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. More particularly, the invention relates to a particular three-dimensional cellular microcompartment comprising in particular at least one cell, at least one transduction agent and / or at least one transfection agent.

Description

[0001] TRANSDUCTION AND / OR TRANSFECTION IN A THREE-DIMENSIONAL MICROCOMPARTMENT

[0002] Technical field

[0003] The present invention relates to the field of genetic and proteomic modification, transiently, constitutively or conditionally, of cells in a three-dimensional cell culture model. The invention relates in particular to a particular cellular microcompartment making it possible to carry out transduction and / or transfection within the internal part of said microcompartment.

[0004] State of the art

[0005] In recent years, cell culture methods have evolved in both complexity and performance. Traditional cell culture methods, which involve growing cells on flat, two-dimensional surfaces, are being replaced by three-dimensional (3D) culture techniques, which offer better reproduction of the in vivo cellular environment.

[0006] 3D cell culture offers an alternative closer to biological reality by reproducing cell-extracellular matrix and cell-cell interactions more precisely, leading to more reliable and relevant results for cell biology studies, medical research and cell therapy.

[0007] Cells grown in these 3D systems can be of any type, including differentiated cells with different phenotypes, progenitor cells, and stem cells.

[0008] However, some constraints associated with 3D cell culture may hinder 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.

[0009] For example, the paper "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 therefore reduces transduction efficiency. Thus, the 3D cell culture systems existing to date do not exploit their performance potential, in particular for carrying out genetic and / or proteomic modification of cells, more particularly by transfection or transduction.

[0010] There is therefore a significant need for a new solution for transfecting and / or transducing cells cultured in 3D so that said cells can express or receive at least one peptide sequence and / or one coding or non-coding RNA sequence and / or one transgene in a transient, constitutive or conditional manner.

[0011] Summary of the invention

[0012] To meet this need, the invention proposes a novel cellular microcompartment comprising an external hydrogel layer defining an internal part, said internal part comprising:

[0013] - at least one human, animal or plant eukaryotic cell;

[0014] - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and

[0015] - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the external layer.

[0016] Preferably, the hydrogel of the internal part is different from the hydrogel constituting the external layer, in that it has at least one different chemical property and / or at least one different physical property.

[0017] Advantageously, the hydrogel of the outer layer maintains the hydrogel of the inner part so as to form a three-dimensional cellular microcompartment. The hydrogel and / or the aqueous solution of the inner part are particularly suitable for maintaining the viability of human, animal or plant eukaryotic cells while allowing the diffusion of said cells and transduction agents and / or transfection agent within the inner part.

[0018] 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 strictly lower than the diffusive conductance of the inner part to this same transduction agent and / or transfection agent.

[0019] Diffusive conductance is the ability of a material, such as a hydrogel layer, to allow the diffusion of an object, such as a transduction agent 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 the material.

[0020] According to one embodiment, the external 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 internal part comprises a hydrogel, the latter comprises pores whose smallest dimension is greater than this dimension d.

[0021] Advantageously, the size of the pores of the outer layer is less than the smallest dimension d of said at least one transduction agent and / or transfection agent, making it possible to prevent the diffusion of at least one transduction agent and / or transfection agent through the outer layer. In other words, the pores of the outer layer make it possible, by their adapted dimensions, to maintain at least one transfection agent and / or transduction agent within the internal part of the microcompartment. Conversely, when the internal part comprises a hydrogel, the latter comprises pores making it possible, by their adapted dimensions, to allow at least one transduction agent and / or transfection agent to diffuse within the internal part.

[0022] In the context of the invention, the pore size can be measured by techniques well known to those skilled in the art, namely:

[0023] - By transmission electron microscopy;

[0024] - By super-resolution optical microscopy;

[0025] - By X-ray diffraction imaging;

[0026] - By neutron diffraction imaging; and

[0027] - By exclusion of molecules of known sizes and conformations.

[0028] Preferably, the external hydrogel layer is characterized by the presence of pores whose largest dimension is less than 25nm, preferably less than 20nm, in particular less than 10nm, even more preferably less than 5nm.

[0029] According to a preferred object of the invention, the hydrogel of the internal 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.

[0030] 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 depending on their size through said solution, in particular soluble particles whose size would be greater than 10 nm, preferably 25 nm.

[0031] 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.

[0032] The invention thus relates to a microcompartment comprising two distinct structural elements allowing transfection and / or transduction within the microcompartment, namely: - an external layer or envelope, formed by a hydrogel layer, preventing cells and transduction and / or transfection agents of the internal part from passing through it. Preferably, said external layer has a diffusive conductance to at least one transduction agent and / or transfection agent strictly lower than the diffusive conductance of the internal part to this same transduction agent and / or transfection agent, and / or comprises pores whose largest dimension is smaller than the smallest dimension d of said transduction agent and / or transfection agent, so that the transduction agent cannot diffuse through this external layer; and

[0033] - an internal part, comprising in particular at least one aqueous solution and / or at least one hydrogel different from that of the external layer, intended to promote the encounter between the cells and the transduction and / or transfection agents comprising at least one molecule of interest, it being understood that said internal part is delimited by the external hydrogel layer. Preferably, said internal part has a diffusive conductance to at least one transduction agent and / or transfection agent strictly greater than the diffusive conductance of the external layer to this same transduction agent and / or transfection agent, and / or when the internal part comprises at least one hydrogel, the latter comprises pores whose smallest dimension is greater than the smallest dimension d of said transduction agent and / or transfection agent, so that the transduction agent can diffuse through this internal part.

[0034] The external hydrogel layer allows, on the one hand, to form the protective external envelope, and therefore to constitute the capsule or microcompartment, on the other hand, the solution and / or the hydrogel of the less rigid, looser internal part, allows cell growth and promotes the movements of cells and transfection and / or transduction agents within the internal part, given the conductance and / or the particularly adapted pore size of the solution and / or the hydrogel.

[0035] Conversely, the known prior art, composed of a single layer of hydrogel, is either too rigid, not allowing the movement of cells and transduction and / or transfection agents and inducing low transfection / transduction efficiency, or on the contrary, too loose and therefore incompatible with a culture in a bioreactor. Indeed, the mechanical constraints of a culture in a bioreactor, in particular due to the significant shear forces generated during the culture, require a suitable cellular microcompartment.

[0036] The external hydrogel layer, different from the internal part comprising a solution and / or a different hydrogel, makes it possible to form the external envelope of the microcompartment or the capsule according to the invention, which makes it possible to protect the contents of the capsule from the external environment, in particular when the capsules are cultured in a bioreactor. Furthermore, the external hydrogel layer guarantees the maintenance of the cells and the transduction / transfection agents inside it. The external layer thus makes it possible to dissociate the concentration of the transduction / transfection agents near the cells, also called "local concentration", from the overall concentration of transduction / transfection agents in the culture system, also called "overall concentration".

[0037] Thus, the structure of the microcompartment optimizes the cell-transfection and / or transduction agent encounters and thus makes it possible to reduce the quantity of transfection and / or transduction agent necessary to obtain the desired transfection and / or transduction efficiency, in particular compared to 2D cultures or 3D cultures described in the prior art.

[0038] According to another preferred object of the invention, the microcompartment comprises at least one transfection agent and / or at least one transduction agent capable of diffusing into the internal part. Preferably, the majority of the transfection and / or transduction agents present in the internal part of the microcompartment are capable of diffusing. According to one embodiment, all the transfection and / or transduction agents present in the internal part of the microcompartment are capable of diffusing.

[0039] Advantageously, the properties of the aqueous solution and / or the 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.

[0040] According to one embodiment, the microcompartment according to the invention comprises at least one transfection agent and / or at least one transduction agent capable of diffusing into the internal part and incapable of diffusing through the external hydrogel layer.

[0041] Advantageously, according to this embodiment, the external hydrogel layer does not allow the diffusion of transfection agent and / or transduction agent, in particular from the internal part to the exterior of the microcompartment but also from the exterior of the microcompartment to the internal part.

[0042] Also, the microcompartment according to the invention comprises four major constituents, namely:

[0043] - at least one external hydrogel layer forming the envelope of said microcompartment;

[0044] - at least one solution and / or one hydrogel, allowing the diffusion of the transduction and / or transfection agents as well as, optionally, the cells present in the internal part of the microcompartment;

[0045] -at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest;

[0046] - at least one human, animal or plant eukaryotic cell.

[0047] 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, even more preferably pluripotent stem cells, progenitors, cells in the process of differentiation and differentiated cells. Preferably, the cells are not cells derived from a human embryo or requiring the destruction of a human embryo.

[0048] The cells present in the inner part may be isolated and / or in the form of at least one layer and / or 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, optionally with at least one lumen.

[0049] According to one embodiment, the microcompartment comprises at least one layer of cells and at least one lumen. When the microcompartment comprises at least one lumen, at least one layer of cells, the transduction agent(s) and / or transfection agent(s), the layer of aqueous solution or the hydrogel of the internal part and the external layer are preferentially successively organized around said lumen.

[0050] According to another aspect, the invention also relates to a set of microcompartments, wherein said set comprises at least one microcompartment according to any of the preceding embodiments.

[0051] 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 transient, constitutive or conditional transgene, capable of being used, for example in the context of cell therapy.

[0052] Advantageously, the conformation of the microcompartment according to the invention makes it possible to carry out transduction and / or transfection in capsulo and consequently produce cells comprising and / or expressing at least one peptide sequence and / or one coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner.

[0053] 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 medicament.

[0054] According to a 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 for inserting at least one peptide sequence and / or a coding or non-coding RNA sequence and / or a transgene transiently, constitutively or conditionally into a eukaryotic cell.

[0055] According to another variant, the invention also relates to the use of the microcompartment according to the invention for manufacturing microtissues comprising and / or expressing at least one peptide sequence and / or one coding or non-coding RNA sequence and / or one transgene in a transient, constitutive or conditional manner. It is understood that in this particular embodiment, the microtissues are preferably not intended to be implanted in a human being or an animal. For example, they can be used as an ex-vivo model. The invention also relates to a transduction method and a transfection method implemented in at least one microcompartment according to the invention.

[0056] On the other hand, the microcompartment according to the invention can be produced in different ways, however according to a particular aspect, the invention relates to a method for preparing the cellular microcompartment according to the invention, comprising the following steps:

[0057] (a) prepare a solution containing cells,

[0058] (b) preparing 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,

[0059] (c) encapsulating the solutions and / or hydrogels of steps (a) and (b) by collinear flow in an outer layer of hydrogel different from that of step (b);

[0060] (d) cultivating 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

[0061] (e) optionally recover the obtained cellular microcompartments.

[0062] According to a particular object of the invention, step (c) comprises the following sub-steps:

[0063] - bringing the solution of step (a) and the solution of step (b) into contact with a layer of hydrogel to form at least one drop, and

[0064] - collect the drop obtained in a calcium bath capable of stiffening said hydrogel solution to form the external layer of each microcompartment.

[0065] Particularly preferably, step (c) is carried out by simultaneous co-injection of the solution from step (a), the solution from step (b) and an external 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 up into drops.

[0066] 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.

[0067] According to a variant of the method 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 external layer of hydrogel having pores whose largest dimension is smaller 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.

[0068] When said injector comprises 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.

[0069] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow.

[0070] Brief description of the Figures:

[0071] [Figure 1] Figure 1 represents the experimental scheme of Example 1 concerning the transduction of primary human CD3+ T lymphocytes.

[0072] [Figure 2A] Figure 2A is a graphical representation of a comparative study of the transduction efficiency of a 2D T cell culture system vs. a 3D system as a function of the MOI of a lentiviral vector.

[0073] [Figure 2B] Figure 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 cell culture system vs. a 3D system.

[0074] [Figure 2C] Figure 2C represents the average number measured by qPCR of GFP transgenes integrated into the genome of T lymphocytes by a Lentiviral vector in a 2D culture system vs a 3D system

[0075] [Figure 2D] Figure 2D is a graphical representation of a comparative study of transduction efficiency in a 2D vs. 3D T cell culture system as a function of the MOI of a Sendai viral vector.

[0076] [Figure 2E] Figure 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.

[0077] [Figure 3A] Figure 3A is a graphical representation of a comparative study of cell viability of a 2D T lymphocyte culture system vs. a 3D culture system in the presence of different MOIs of a Sendai viral vector.

[0078] [Figure 3B] Figure 3A is a graphical representation of a comparative study of cell viability of a 2D T lymphocyte culture system vs. a 3D culture system in the presence of different MOIs of a Sendai viral vector.

[0079] [Figure 4] Figure 4 represents the experimental design of Example 2 for the transduction of human induced pluripotent stem (hIPS) cells. [Figure 5] Figure 5 is a graphical representation of a comparative study of the transduction efficiency of a 2D vs. a 3D human induced pluripotent stem cell culture system as a function of the MOI of several different transduction agents.

[0080] [Figure 6] Figure 6 is a graphical representation showing the co-transduction efficiency of 1, 2 or 3 lentiviral vectors into T lymphocytes.

[0081] [Figure 7A] Figure 7A is a graphical representation of a comparative study of transduction efficiency in microcompartments according to the invention vs. microcompartments without the invention.

[0082] [Figure 7B] Figure 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.

[0083] [Figure 7C] Figure 7C is a graphical representation of a comparative study of the cell viability of microcompartments according to the invention vs. microcompartments outside the invention after transduction.

[0084] [Figure 7D] Figure 7D is a graphical representation of a comparative study showing the amplification factor of microcompartments according to the invention vs. microcompartments without the invention after transduction.

[0085] [Figure 8] Figure 8 is a simplified experimental diagram of Example 5.

[0086] [Figure 9] Figure 9 is a graphical representation of a comparative study showing the ability of a transduction agent to pass through the outer hydrogel layer of the microcompartment according to the invention from the outside to the inside.

[0087] [Figure 10] Figure 10 is a simplified experimental diagram of Example 6.

[0088] [Figure 11] Figure 11 is a graphical representation of a comparative study showing the ability of a transduction agent to pass through the outer hydrogel layer of the microcompartment according to the invention from the inside to the outside.

[0089] [Figure 12] Figure 12 is a graphical representation of a comparative study of the transduction efficiency of a Sendai vector and a lenviral vector, particularly during co-transduction.

[0090] Detailed description of the invention

[0091] Definitions

[0092] For the purposes of the invention, "differentiated cells" means cells that have a particular phenotype, as opposed to pluripotent stem cells that are not differentiated or progenitor cells that are in the process of differentiation. For the purposes of the invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention consist of or are obtained from human cells or from immunologically humanized non-human mammalian cells. For the purposes of the invention, "mutant cell" means a cell carrying at least one mutation.

[0093] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in cell differentiation but not yet differentiated.

[0094] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA4 / 5, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, embryonic stem cells of human beings can be excluded.

[0095] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire original organism, without 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. In particular, they may be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0096] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106).

[0097] For the purposes of the invention, "diffusive conductance" means the capacity of a material to allow the diffusion of an object in solutions. Diffusive conductance can be measured experimentally using the following formula:

[0098] [Math 1] dn = —AC * \ object * dt

[0099] Where dn is the number of objects having crossed the length L in a time dt; AC is the difference in concentration between two points of the material separated by a length L (assumed stable in time dt) pobject is the diffusive conductance of the material to the object

[0100] [Math2]

[0101] Where D is the effective diffusion coefficient of objects in the material

[0102] L is the length of the material through which objects diffuse

[0103] JJdS corresponds to the integration over the entire surface of the material through which the objects diffuse.

[0104] In the context of the invention, the diffusive conductance of the hydrogel of the external part can be measured using two compartments separated by the hydrogel of the external layer whose diffusive conductance is to be measured. To measure its diffusive conductance, it is sufficient to place in the first compartment the solution containing the transfection agent or the transduction agent in question and in the other compartment, the solution without the agent. It is then sufficient to measure with a suitable method, such as counting, fluorescence or dosage, the evolution of the quantity of agent in the compartment as a function of time to deduce the diffusive conductance. For example, the two compartments can for example be two reservoirs separated by a membrane made of the hydrogel of the external layer whose diffusive conductance is to be measured.

[0105] Alternatively, the diffusive conductance of the hydrogel from the outer layer to an agent can be measured locally using the FRAP (Fluorescence Recovery After Photobleaching) technique, for fluorescent agents of interest. This technique consists of irradiating an area of ​​a material with a laser, allowing the photobleaching of the fluorescent agents in the irradiation zone, and observing the return to a normal fluorescence level. The time taken to return to normal is directly related to the diffusion coefficient, which then allows the calculation of a diffusive conductance.

[0106] In the context of the invention, the diffusive conductance of the hydrogel from the internal part to a transfection or transduction agent is defined as follows:

[0107] [Math3] pintmin = Dint*n(R) 2 / r

[0108] 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 to the inner part.

[0109] For the purposes of the invention, the term "cell layer" or "cell base" means several cells forming a layer or base that can be structured around a lumen; this may be, for example, a cellular tissue or micro-tissue or a three-dimensional grouped culture. The thickness of the cell layer may be variable. This layer is organized in three dimensions in the microcompartment.

[0110] For the purposes of the invention, "density of the hydrogel of the outer layer" or "density of the hydrogel of the inner part" means the mass of the hydrogel in its hydrated state divided by its total volume, including absorbed water.

[0111] For the purposes of the invention, the term “Feret diameter” means the distance, in particular “d” or “D”, between two tangents, these two tangents being parallel, such that the entire projection is between these two parallel tangents.

[0112] For the purposes of the invention, the term "drop" also means a three-dimensional structure formed from at least one liquid solution comprising the constituents of a non-rigidified hydrogel (polymerization precursors, non- or partially crosslinked polymer chains), hydrogel precursor elements. Also, the drop constitutes a transitional state between the co-injection of the different constituents and the microcompartment according to the invention.

[0113] By "hydrogel of plant or synthetic origin" we mean a hydrogel that is not of animal origin and / or derived from cancer cell lines such as Matrigel®. This may include, for example, but is not limited to, alginate.

[0114] For the purposes of the invention, "the smallest dimension" of X means the value of the smallest Feret diameter of X.

[0115] For the purposes of the invention, "the largest dimension" of X means the value of the largest Feret diameter of X.

[0116] For the purposes of the invention, the term "light" or "lumen" means 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.

[0117] For the purposes of the invention, the term “microcompartment” or “capsule” means a partially or totally closed three-dimensional structure containing several cells.

[0118] By “MOI or multiplicity of infection” within the meaning 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.

[0119] For the purposes of the invention, the term "pore" means a sub-volume within the general hydrogel volume in which monomers and / or polymers of the hydrogel do not have covalent or ionic chemical bonds in continuity with the three-dimensional network of the general hydrogel volume. For the purposes of the invention, the term "diffusion resistance" means a parameter characterizing the difficulty that a substance encounters in moving by Brownian motion through a material or medium. Diffusion is the process by which molecules move from a region of high concentration to a region of low concentration, often described by Fick's law. In the context of the invention, diffusion resistance can be influenced by several factors:

[0120] * The porosity of the polymer network: A denser network will offer greater resistance to diffusion.

[0121] * Pore size: Smaller pores increase the resistance to diffusion of larger molecules.

[0122] * The interaction between diffusing molecules and the material: Chemical or physical interactions can slow down diffusion.

[0123] Diffusion resistance can be measured using the following formula:

[0124] R=d / D-AR where: d is the thickness of the material,

[0125] D is the diffusion coefficient of the substance through the material,

[0126] A is the surface through which diffusion occurs.

[0127] This relationship shows that diffusion resistance increases with material thickness and decreases with increasing diffusion coefficient and surface area.

[0128] By "tissue" or "biological tissue" within the meaning of the invention, we mean the common meaning of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a set of similar cells of the same origin (most often from a common cell lineage, although they can find their origin by association of distinct cell lineages), grouped in clusters, networks or bundles (fibers). A tissue forms a functional whole, that is to say that its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs.

[0129] Microcompartment according to the invention

[0130] The present invention relates to a three-dimensional cellular microcompartment comprising an external hydrogel layer defining an internal part, said internal part comprising:

[0131] - 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

[0132] - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the external layer.

[0133] The microcompartment according to the invention is a three-dimensional microcompartment, delimited by the external hydrogel layer and inside said external layer, an internal 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.

[0134] Advantageously, the external layer delimits an internal part and makes it possible to contain the elements present within the internal part, in particular the cells and the transfection and / or transduction agents.

[0135] Preferably, the hydrogel of the internal part is different from the hydrogel constituting the external 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.

[0136] Advantageously, the hydrogel of the internal part, different from that of the external layer, allows the transfection and / or transduction agents to diffuse into the internal part and consequently improves the transduction / transfection efficiency, in particular compared to capsules or microcompartments constituted by a single hydrogel.

[0137] According to one embodiment, the hydrogel of the external layer has a density and / or viscosity strictly greater than the density and / or viscosity of the solution and / or the hydrogel present within the internal part.

[0138] According to another embodiment, the hydrogel of the outer layer has a molecular weight different from the hydrogel present within the inner part. Preferably, in this embodiment, the cellular 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 of molecular weight lower than the hydrogel of the outer layer. Thus, in this embodiment, the molecular weight of the hydrogel of the outer layer is greater than the molecular weight of the hydrogel of the inner part.

[0139] Advantageously, the hydrogel and / or the solution of the internal part allows the cells and the transfection and / or transduction agents to diffuse within the internal part. Furthermore, the external layer has a greater resistance to diffusion than the solution and / or the hydrogel of the internal part, which makes it possible to improve the transfection and / or transduction within the microcompartment. Furthermore, the external hydrogel layer also makes it possible to protect the cells from the external environment, to limit the uncontrolled proliferation of the cells, and their differentiation in the event of differentiation. Preferably, the microcompartment according to the invention is characterized by a diffusive conductance of the external layer to at least one transduction agent and / or transfection agent strictly lower than the diffusive conductance of the internal part to this same transduction agent and / or transfection agent.

[0140] According to one embodiment, the microcompartment according to the invention has a maximum diffusive conductance of the hydrogel of the external layer to at least one transduction agent and / or transfection agent of less than 0.1 mm. 3 / h, preferably less than 0.05mm 3 / h, even more preferably less than 0.01mm 3 / h.

[0141] According to one embodiment, the microcompartment according to the invention has a maximum diffusive conductance of the hydrogel from the internal part to at least one transduction agent and / or transfection agent greater than 0.1 mm 3 / h, preferably greater than lmm 3 / h, even more preferably greater than 10 mm 3 / h, especially greater than 50 mm 3 / h.

[0142] According to a preferred embodiment, the microcompartment according to the invention has a maximum diffusive conductance of the hydrogel of the external layer to at least one transduction agent and / or transfection agent of less than 0.003 mm. 3 / h, especially less than 0.001mm 3 / h, preferably less than 0.0001mm 3 / h, even more preferably less than 0.00001mm 3 / h.

[0143] According to a preferred embodiment, the microcompartment according to the invention has a maximum diffusive conductance of the hydrogel from the internal part to at least one transduction agent and / or transfection agent greater than 0.003 mm 3 / h, preferably greater than 0.01 mm 3 / h, even more preferably greater than 0.1 mm 3 / h, especially greater than 1 mm 3 / h.

[0144] According to another embodiment, the microcompartment according to the invention has a ratio of the diffusive conductance of the hydrogel of the internal part to the diffusive conductance of the hydrogel of the external layer relative 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.

[0145] In the context of the invention, the diffusive conductance is preferably measured at 25°C and at a pH of 7.

[0146] Advantageously, the microcompartment according to the invention, by its external hydrogel layer, makes it possible both to protect the cells from physical constraints during culturing, and also to limit the diffusion in the capsule of transduction agents and / or transfection agents present in the medium which would not be encapsulated in the internal part.

[0147] In a complementary manner, the microcompartment according to the invention allows, thanks to its external layer, to maintain the transduction agents and / or transfection agents within the internal part. This physical barrier makes it possible to bring the transduction agents and / or transfection agents closer to the cells by reducing the volume available for the diffusion of said agents and therefore to increase the efficiency of transduction and / or transfection while reducing the quantity of transfection and / or transduction agents for similar efficiency.

[0148] According to one embodiment of the invention, the three-dimensional cellular microcompartment comprises an external hydrogel layer defining an internal part, said internal part comprising:

[0149] - at least one human, animal or plant eukaryotic cell;

[0150] - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and

[0151] - at least one aqueous solution and / or at least one hydrogel, in which the porosity of the external 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, when the internal part comprises a hydrogel, the latter comprises pores whose smallest dimension is greater than this dimension d.

[0152] In the context of the invention, the pore size can be measured by techniques well known to those skilled in the art, namely:

[0153] - By transmission electron microscopy;

[0154] - By super-resolution optical microscopy;

[0155] - By X-ray diffraction imaging;

[0156] - By neutron diffraction imaging;

[0157] - By exclusion of molecules of known sizes and conformations.

[0158] Preferably the pore size of the hydrogel of the outer layer and the hydrogel of the inner part is measured by neutron scattering or small angle X-rays (SANS / SAXS) and by diffusion test of polymers of known molecular mass.

[0159] Preferably, the hydrogel of the outer layer is characterized by the presence of pores whose largest dimension is less than the smallest dimension d of at least one transduction agent 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 less than the smallest dimension d of the majority of the transduction agents 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 less than the smallest dimension d of all the transduction agents and / or all the transfection agents present in the inner part.According to a particularly suitable embodiment, the hydrogel constituting the external layer is characterized by the presence of pores whose largest dimension is less than the smallest dimension d of the transduction agent or of the transfection agent having the smallest dimension d within the internal part.

[0160] Preferably, the hydrogel of the outer layer is characterized by the presence of pores whose largest dimension is less than 25nm, preferably less than 20nm, in particular less than 10nm, even more preferably less than 5nm. Advantageously, the particular pore size of the outer hydrogel layer makes it possible to maintain the cells and the transduction agents and / or transfection agents in the inner part.

[0161] 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 25nm, 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 25nm, preferably less than 10 nm.

[0162] According to one embodiment, the hydrogel of the internal part comprises pores whose smallest dimension is greater than the smallest dimension d of at least one transduction agent and / or transfection agent present in the internal part. Preferably, the hydrogel constituting the internal part comprises pores whose smallest dimension is greater than the smallest dimension d of the majority of the transduction agents and / or transfection agents present in the internal part. According to one embodiment, the hydrogel of the internal 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 internal part.

[0163] Preferably, the hydrogel of the internal 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 internal part.

[0164] According to a particularly suitable embodiment, the smallest dimension of the pores of the hydrogel of the internal part is greater than 100 nm, preferably greater than 25 nm, even more preferably between 25 and 500 nm.

[0165] Preferably, the microcompartment according to the invention is characterized in that it comprises at least one continuous diffusion path within the hydrogel of the internal part between a cell and a transfection and / or transduction agent for which the smallest dimension is greater than 100 nm, preferably between 25 and 500 nm.

[0166] 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 internal 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 internal part, and in that the hydrogel of the external layer is characterized by the presence of pores whose largest dimension is less than that of the dimension D so that the transduction agent cannot diffuse through this external layer.

[0167] Preferably, the hydrogel of the internal 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 internal part.

[0168] According to a particular embodiment, the microcompartment according to the invention comprises an external hydrogel layer defining an internal part, said internal part comprising at least one hydrogel, in which the Young's modulus of the hydrogel of the internal part is strictly lower than the Young's modulus of the hydrogel of the external layer. Thanks to this Young's modulus of the hydrogel of the internal part, the external layer will be more rigid than the hydrogel of the internal part, the hydrogel of which is looser, more loose, in particular due to the presence of a shorter chain, facilitating the growth of the cells and the movement of the transduction and / or transfection agents.

[0169] According to an object of the invention, the advantages cited above can be improved when the Young's modulus of the hydrogel of the internal part is 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 hydrogel of the external layer is greater than 10 kPa, more preferably greater than 60 kPa, even more preferably greater than 100 kPa.

[0170] According to one embodiment, the hydrogel of the inner part is entangled with the hydrogel of the outer layer, in particular on the inner face of the outer layer. Also, the delimitation between the two hydrogels despite a different Young's modulus may not be perfectly clear. Consequently, at least a portion of the hydrogel of the layer or of the mesh of the inner part may be entangled with the inner face of the outer layer.

[0171] The hydrogel of the internal part with a low Young's modulus then presents viscoelastic and viscoplastic properties which are particularly advantageous for obtaining an extracellular matrix substitute, in which the cells will be able to lodge, grow, move and be transfected and / or transduced in a satisfactory manner.

[0172] Advantageously, a hydrogel, for example an alginate hydrogel, with a low Young's modulus, has faster relaxation properties, also known as "fast-relaxing", which allows cells to move, change shape, expand, proliferate and mechanically remodel the hydrogel-based matrix and also allows the diffusion of transfection and / or transduction agents, making the microcompartment according to the invention particularly suitable for in capsulo transfection and / or transduction. Preferably, the hydrogel(s) used in the microcompartments according to the invention are biocompatible, i.e. they are not toxic to the cells. They must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival.

[0173] The hydrogel of the outer layer and / or the inner part may comprise or consist of alginate.

[0174] The 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 microfluidic injector to form the microcompartments according to the invention.

[0175] According to a preferred embodiment, the external hydrogel layer comprises at least alginate. It may consist exclusively of alginate.

[0176] The alginate constituting the outer layer may in particular be a sodium alginate, composed of 80% a-L-guluronate and 20% pD-mannuronate, having a Young's modulus greater than 10 kPa, preferably greater than 60 kPa, more preferably greater than 100 kPa.

[0177] 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.

[0178] When the concentration of the alginate solution intended to form the external layer of the microcompartment is equal to 2%, the viscosity of the alginate forming the external layer is preferably between 100 and 200mPa / s.

[0179] Preferably, the outer hydrogel layer is cell-free.

[0180] The hydrogel of the inner portion may be in the form of a layer or a mesh. According to one embodiment, the hydrogel of the inner portion is juxtaposed at least partially with the inner face of the outer layer.

[0181] According to one embodiment, the microcompartment according to the invention comprises in its internal part:

[0182] - a layer of aqueous solution; and / or a hydrogel layer and / or mesh, or

[0183] - an aqueous solution; and / or a hydrogel layer and / or mesh, or

[0184] - a layer of aqueous solution; and / or a hydrogel.

[0185] Preferably, the microcompartment according to the invention comprises in its internal part:

[0186] - at least one layer of aqueous solution; and / or - at least one layer and / or a hydrogel mesh.

[0187] Preferably, the hydrogel(s) used in the internal part of the microcompartments according to the invention are biocompatible, that is to say they are not toxic to the cells. They must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival. These hydrogels can be chosen from all the biocompatible hydrogels having the characteristics of the invention. They can be, for example, alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid and / or collagen. They can also be an extracellular matrix or an extracellular matrix substitute such as Matrigel®.

[0188] 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 made of alginate. This difference can be characterized in particular by a difference in diffusive conductance, viscosity, density, molecular weight, Young's modulus and / or pore sizes. According to one variant, the inner part comprises a layer and / or a mesh of hydrogel comprising at least alginate. The layer and / or mesh can consist exclusively or partially of alginate. The alginate present in the inner part can be in particular a sodium alginate, composed of 80% α-L-guluronate and 20% pD-mannuronate.The alginate of the internal 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 of between 0.01 kPa and 200 kPa, preferably between 0.1 kPa and 60 kPa, more preferably between 0.1 kPa and 5 kPa.

[0189] Preferably, the alginate present in the internal part has an average molecular weight of at most 75 kDa. When the hydrogel of the internal part is alginate, the concentration of the alginate solution intended to form the internal part of the microcompartment is preferably between 0.25 and 2%, more preferably between 0.25 and 1%, even more preferably 0.5% (percentage by mass). When the concentration of the alginate solution intended to form the internal part of the microcompartment is 0.5%, the viscosity of the alginate is preferably 3 mPa / s.

[0190] Advantageously, although mammalian cells are unable to interact with alginate, particularly because it allows minimal protein adsorption, alginate is well characterized, easy to sterilize and store, can possibly be chemically modified, and offers interesting mechanical properties. Also, alginate allows for good growth, little cell death, good amplification, and a good proportion of transfected and / or transduced cells.

[0191] According to one embodiment, the hydrogel of the inner part comprises fibrin or is fibrin, the fibrin is preferentially obtained from the polymerization of fibrinogen by a fibrinogen polymerization agent, such as thrombin. This polymerization agent can be added during encapsulation and / or after encapsulation. Also, the polymerization of the fibrinogen solution by the thrombin solution takes place during encapsulation and / or after it. When it takes place after encapsulation, the polymerization takes place within the newly formed drop or capsule, i.e. after the rigidification of the outer layer.

[0192] The hydrogel of the internal 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. For example, the peptide sequence may be a peptide or a protein. The peptide sequence may be, for example, a YIGSR motif and / or an RGD motif. The YIGSR motif is a peptide derived from the laminin pi chain 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.

[0193] According to a particular embodiment of the invention, the internal part comprises at least two different hydrogels, each being different from the hydrogel of the external layer. These at least two hydrogels present in the internal part are preferably chosen from alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid, and collagen.

[0194] When the inner part comprises at least two hydrogels, the second hydrogel may be in the form of particles.

[0195] Advantageously, this form of the second hydrogel does not prevent the diffusion of transfection and / or transduction agents within the internal part while providing a beneficial effect on the cells, preferentially by improving cell survival.

[0196] 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.

[0197] According to another embodiment, when the internal part comprises at least one aqueous solution, the latter has a viscosity of between 0.1 and 10 mPa / s.

[0198] Preferably, when the internal part comprises an aqueous solution and a hydrogel, the internal part has a viscosity of between 0.1 and 50 mPa / s.

[0199] Preferably, the viscosity of the internal part is between 0.1 and 50 mPa / s.

[0200] Advantageously, the particular viscosity of the aqueous solution and / or hydrogel facilitates the encounter of the transfection agents and / or transduction agents and the cells within the internal part of the microcompartment and consequently increases the proportion of transduced / transfected cells compared to the 2D cultures or 3D cultures described in the prior art. In the context of the invention, the viscosity of the aqueous solution and / or hydrogel can be measured using a viscometer at 20°C.

[0201] Advantageously, the hydrogel and / or the aqueous solution of the internal part make it possible to promote the encounter with the cells and consequently improve the efficiency of transduction / transfection.

[0202] Preferably, when the internal part comprises an aqueous solution, this is preferably a culture medium adapted to the cells of the microcompartment. Typically, the culture medium is chosen from culture media usually compatible with the culture of the cells present within the microcompartment, for example from culture media used in two-dimensional culture or in spheroid culture.

[0203] In addition to the hydrogel and / or the 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.

[0204] According to one embodiment, the cellular microcompartment according to the invention comprises:

[0205] - an outer hydrogel layer defining an inner portion, said inner portion comprising:

[0206] - at least one human, animal or plant eukaryotic cell;

[0207] - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and

[0208] - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the external layer in which said at least one transduction agent and / or at least one transfection agent maintains the cell type of the cells present within the internal part.

[0209] In other words, according to this embodiment, said at least one transfection agent and / or at least one transfection agent does not modify the cell type of the cells present within the internal part of the microcompartment.

[0210] Preferably, 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 in the hydrogel of the internal part.

[0211] When the microcompartment according to the invention comprises at least one transduction agent, said agent may be chosen from a viral particle, a pseudo-viral particle or their combination.

[0212] Preferably, the transduction agent is chosen from an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a baculovirus and combinations thereof. 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 0.01 and 50, even more preferably between 0.01 and 10.

[0213] According to one embodiment, when the microcompartment comprises at least one transduction agent, the MOI (“multiplicity of infection”) of said microcompartment is between 0.01 and 5.

[0214] According to another embodiment, when the microcompartment comprises at least one transduction agent, the MOI (“multiplicity of infection”) of said microcompartment is between 5 and 100, in particular between 5 and 50, preferably between 5 and 25, more preferably between 5 and 15.

[0215] According to a variant, when the microcompartment comprises at least one transduction agent, the MOI of said microcompartment is less than 10.

[0216] Advantageously, the microcompartment according to the invention, by its particular conformation, makes it possible to promote the encounter between the transduction agents and the cells. Consequently, the MOI necessary to obtain satisfactory transduction is lower than those of two-dimensional systems or three-dimensional systems not having the characteristics of the microcompartment according to the invention.

[0217] The microcompartment according to the invention thus makes it possible to reduce the MOI necessary to obtain satisfactory transduction, thereby reducing the costs associated with transduction.

[0218] When the microcompartment according to the invention comprises at least one transfection agent, said agent may be chosen 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.

[0219] According to a particular embodiment, the microcompartment comprises at least one transfection agent, said agent being a derivative of the plasma membrane comprising proteins of viral origin.

[0220] 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 comprising and / or expressing at least one peptide sequence and / or one coding or non-coding RNA sequence and / or one transgene in a transient, constitutive or conditional manner. Furthermore, the microcompartment according to the invention requires a lower concentration of transduction and / or transfection agent than the solutions proposed by the prior art while obtaining a similar transfection / transduction efficiency. According to one embodiment, when the microcompartment comprises at least one transfection agent, the ratio of quantity of DNA or quantity of RNA / cell is less than 10, preferably less than 5, even more preferably less than 2.

[0221] Advantageously, the microcompartment according to the invention, by its particular conformation, makes it possible to promote the encounter between the transfection agents and the cells. Consequently, the ratio of quantity of DNA or quantity of RNA / cell necessary to obtain a satisfactory transfection is lower than those of two-dimensional systems or three-dimensional systems not having the characteristics of the microcompartment according to the invention.

[0222] The microcompartment according to the invention thus makes it possible to reduce the quantity of DNA or RNA necessary to obtain satisfactory transfection, thereby reducing the costs associated with transfection.

[0223] 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 microcompartment is greater than 10 nm, preferably between 15 and 25 nm. According to a variant, the majority of the transfection agents and / or the transduction agents present in the internal part of the microcompartment, preferably all of them, have a smallest dimension d of less than 25 nm, preferably between 15 and 25 nm.

[0224] According to another embodiment, the largest dimension D of at least one transfection agent and / or at least one transduction agent present in the internal part of the microcompartment is greater than 25nm, preferably greater than 100nm, in particular greater than 500nm, even more preferably greater than 1pm. According to a variant, the majority of the transfection agents and / or the transduction agents present in the internal part of the microcompartment, preferably all, have a largest dimension D greater than 50nm, in particular greater than 100nm, even more preferably between 300 and 500nm.

[0225] 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 internal part.

[0226] Advantageously, said transduction agent and / or transfection agent can diffuse into the aqueous solution and / or into the hydrogel of the internal part so as to increase its chances of encountering a cell and inserting at least one molecule of interest therein.

[0227] 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 chosen from a nucleic acid, a ribonucleic acid, a protein, a peptide and their combinations. 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 therein.

[0228] In addition to the hydrogel and / or the 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.

[0229] In the context of the invention, the cells present in the microcompartment may be any type of cell, in particular the cells are eukaryotic cells. More preferably, the cells are human or plant or animal cells.

[0230] Preferably, the cells present in the internal part are chosen from pluripotent cells, progenitors, cells in the process of differentiation, differentiated cells and their mixtures.

[0231] Alternatively, the cells present in the internal part are chosen from pluripotent cells, progenitors, cells in the process of differentiation and their mixtures.

[0232] According to another variant, the microcompartment according to the invention does not comprise a differentiated cell.

[0233] In a particular embodiment, the microcompartment comprises pluripotent stem cells. A pluripotent stem cell, or pluripotent cell, is understood to mean a cell that has the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such. The pluripotent stem cells may in particular be induced pluripotent stem (iPS) cells, MUSE (Multi-lineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells. According to one embodiment, the microcompartment according to the invention does not comprise embryonic stem (ES) cells.

[0234] According to a particularly suitable variant of the invention, the microcompartment according to the invention comprises human or animal induced pluripotent stem cells.

[0235] According to one embodiment, the microcompartment according to the invention comprises:

[0236] - an outer hydrogel layer defining an inner portion, said inner portion comprising:

[0237] - at least one cell chosen from a human or animal induced pluripotent stem cell or a lymphocyte, in particular a CD3+ T lymphocyte;

[0238] - at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest, and

[0239] - at least one aqueous solution and / or at least one hydrogel different from the hydrogel constituting the external layer. 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 and / or progenitor cells have preferably been obtained from pluripotent stem cells, in particular human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile has been 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, the multipotent and / or progenitor cells have been obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells. According to another variant, the microcompartment according to the invention comprises differentiated human or animal cells. The differentiated cells have preferably been obtained from pluripotent stem cells or progenitor cells, in particular 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 or progenitor cells after contact with a solution capable of initiating the differentiation of said stem cells. According to a variant, the cellular content of the microcompartment comprises homogeneous or mixed cellular identities.

[0240] In particular, differentiated cells may be present as at least one layer of cells or as a three-dimensional tissue, cell aggregate or microtissue or as several tissues or microtissues in the microcompartment. It may be a compacted or non-compacted tissue or microtissue, with or without a lumen.

[0241] 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 in its internal part at least two different cell types. In particular, the microcompartment according to the invention can comprise, for example, stem cells induced to pluripotency and / or multipotent cells and / or progenitor cells and / or cells in the process of differentiation and / or differentiated cells.

[0242] When the microcompartment according to the invention comprises at least one layer of cells or cell base, the aqueous solution and / or the hydrogel are preferably arranged between the external hydrogel layer and said layer or cell base. It is understood that the microcompartment may also comprise in its internal part cells suspended in the aqueous solution or the hydrogel of the internal part. When the microcompartment according to the invention comprises at least one cellular aggregate and / or tissue and / or microtissue, the aqueous solution and / or the hydrogel are preferably arranged between the external hydrogel layer and said cell aggregate and / or tissue and / or microtissue. It is understood that the microcompartment may also comprise in its internal part cells suspended in the aqueous solution or the hydrogel of the internal part.

[0243] According to another preferred object of the invention, the aggregate and / or the cell layer and / or the cell base and / or the tissue and / or the microtissue comprises at least one light or lumen. Said light may contain a liquid, in particular culture medium and / or a liquid secreted by the cells.

[0244] Advantageously, the presence of this hollow part allows the cells to have a small diffusive volume whose composition they can control, promoting cellular communication.

[0245] According to one embodiment, the cell layer and the hydrogel layer and / or mesh and / or aqueous solution layer of the internal part are organized around at least one lumen, more preferably they are organized successively around at least one lumen.

[0246] According to one embodiment, the cell layer and the hydrogel layer and / or mesh and / or aqueous solution layer of the internal part are successively organized around a lumen. This is referred to as a cyst-shaped conformation.

[0247] According to an embodiment in which the microcompartment comprises only a single cyst, the cell layer, the hydrogel layer and / or mesh and / or the aqueous solution layer of the internal part, and the external layer are organized successively around a lumen.

[0248] This cyst-shaped conformation reduces the pressures experienced by the cells. This configuration also reduces cell mortality and increases the culture amplification factor. Consequently, this reduces the number of passages and dissociations required; and reduces the culture time required to reach the final number of cells required.

[0249] According to one embodiment, the microcompartment may comprise one or more cysts, and / or one or more tissues and / or microtissues and / or cell aggregates with or without lumen(s).

[0250] Advantageously, the microcompartment according to the invention is suitable for the transduction and / or transfection of any type of eukaryotic cell.

[0251] Such a microcompartment is thus particularly suitable for carrying out a genetic and / or transcriptomic and / or protein modification of cells of interest, preferably so that said cells can comprise and / or express at least one peptide sequence and / or one coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner.

[0252] According to a particular object 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 may be cells obtained by amplification, from at least one cell.

[0253] 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 external layer of hydrogel of at least one cell.

[0254] Preferably, the cells present in the microcompartment according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28 or 30 cycles of cell division after encapsulation in an external 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 external hydrogel layer of at least 1 cell, preferably between 1 and 50 cells.

[0255] 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.

[0256] Preferably, all of the cells initially encapsulated in the microcompartment before the first cycle of cell division represent a volume less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0257] Thus, according to one embodiment, the cells present in the microcompartment according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division, after encapsulation in an external layer of hydrogel of cell(s) representing a volume less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0258] 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.

[0259] 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. The microcompartment according to the invention can be obtained by encapsulation carried out by means of a co-injection carried out concentrically via a microfluidic injector forming a jet at the injector outlet consisting of the mixture of the different useful solutions, said jet breaking up into drops. The drops are then collected in a bath, in particular a calcium bath, capable of stiffening the hydrogel solution to form the external layer of each microcompartment.

[0260] According to a variant allowing 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.

[0261] The cellular microcompartment according to the invention is preferably closed or partially closed, that is to say that the external layer is closed or partially closed. More preferably the microcompartment is closed.

[0262] The microcompartment according to the invention can be in any three-dimensional form, that is to say it can have the shape of any object in space. The microcompartment can have any shape compatible with the encapsulation of cells. Preferably, the microcompartment according to the invention is in a spherical or elongated form. It can have the shape of an ovoid, a cylinder, a spheroid or a sphere. In particular, it can be in the form of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0263] It is the outer layer of the microcompartment, i.e. 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 μm and 1 mm, preferably between 100 μm and 700 μm. It may be between 200 μm and 600 μm, in particular between 300 μm and 500 μm.

[0264] Its largest dimension is preferably greater than 10 pm, more preferably between 10 pm and 1 m, even more preferably between 10 pm and 50 cm.

[0265] The microcompartment according to the invention can optionally be frozen for storage. It will then have to be defrosted before use.

[0266] The invention also relates to several microcompartments together. Also, the invention also relates to a set or series of cellular microcompartments as described above comprising at least one cellular microcompartment according to the invention.

[0267] The invention also relates to a set or series of microcompartments of at least two cellular microcompartments in three dimensions, in which at least one microcompartment is a microcompartment according to the invention, preferably the majority of the microcompartments of the set are microcompartments according to the invention. According to a particular embodiment, all of the microcompartments of the set are microcompartments according to the invention.

[0268] The set of microcompartments according to the invention preferably comprises between 2 and 1016 microcompartments, preferably between 1000 and 109 microcompartments according to the invention.

[0269] Preferably, the series of microcompartments according to the invention is in a culture medium, in particular in an at least partially convective culture medium.

[0270] 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.

[0271] The presence of an outer hydrogel layer and the aqueous solution and / or the hydrogel of the inner part, allow a uniform distribution of cells between the microcompartments. Furthermore, the outer hydrogel layer makes it possible to avoid fusions of microcompartments which are a major source of variability unfavorable for the phenotypic homogeneity of the cells.

[0272] According to one embodiment, the microcompartment according to the invention can be obtained by a method comprising the implementation of the following steps:

[0273] (a) prepare a solution containing cells,

[0274] (b) preparing 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,

[0275] (c) encapsulating the solutions and / or hydrogels of steps (a) and (b) by collinear flow in an external hydrogel layer having pores whose largest dimension is smaller than the smallest dimension d of said transduction agent and / or transfection agent of step (a);

[0276] (d) cultivating 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

[0277] (e) optionally recover the obtained cellular microcompartments.

[0278] Thus, the microcompartment according to any of the embodiments previously described or the set of microcompartments according to any of the embodiments previously described, is also particularly suitable for use in cell culture, in particular in three-dimensional cell culture. These allow the production of cells of interest in large quantities, in particular cells, micro-tissue or organoid expressing at least one peptide sequence, a coding or non-coding RNA sequence, or a transgene in a transient, constitutive or conditional manner, capable of being used, for example in the context of cell therapy. 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 medicament.

[0279] This makes the microcompartment particularly suitable for clinical use.

[0280] According to another object, the invention relates to the use of the microcompartment according to any of the preceding objects, for inserting at least one peptide sequence, a coding or non-coding RNA sequence, or a transgene transiently, constitutively or conditionally into at least one eukaryotic cell.

[0281] The invention also relates to the use of the microcompartment according to the invention for implementing transfection and / or transduction method(s).

[0282] According to another object, the invention relates to the use of the microcompartment according to any 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.

[0283] Preparation process

[0284] The microcompartment can be obtained by different means known to those skilled in the art for preparing microcompartments or capsules.

[0285] The invention also relates to a particular method for preparing microcompartments.

[0286] According to one embodiment, the invention relates to a method for preparing a microcompartment comprising the following steps:

[0287] (a) prepare a solution containing cells,

[0288] (b) preparing at least one aqueous solution and / or at least one hydrogel, said solution and / or said hydrogel comprising at least one transduction agent and / or at least one transfection agent comprising at least one molecule of interest,

[0289] (c) encapsulating the solutions and / or hydrogel(s) of steps (a) and (b) by collinear flow in an external layer of hydrogel, said hydrogel being different from the possible hydrogel of step (b).

[0290] According to one embodiment, the preparation method according to the invention also comprises 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.

[0291] 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 external layer of hydrogel, said hydrogel having a diffusive conductance to at least one transduction agent and / or transfection agent strictly lower than the diffusive conductance of the internal part to this same transduction agent and / or transfection agent.

[0292] According to one embodiment, the method for preparing a microcompartment according to the invention comprises the implementation of the following steps:

[0293] (a) prepare a solution containing cells,

[0294] (b) preparing 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.

[0295] (c) encapsulating the solutions and / or hydrogels of steps (a) and (b) by collinear flow in an external hydrogel layer having pores whose largest dimension is smaller than the smallest dimension d of said transduction agent and / or transfection agent of step (a).

[0296] According to one embodiment, the solution of step (a) is produced 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.

[0297] 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 at the time of formation of the external hydrogel layer.

[0298] The method according to the invention may comprise a step of dissociating the cells by chemical, enzymatic or mechanical dissociation, carried out before or simultaneously with the step of incubating the cells, itself carried out before step a) of mixing. This step is particularly important in the case of adherent cells.

[0299] The encapsulated cells are suspended as single cells and / or cell clusters. Preferably, the single cells represent less than 50% in number of the total encapsulated cells, more preferably the single cells are hPSC cells. Indeed, it is preferable to encapsulate cell clusters because this reduces apoptosis.

[0300] Preferably, the cell density of the solution from step (a) is between 0.1 and 40 million cells per mL, preferably between 0.8 and 20 million.

[0301] The solution of step (a) preferably comprises at least one cell chosen from pluripotent cells, progenitors, cells in the process of differentiation, differentiated cells and mixtures thereof.

[0302] According to a preferred embodiment, the solution of step (a) comprises at least 2 different cell types. Preferably, the transduction agent is chosen from an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a Sendai virus, a baculovirus and combinations thereof.

[0303] According to another embodiment, the solution of step (b) comprises at least one transfection agent comprising at least one molecule of interest, said agent can be chosen 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 their combinations.

[0304] Preferably, the encapsulation step (c) comprises the following sub-steps:

[0305] - bringing into contact the solution of step (a), the solution of step (b) and a hydrogel solution intended to form the external layer to form at least one drop, and

[0306] - collect the drop obtained in a calcium bath capable of stiffening said hydrogel solution to form the external layer of each microcompartment.

[0307] Once the outer hydrogel layer has been stiffened 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 outer layer, this makes it possible to avoid / limit the stiffening of the hydrogel in the inner part by the calcium bath. In particular, 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 stiffening process by the calcium bath does not make it possible to stiffen the hydrogel in the inner part during the period of use of the capsules and therefore to facilitate cell multiplication.

[0308] Preferably, step (c) is carried out by simultaneous co-injection of the hydrogel solution intended to form the external 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 up into drops.

[0309] According to an 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.

[0310] According to a variant, step (c) is carried out by a simultaneous co-injection of the hydrogel solution intended to form the external 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, 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. 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 μm, more preferably between 80 and 300 μm, and the flow rate of each of the solutions is between 1 and 100 mL / h.

[0311] According to another variant of the invention, the internal 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 the mesh of the internal part.

[0312] When at least one hydrogel of the internal part is fibrin, it is preferentially obtained from the polymerization of fibrinogen by a fibrinogen polymerization agent, advantageously said agent is thrombin, said agent can be added during encapsulation and / or after encapsulation.

[0313] Also, according to a variant the thrombin solution is co-injected with the other solutions, this is preferentially mixed with the isotonic intermediate solution and the fibrinogen solution is mixed with the solution from step (b).

[0314] Preferably, the concentration of fibrinogen is between 10 and 25 mg / mL, preferably 14-20 mg / mL, more preferably 20 mg / mL.

[0315] According to another object of the invention, the concentration of thrombin is preferably between 0.001U / mL and 2U / ml, more preferably between 0.01U / mL and 1U / ml, between 0.01U / mL and 0.05U / ml, even more preferably 0.04U / ml. By "U" is meant a unit of enzymatic activity (i.e. the concentration for an enzyme) which represents the quantity of enzyme necessary to treat one micromole of substrate in 1 minute. It being understood that the concentration indicated is that in the mixture. Indeed, advantageously the thrombin is mixed with the other constituents according to a ratio of 1:1. Also, within the capsule, when the concentration of thrombin, before mixing, is 0.01U / ml, the concentration in the capsule is of the order of 0.01U / ml.

[0316] The steps following encapsulation can be carried out without agitation or with agitation. Preferably, the steps following encapsulation are carried out with continuous or sequential agitation. This agitation is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusive gradient. For example, it allows homogeneous control of the level of cellular oxygenation; thus avoiding the phenomena of necrosis linked to hypoxia, or oxidative stress linked to hyperoxia. Consequently, it avoids an increase in cell mortality and / or oxidative stress.

[0317] Preferably, after the step of culturing the capsules obtained, the method comprises a step which consists of rinsing the capsules resulting from step (d), advantageously so as to eliminate the cytoprotective factor, such as the apoptosis inhibitor. When the method according to the invention comprises a step of rinsing the capsules obtained, the solution constituting the calcium bath is eliminated 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).

[0318] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the cells after step (d), preferably after the rinsing step if such a step is present after step (d). By "at least one re-encapsulation of the cells" is meant at least two encapsulation cycles. Preferably each encapsulation cycle corresponds to one passage. In this variant of the method (at least one re-encapsulation of the cells after step (d) the number of cell divisions of the entire method (for all the passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0319] In a method according to the invention there may be several re-encapsulations, preferably between 1 and 100, in particular between 1 and 10 re-encapsulations.

[0320] Each re-encapsulation may include:

[0321] - a step of dissociating the microcompartment or series of microcompartments to obtain a cell suspension or a cell cluster suspension; the removal of the external hydrogel layer may be achieved in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, the removal may be achieved using a saline phosphate buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection, and

[0322] - a step of re-encapsulation of all or part of the cells or clusters of cells in a hydrogel capsule.

[0323] Re-encapsulation is a suitable means for increasing cellular amplification obtained from the pluripotent stage, and reducing the risks of mutation.

[0324] According to a particular embodiment, the re-encapsulation comprises the following steps:

[0325] - remove the outer hydrogel layer,

[0326] - resuspending the cells which were contained in the microcompartment so as to obtain single cells and / or at least one set or cluster of cells in an isotonic medium, preferably a culture medium containing an apoptosis inhibitor,

[0327] - encapsulate the cell suspension in a hydrogel layer;

[0328] - preferably, culturing the microcompartments obtained in an isotonic solution containing an apoptosis inhibitor, preferably a culture medium containing an apoptosis inhibitor; - preferably, rinsing the microcompartments, advantageously, so as to eliminate the apoptosis inhibitor;

[0329] - cultivate the microcompartments in an isotonic solution, preferably a culture medium, for at least one cell division cycle, and

[0330] - optionally recover the cellular microcompartments obtained.

[0331] The invention also relates to a transduction method implemented in at least one cellular microcompartment defined in any of the embodiments previously described.

[0332] According to one embodiment, the transduction method according to the invention does not modify the cell type of the transduced cells.

[0333] Finally, the invention also relates to a transfection method implemented in at least one cellular microcompartment defined in any of the embodiments previously described.

[0334] According to one embodiment, the transfection method according to the invention does not modify the cell type of the transfected cells.

[0335] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results.

[0336] Example 1: Transduction of primary human CD3+ T lymphocytes

[0337] The experimental scheme of Example 1 is described in Figure 1.

[0338] Primary human CD3+ T lymphocytes are thawed, counted and seeded at 1 million cells per ml in TexMACS medium (Mi Itenyi Biotec).

[0339] After seeding, cells are activated by adding "T Cell TransAct human" to the cell culture medium (1 / 100 dilution) and IL-2 (final concentration of 300 U / mL).

[0340] Cells are cultured in a T75 cell culture flask (kept upright) at 37°C, 5% CO2.

[0341] 48 hours after activation, cells are collected and concentrated to 10 million cells per ml in TexMACS medium supplemented with IL-2 (300 U / ml).

[0342] 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.

[0343] [Table 1]

[0344] The obtained cell population (containing viral particles) is divided into 2 samples, one for 2D cell culture in a 12-well plate (final medium volume of 1 mL at 0.5 million cells per mL) and the other for 3D cell encapsulation.

[0345] For encapsulation, 400 μl of the cell sample and viral vectors (at a rate of 5 million cells per ml) are 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).

[0346] Cells pass through the encapsulation device and capsules are collected in a calcium bath (25 mM HEPES, 100 mM CaCl2, Tween80) located below the outlet nozzle of the encapsulation chip (large capsule configuration). Capsules are then collected and rinsed with DMEM / F12 medium supplemented with CaCl2 (3 mM final) on a 100 µm filter. Rinsed capsules are resuspended in 4 volumes (using the capsule volume as a reference) of TexMACS medium supplemented with CaCl2 (3 mM final).

[0347] 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 to a T75 flask and incubated at 37°C, 5% CO2.

[0348] 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 composed of TexMACS culture medium.

[0349] 2 days (for SeV vectors) or 5 days (for LV vectors) after transduction / encapsulation, cells are decapsulated using 10 volumes of ReLeSR, pelleted and resuspended in TexMACS medium to be counted and further processed for flow cytometry.

[0350] Cells to be analyzed by flow cytometry were incubated with LIVE / DEAD™ Fixable Near IR (780) (Thermo Fisher Scientific, catalog # L34994) for 30 minutes at room temperature (RT) following the manufacturer's instructions. Cells were then incubated with different fluorescent antibodies (see Table 2), fixed in PBS / 4% PFA, and analyzed by flow cytometry (LSR Fortessa 5L). Flow cytometry results obtained for GFP expression are shown in Figure 2A and 2B for the Lentiviral vector and in Figure 2D and 2E for the Sendai viral vector. As a complement, cell viability results comparing the 2D and 3D systems are shown in Figure 3A for the Lentiviral vector and 3B for the Sendai viral vector.

[0351] [Table 2]

[0352] The transduction efficiency (% of GFP+ cells) of T lymphocytes by lentiviruses is better for 3D cell encapsulation conditions than for 2D conditions, especially for the lowest MOI tested (0.1 and 1), and this without affecting cell viability (Figure 3A and 3B). Moreover, in addition to transducing more cells, 3D cell encapsulation allows better transgene expression in transduced cells as demonstrated by higher fluorescence intensities (Figure 2B and 2E). This better transgene expression is associated with a greater number of transgenes inserted into the T lymphocyte genome in 3D conditions than in 2D at equivalent MOI (Figure 2C).

[0353] Similarly, the transduction efficiency (% of GFP+ cells) of T lymphocytes by Sendai viral vectors is better for 3D cell encapsulation conditions than for 2D conditions for all MOIs tested (0.3, 1 and 5) (Figure 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 higher fluorescence intensities.

[0354] Example 2: Transduction of human induced pluripotent stem (hIPS) cells

[0355] For hiPSCs, cell culture conditions for 2D and 3D configurations (before cell encapsulation) are different from Example 1.

[0356] The experimental scheme of Example 2 is described in Figure 4.

[0357] The day before encapsulation, cells for 2D transduction experiments are seeded at 25,000 cells / cm 2 in a 12-well plate in mTeSRl supplemented with Rock inhibitor (10 pM final).

[0358] 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, 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) for a final density of 5 million cells / mL. 400 μl of the cell suspension and viral particles are then injected into the encapsulation system programmed with the following parameters: Alginate syringe flow rate (100 mL / h); Cell and intermediate solutions flow rate (50 mL / h); Electric field (2000V).Cells pass through the encapsulation device and capsules are collected in a calcium bath (25 mM HEPES, 100 mM CaCl2, Tween80) located below the outlet nozzle of the encapsulation chip (large capsule configuration).

[0359] The capsules are then collected and rinsed with DMEM / F12 medium supplemented with CaCl2 (3mM final) 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 CaCl2 (3mM final). Once the capsules settle (~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), transferred to a T75 flask and incubated at 37°C, 5% CO2.

[0360] 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 composed of mTeSRl culture medium.

[0361] On the same day, the medium of cells cultured for 2D cell transduction (seeded the day before in a 12-well plate) was replaced with mTeSRl medium supplemented with Rock inhibitor (10 pM final) and transduced with viral vectors at the same MOI as those used in the 3D configuration (see Table 3).

[0362] [Table 3]

[0363] The day after transduction, the medium of 2D-grown cells was replaced with mTeSRl without Rock inhibitor and the medium of 3D-grown cells was replaced with mTeSRl supplemented with Rock inhibitor (10 μM). Two days after transduction, the medium was again replaced with mTeSRl without Rock inhibitor for both 2D- and 3D-grown cells. Three days after transduction, 3D-grown cells were decapsulated by removing the growth medium and adding 10 volumes of ReLeSR (for each volume of capsules). After decapsulation, cells were dissociated using 0.5X TrypLE, resuspended in mTeSRl medium and subjected to flow cytometry. Cells to be analyzed by flow cytometry were incubated with the fixative lime for 30 minutes at room temperature (RT) following the manufacturer's instructions.The cells were then incubated with different 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 Figure 5.

[0364] [Table 4]

[0365] 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.

[0366] Example 3: Transduction of encapsulated cell according to the invention with several transduction agents.

[0367] The experimental conditions of this example are identical to those of example 1.

[0368] However, transduction is performed with 3 lentivectors instead of just one in Example 1. The MOIs shown correspond to the total MOI used for the combination of the 3 lentivectors. The results are shown in Figure 6. These results show that it is possible to co-transduce cells using multiple vectors in a 3D microcompartment.

[0369] Example 4: Comparative study of transduction efficiency within a microcompartment comprising a single hydrogel vs. a microcompartment according to the invention

[0370] To carry out this comparative study, this example includes 3 specific conditions:

[0371] *Condition 1: a microcompartment according to the invention comprising an external hydrogel layer composed of 2% high molecular weight alginate (150-250 kDa) and an internal part comprising an aqueous solution (mTeSRl medium).

[0372] *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.

[0373] *Condition 3: a “full” microcompartment, outside the invention, comprising a 1.2% high molecular weight alginate hydrogel (150-250 kDa). To obtain condition 3, the protocol described by Neumann et al., 2013 (DOI 10.1007 / sl2033-012-9522-y) was reproduced.

[0374] Results :

[0375] Three days after transduction, it can be observed in Figure 7A that transduction is more efficient in condition 1 (82.5% of GFP+ cells with a fluorescence intensity of 32338) than in condition 3 (37.1% of GFP+ cells with a fluorescence intensity of 11317). Condition 2 has an intermediate transduction rate (73.3% of GFP+ cells with a fluorescence intensity of 20909). In addition to improving transduction efficiency, condition 2 demonstrates better viability as well as better amplification of cells compared to condition 3 (Figure 7C).

[0376] Transduction within the microcompartments generated under conditions 1 and 2 is therefore more efficient and less toxic to cells than transduction performed under condition 3 (Figure 7A and Figure 7B). Furthermore, the fact that condition 2 is more efficient than condition 3 (Figure 7D) demonstrates that the physical properties necessary for microcompartment formation can be decorrelated from those promoting optimal transduction and cell culture, particularly in a bioreactor. Indeed, it would be impossible to generate / maintain the physical integrity of capsules full of 0.5% alginate.

[0377] Example 5: Measurement of the infection capacity of a transduction agent to diffuse through the external hydrogel layer of the microcompartment according to the invention from the outside to the inside.

[0378] To carry out this example, human cancer cells are encapsulated according to the protocol described in example 1, at 3 million cells per ml in a microcompartment according to the invention.

[0379] For 24 hours, the encapsulated cells are placed in a ventilated tube, in 500 µl of medium: either in uninfected medium (condition 1), or in infected medium (lentiviral vector coding for GFP at MOI 10) (condition 2).

[0380] In each condition, uninfected medium is added so that the final volume is equal to 5 mL.

[0381] The fluorescence of the cells is then observed according to the same protocol as example 1 by flow cytometry in order to observe or not the transfection of the cells.

[0382] A simplified experimental scheme of this example is shown in Figure 8.

[0383] The results of this example are presented in Figure 9 and show that transduction agents such as lentiviral vectors are not capable of crossing the external hydrogel layer from the outside to the inside of the microcompartment according to the invention, in particular after 24 hours of incubation. Advantageously, the microcompartment according to the invention, by its external hydrogel layer, makes it possible both to protect the cells from physical constraints during culturing, but also to prevent the diffusion into the capsule of transduction agents present in the medium which would not be encapsulated in the internal part.

[0384] The microcompartment according to the invention thus guarantees transduction carried out solely by transduction agents of interest and encapsulated within the internal part of the microcompartment according to the invention.

[0385] Example 6: Measurement of the capacity of a transduction agent to diffuse through the external hydrogel layer of the microcompartment according to the invention from the inside to the outside.

[0386] To carry out this example, human cancer cells are seeded at 80,000 cells per well on a 12-well plate for 24 hours.

[0387] In a complementary manner, lentiviral vectors encoding GFP are encapsulated according to the same protocol as example 1 in microcompartments at 1x108 TU / mL (encapsulation volume = 400 μl).

[0388] Following the same protocol as Example 1, empty microcompartments, i.e. without cells or lentiviral vectors, are produced to represent a negative control for this example.

[0389] Empty microcompartments (condition 1 - negative control) or microcompartments containing lentiviral vectors (condition 2) are then placed on top of the previously obtained cells.

[0390] After a rinsing step, conditions 1 and 2 are incubated for 48 hours at 37°C at 5% CO2 before removing the microcompartments.

[0391] The fluorescence of the cells is then observed according to the same protocol as example 1 by flow cytometry in order to observe or not the transduction of the cells in conditions 1 and 2 48 hours after the removal of the microcompartments.

[0392] To perform a positive control (condition 3), the microcompartments obtained after rinsing and used for incubation for 48 hours of condition 2, are collected and the encapsulated lentiviral vectors are decapsulated and then transferred into DMEM medium.

[0393] New human cancer cells are seeded at 160,000 cells per well to which 1 ml of decapsulated lentiviral vector (diluted 1 / 10) is added.

[0394] The mixture of human cancer cells and decapsulated lentiviral vectors is incubated for 48 hours before analysis by flow cytometry in order to observe or not the transduction of the cells in condition 3 (positive control).

[0395] A simplified experimental scheme of this example is shown in Figure 10.

[0396] The results of this example are presented in Figure 11 and show that the transduction agents are functional (condition 3) but are not capable of diffusing from the inside to the outside of the outer layer of the microcompartment according to the invention, in particular after 48 hours of incubation (condition 2). Advantageously, the microcompartment according to the invention makes it possible to maintain the transduction agents within the internal part of the microcompartment, thus allowing better control of the concentration of the transduction agents as well as a greater probability of encounter between the cells and the transduction agents.

[0397] Example 7: Comparative study of the transduction efficiency of two transduction agents

[0398] The objective of this example is to study the transduction efficiency of two transduction agents (a lentiviral vector encoding GFP and a Sendai vector encoding mCherry) on CD3+ T lymphocytes.

[0399] Thus, for the realization of this example several conditions were met, namely:

[0400] Condition 1: Negative control corresponding to non-translated cells;

[0401] Condition 2: CD3+ T lymphocytes transduced using Sendai vectors at MOI 1;

[0402] Condition 3: CD3+ T lymphocytes transduced using lentiviral vectors at MOI 1.5;

[0403] Condition 4: Co-transduced CD3+ T lymphocytes with an MOI of 1.5 for the lentiviral vectors and an MOI of 1 for the Sendai vector.

[0404] All the conditions were prepared according to the same protocol as example 1, thus forming on the one hand a 3D system with the microcompartments according to the invention and on the other hand a 2D culture system.

[0405] All conditions were incubated for 48 hours and then flow cytometry analysis was performed to compare the transduction efficiency of the two transduction agents, including co-transduction.

[0406] The results of co-transduction of the lentiviral vector and the Sendai vector 48 hours after encapsulation are presented in Figure 12.

[0407] These results demonstrate that compared to a 2D system, the microcompartment according to the invention allows a similar or higher transduction efficiency for both transduction agents. Furthermore, these results also indicate that it is possible to obtain a higher proportion of co-transduced cells using the microcompartment according to the invention.

[0408] Example 8: Measuring the transfection efficiency of human cancer cells using exosomes

[0409] The objective of this example is to compare the efficiency of exosome transfection in a 3D system via the use of microcompartments according to the invention.

[0410] To carry out this example, the exosomes were obtained according to the following protocol:

[0411] * Cultivation of human cancer cells to obtain approximately 80,000 cells per well; *Recovery of 10 ml of culture medium after 72 hours of culture and centrifugation at 3,000 g for 15 minutes to remove cells and cell debris;

[0412] *Transfer the supernatant to a new tube and add 2mL of ExoQuick-TC to the medium. Exo-Quick-TC is a commercial solution for precipitating extracellular vesicles such as exosomes;

[0413] *Refrigerate overnight (at least 12 hours) at +4°C;

[0414] *Centrifuge the ExoQuick-TC / medium mixture at 1500g for 30 minutes. After centrifugation, exosomes may appear as a beige or white pellet at the bottom of the container;

[0415] *Removal of supernatant and centrifugation of residual ExoQuick-TC solution by centrifugation at 1500 g for 5 minutes;

[0416] *Removal of all traces of liquid by suction;

[0417] *Resuspend the pellet including exosomes in 100 to 500 µl using sterile 1X PBS.

[0418] The exosomes thus obtained were then prepared according to different conditions, namely:

[0419] *Condition 1: Exosomes transfected with a TX-Red SiRNA (200pl);

[0420] *Condition 2: Exosomes transfected with a GFP lentiviral plasmid (200pl).

[0421] In parallel, a negative control was performed using empty exosomes.

[0422] To carry out this example, the exosomes were transfected as follows:

[0423] *Preparation of a 0.15mL mixture comprising:

[0424] -10 µl of Exo-Fect solution + 20 µl of nucleic acid (20 pmolsi or 5 µg of plasmid DNA);

[0425] -70 ul sterile lx PBS;

[0426] - 50 ul of purified exosomes in 1x PBS suspension;

[0427] - 150 ul of total transfection reaction;

[0428] *Incubate the exosome transfection solution at 37°C for 10 minutes and then immediately place the tube on ice.

[0429] *To stop the reaction 30 µl of the ExoQuick-TC reagent provided in the kit to the sample suspension of transfected exosomes is added to the mixture;

[0430] *Cooling the transfected exosome sample on ice or at 4°C for 30 minutes;

[0431] *Centrifugation of the transfected exosome sample for 3 minutes at 13,000-14,000 rpm; * Removal of the supernatant and resuspension of the transfected exosome pellet in 300 µl of 1x PBS.

[0432] Transfected exosomes are ready to be added to target cells or used in vivo.

[0433] The transfected exosomes thus obtained are then added to the target cells according to the following protocol:

[0434] *Mixture of 150 µl of untransfected exosomes with approximately 2 xl0 6 encapsulated human cancer cells; OR Co-encapsulation according to the protocol of Example 1 of 150 μl of transfected exosomes with approximately 2 xl0 6 human cancer cells so as to form microcompartments according to the invention *incubation of the cells for 24 hours for exosomes transfected with SiRNA and 48 hours for exosomes transfected with the lentiviral plasmid GFP);

[0435] *Visualization by fluorescence microscopy and flow cytometry according to the protocol of Example 1 following the following excitation / emission guidelines for the fluorophore used.

[0436] Table 5 below describes the ratio of quantity of siRNA or plasmid DNA per cell as well as the efficiency of transfection in the microcompartment according to the invention.

[0437] [Table 5]

[0438] Table 5 thus makes it possible to demonstrate that the microcompartment according to the invention is suitable for carrying out transfection using a transfection agent such as exosomes.

Claims

CLAIMS

1. A three-dimensional cellular microcompartment comprising an outer hydrogel layer defining an inner portion, said inner portion 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 external layer.

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

3. Cellular microcompartment according to one of the preceding claims, in which the diffusive conductance of the outer layer to at least one transduction agent and / or transfection agent is strictly lower than the diffusive conductance of the inner part to this same transduction agent and / or transfection agent.

4. Cellular microcompartment according to 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. Cellular microcompartment according to one of the preceding claims, in which the maximum diffusive conductance of the hydrogel of the internal 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. Cellular microcompartment according to one of the preceding claims, in which the ratio of the diffusive conductance of the hydrogel of the internal part to the diffusive conductance of the hydrogel of the external 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. Cellular microcompartment according to one of the preceding claims, in which the outer layer comprises pores whose largest dimension is less than the smallest dimension d of said at least one agent transduction and / or transfection agent and, when the internal part comprises a hydrogel, the latter comprises pores whose smallest dimension is greater than this dimension d.

8. Cellular microcompartment according to claim 7, characterized in that the smallest dimension of the pores of the hydrogel of the internal part is greater than 100 nm, preferably between 25 and 500 nm.

9. Cellular microcompartment according to claim 7 or 8, characterized in that the external layer comprises pores whose largest dimension is less than 25nm, preferably less than 10nm.

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

11. Cellular microcompartment according to one of the preceding claims, characterized in that the hydrogel of the external layer has a molecular weight different from the hydrogel present within the internal part.

12. Cellular microcompartment according to one of the preceding claims, characterized in that the external layer has a greater resistance to diffusion than the solution and / or the hydrogel of the internal part.

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

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

15. Cellular 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 internal part.

16. Cellular microcompartment according to one of the preceding claims, characterized in that it comprises at least one transduction agent and in that the MOI (“multiplicity of infection”) of said microcompartment is between 0.01 and 100.

17. Cellular microcompartment according to one of the preceding claims, characterized in that it comprises at least one transfection agent and in that the ratio of quantity of DNA or quantity of RNA / cell of said microcompartment is less than 10.

18. Cellular microcompartment according to 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.

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

20. Cellular microcompartment according to 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 chosen from a nucleic acid, a ribonucleic acid, a protein, a peptide and their combinations.

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

22. Cellular microcompartment according to one of the preceding claims, characterized in that the eukaryotic cells present in the internal part are chosen from pluripotent cells, progenitors, cells in the process of differentiation, differentiated cells and their mixtures.

23. Cellular microcompartment according to one of the preceding claims, characterized in that the internal part comprises at least one aqueous solution and / or a hydrogel whose Young's modulus is strictly lower than the Young's modulus of the hydrogel of the external layer.

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

25. Cellular microcompartment according to one of the preceding claims, characterized in that the Young's modulus of the hydrogel in the internal part is between 0.1 and 5 kPa.

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

27. ​​Cellular microcompartment according to one of the preceding claims, characterized in that the hydrogel of the external layer and / or in the internal part is chosen from fibrin, collagen, fibronectin, entactin, hyaluronic acid, alginate, laminin, an extracellular matrix substitute and mixtures thereof.

28. Cellular microcompartment according to one of the preceding claims, characterized in that the aqueous solution of the internal part is a culture medium suitable for the culture of the cells present in the internal part.

29. Cellular microcompartment according to one of the preceding claims, characterized in that the microcompartment is closed.

30. Cellular microcompartment according to 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.

31. Cellular microcompartment according to 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.

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

33. Cellular microcompartment according to one of claims 1 to 31 or set of cellular microcompartments according to claim 32 for use as a medicament.

34. Use of a cellular microcompartment according to one of claims 1 to 31 or set of cellular microcompartments according to claim 32 for producing on a large scale cells and / or micro-tissues and / or organoids expressing at least one peptide sequence and / or one coding or non-coding RNA sequence and / or a transgene in a transient, constitutive or conditional manner.

35. Cellular microcompartment according to one of claims 1 to 31 or set of cellular microcompartments according to claim 32 for inserting at least one peptide sequence and / or one coding RNA sequence or non-coding and / or a transgene transiently, constitutively or conditionally in at least one cell.

36. Microcompartment according to one of claims 1 to 31, for use in a transduction method.

37. Microcompartment according to one of claims 1 to 31, for use in a transfection method.

38. A method of preparing a cellular microcompartment according to one of claims 1 to 31, comprising the following steps: (a) prepare a solution containing cells, (b) preparing at least one aqueous solution and / or at least one hydrogel, said solution and / or said 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 external layer of hydrogel, said hydrogel being different from the possible hydrogel of step b).

39. Method according to the preceding claim, characterized in that it also comprises a step (d) which consists of cultivating 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.

40. Method according to one of claims 38 or 39, characterized in that the cell density of the solution of step (a) is between 0.1 and 40 million cells per mL, preferably between 0.8 and 20 million.

41. Method according to one of claims 38 to 40, characterized in that the solution of step (a) comprises at least 2 different cell types.

42. Method according to one of claims 38 to 41, characterized in that step (c) is carried out by simultaneous co-injection of a hydrogel solution intended to form the external 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 up into drops.

43. 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.

44. Method according to one of claims 38 to 43, characterized in that step (c) is carried out by simultaneous co-injection of the hydrogel solution intended to form the external 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.

45. 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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