.

27. ​​Cell microcompartment according to any one of the preceding claims, characterized in that the microcompartment is closed.

28. Cell microcompartment according to any one of the preceding claims, characterized in that the microcompartment has the shape of an ovoid, a cylinder, a spheroid, a sphere or a teardrop.

29. Cell microcompartment according to any one of the preceding claims, characterized in that the internal part of the microcompartment comprises at least one layer of cells and / or at least one aggregate of cells and / or at least one three-dimensional cellular micro-tissue, said aggregate and / or micro-tissue optionally comprising at least one lumen.

30. A set of three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to one of the preceding claims.

31. Cell microcompartment according to any one of claims 1 to 29 or set of cell microcompartments according to claim 30 for its use as a drug.

32. Use of a cell microcompartment according to any one of claims 1 to 29 or a set of cell microcompartments according to claim 30 to produce on a large scale cells and / or micro-tissues and / or organoids expressing at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner.

33. A method for preparing a cell microcompartment according to any one of claims 1 to 29, comprising the following steps: (a) preparing a solution containing cells, (b) preparing at least one aqueous solution and / or at least one hydrogel, said solution and / or hydrogel comprising at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, (c) encapsulating the solutions and / or hydrogel(s) of steps (a) and (b) by collinear flow in an outer layer of hydrogel, said hydrogel being different from the possible hydrogel of step b).

34. A method according to the preceding claim, characterized in that it also comprises a step (d) which consists of culturing the microcompartments obtained in step (c) in a culture medium, preferably in a bioreactor, preferably for at least 1 day, preferably from 3 to 50 days.

35. A method according to any one of claims 33 or 34, characterized in that the cell density of the solution in step (a) is between 0.1 and 40 million cells per mL, preferably between 0.8 and 20 million.

36. A method according to any one of claims 33 to 35, characterized in that the solution in step (a) comprises at least 2 different cell types.

37. A method according to any one of claims 33 to 36, characterized in that step (c) is carried out by simultaneous co-injection of a hydrogel solution intended to form the outer layer, of the solution of step (a) and of the solution of step (b); said co-injection is carried out concentrically via a microfluidic or millifluidic injector forming a jet at the injector outlet consisting of the mixture of said solutions, said jet breaking into drops.

38. A method according to the preceding claim, characterized in that the final opening diameter of the microfluidic injector is between 50 and 800 pm, preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 10 and 2000 mL / h, preferably between 11 and 100 mL / h.

39. A method according to any one of claims 33 to 36, characterized in that step (c) is carried out by simultaneous co-injection of the hydrogel solution intended to form the outer layer, the solution of step (a) and the solution of step (b), said co-injection is carried out concentrically via a microfluidic or millifluidic injector, said injector comprising a tip, said tip being in contact with a calcium solution, forming a jet at the injector outlet consisting of the mixture of said solutions, said jet forming a tube.

40. A method according to the preceding claim, characterized in that the final opening diameter of the microfluidic injector is between 50 and 1000 pm, preferably between 80 and 300 pm, and the flow rate of each of the solutions is between 1 and 100 mL / h.

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