Cellular microcompartments containing lymphocytes forming grouped 3D cultures and with low TNFα content

Abstract

The present invention relates to a closed three-dimensional cellular microcompartment or assembly of three-dimensional cellular microcompartments having an ovoid, cylindrical, spheroidal or spherical shape or a substantially ovoid, cylindrical, spheroidal or spherical shape, the smallest dimension being between 200 and 400 μm, comprising an outer hydrogel layer defining an inner portion, the inner portion comprising 500 to 5000 lymphocytes forming a grouped three-dimensional culture in a medium, the TNFα content being less than 100 ng / mL of medium. The present invention also relates to a method for producing such a microcompartment or microcompartment assembly.

Description

[Technical field]

[0001] The present invention relates to the field of large scale lymphoid cell cultures. In particular, the present invention relates to specific cell microcompartments, methods for their preparation and uses thereof. [Background technology]

[0002] Advances made over the past few decades have highlighted the benefits of cell therapy in the treatment of a wide range of disease conditions.

[0003] Cell therapy is defined as the administration of live cells to a patient for the purpose of preventing, treating or ameliorating a medical condition. The goal is to achieve a lasting effect by injecting the therapeutic cells.

[0004] A number of cell therapy approaches can be envisaged, including: - Autologous approach, where the patient is his own donor. -Allogeneic approach where the donor is different from the patient.

[0005] Cell therapy encompasses a broad therapeutic area and is applied to numerous cell types ranging from pluripotent cells to specialized adult cells with metabolic (e.g., hepatocytes) or mechanical functions (e.g., myoblasts, chondrocytes), as well as hematopoietic lineages such as mononuclear blood cells (e.g., T lymphocytes or NK lymphocytes).

[0006] One of the most promising treatments to date is CAR (chimeric antigen receptor) cell therapy. CAR-T cells are T lymphocytes that express chimeric antigen receptors, artificial receptors designed to recognize antigens present on the surface of tumor cells. CAR-T cells induce the immune system to target and eradicate cancer cells. This approach is considered a major advance in immunotherapy, and many CAR-T cells are currently being developed to expand their use. CAR-T cells are generally generated using an autologous approach. However, it is also possible to generate allogeneic CAR-T cells.

[0007] An alternative approach is to use NK (natural killer) lymphocytes, which are highly potent cytotoxic effector cells with proven antitumor effects.

[0008] In order to develop the potential for cell-based therapies using lymphocytes, particularly CAR-T or CAR-NK therapies, it is necessary to be able to rely on large-scale lymphocyte cell culture techniques.

[0009] The ex vivo production of immune cells such as T or NK lymphocytes is particularly complicated, especially on a large scale.In fact, the in vitro culture methods developed to date are too stressful, leading to rapid cell exhaustion and making it impossible to produce lymphocytes on a large scale.In vivo, immune cell proliferation mainly occurs in mechanically stable environments such as solid tissues.Because bioreactor environments are filled with non-physiological shear stress, the proliferation rate obtained is generally still insufficient compared to in vivo proliferation capacity.

[0010] Currently, there is no satisfactory solution for the rapid large-scale production of functional lymphocytes, particularly CAR-T cells or CAR-NK cells.

[0011] For CAR cell expansion, several culture methods are mainly used: static flask culture, static gas permeable bag culture and rocking bioreactor culture (Vormittag P, Gunn R, Ghorashian S, Veraitch FS. Curr Opin Biotechnol. 2018), the G-rex® platform (Ludwig et al. "Methods and Process Optimization for Large-Scale CAR T Expansion Using the G-Rex Cell Culture Platform" Methods Mol Biol 2020;2086:165-177).

[0012] However, these culture methods are not suitable for large-scale lymphocyte production due to exposure of the cells to numerous handling steps that increase the risk of contamination, exposure of the cells to excessive stress, and / or limitations on the development and capabilities of the platforms and methods used. Moreover, the operations involved in culture and storage are too demanding for large-scale production.

[0013] To remedy this problem, the prior art has proposed activating lymphocytes before culturing them in order to achieve rapid lymphocyte proliferation.

[0014] Nevertheless, early lymphocyte activation leads to the physiological process of cell depletion, which corresponds to the early maturation of naive T cells into a subpopulation of effector T cells and limits graft persistence in vivo.

[0015] Another solution proposed by the prior art is the use of hydrogel and alginate microtubes suspended in cell culture medium. This method reported a lymphocyte proliferation rate of 320 on day 14 of culture (Lin et al. Adv. Healthcare Mater. 2018, 1701297).

[0016] However, although this method provides sufficient growth rates, it is not suitable for large-scale cultivation: it requires the development of a dedicated culture system and is too complex to be adapted to large scale.

[0017] On the other hand, this method does not guarantee a functional phenotype of the lymphocytes and, consequently, a long-lasting effect of the lymphocytes.

[0018] The complexity of the differentiation and function of each lymphocyte subpopulation means that analysis of the phenotype during expansion is essential for the success of any lymphocyte-based therapy, particularly those using CAR-T or CAR-NK cells.

[0019] Less differentiated T cell subpopulations such as naive T lymphocytes and Tscm (stem cell-like memory T cell) lymphocytes appear to be particularly important in ensuring sustained efficacy in patients (Vita Golubovskaya and Lijun Wu (Cancers 2016, 8, 36). Furthermore, memory-like NK subpopulations allow for sustained clinical responses (Romee et al. "Cytokine activation induces human memory-like NK cells" Blood 2012 Dec 6;120(24):4751-60).

[0020] On the other hand, the inventors also know that effector T lymphocyte subpopulations are essential to ensure a significant effect on target cells.

[0021] It therefore appears necessary to control cell subpopulations during large-scale lymphocyte culture to obtain an optimal ratio between persistence and in vivo efficacy.

[0022] Current production processes involving lymphocytes are very costly, difficult to scale up, and result in poor mixed quality and persistence of lymphocytes.

[0023] Therefore, there is a strong need for solutions that allow for the large-scale production of lymphocytes with a functional phenotype to meet the much needed demand for lymphocyte-based cell therapies.

[0024] Therefore, it is an object of the present invention to fulfill all of these needs and overcome the disadvantages and limitations of the prior art. Summary of the Invention

[0025] To this end, the present invention proposes the use of specific microcompartments to obtain large-scale production of lymphocytes suitable for use in cell therapy.

[0026] To this end, an object of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part comprising 500 to 5000 lymphocytes forming a grouped three-dimensional culture in a medium, and having an endogenous tumor necrosis factor (TNFα) content of less than 100 ng / mL of medium.

[0027] Preferably, the inner portion of the microcompartment comprises a perforin content of less than 0.5 μg / mL of medium, and / or a granzyme B content of less than 1 μg / mL of medium, and / or an IL6 content of less than 0.5 μg / mL of medium, and / or a GM-CSF content of less than 0.5 μg / mL of medium.

[0028] Advantageously, the microcompartments according to the invention allow the production of lymphocytes with a functional phenotype, whilst ensuring rapid large-scale production.

[0029] According to a particularly preferred embodiment, the microcompartments according to the invention contain lymphocytes which form grouped three-dimensional aluminal cultures.

[0030] The microcompartments according to the invention can be obtained after encapsulation of lymphocytes with or without the addition of extracellular matrix, and according to variants with or without the addition of extracellular matrix elements.

[0031] Advantageously, the absence of addition of extracellular matrix or matrix elements during lymphocyte encapsulation improves their proliferation rate after 3 days in culture.

[0032] According to a preferred object of the invention, the microcompartments according to the invention are obtained after encapsulation of preactivated lymphocytes. Lymphocyte activation advantageously stimulates lymphocyte proliferation in the inner part of the microcompartments according to the invention. According to one variant, lymphocyte activation can be carried out in the microcompartments after encapsulation.

[0033] According to another particularly preferred embodiment, the microcompartment according to the invention comprises lymphocytes expressing a chimeric antigen receptor. When the microcompartment according to the invention comprises T lymphocytes, it preferably comprises naive and / or Tscm lymphocytes at a concentration of more than 20%, even more preferably more than 40%.

[0034] Advantageously, such a high concentration of naive and / or Tscm lymphocytes ensures a long-lasting effect and avoids the phenomenon of early lymphocyte depletion.

[0035] According to a preferred object of the invention, the invention relates to a set of microcompartments.

[0036] According to another aspect, the present invention relates to a process for preparing a microcompartment or a set of microcompartments, comprising carrying out at least the following steps: incubation, encapsulation and culture. Finally, the present invention is directed to a microcompartment according to the invention for use as a medicament, preferably in the prevention or treatment of an autoimmune disease, an immune deficiency syndrome, a cancer, a viral disease or an inflammatory disease.

[0037] Other features and advantages will become apparent from the detailed description of the invention and the examples that follow. [Brief description of the drawings]

[0038] [Figure 1a] 1 is a phase contrast microscope image at ×20 magnification of a set of microcompartments according to the invention containing T lymphocytes after 3 days of encapsulation culture. [Figure 1b] 1 is a phase contrast microscope image at magnification x10 of a set of microcompartments according to the invention containing T lymphocytes after 3 days of encapsulation culture. [Diagram 2] 1 shows the results of a test of lymphocyte expansion in microcompartments according to the invention. [Diagram 3] Figure 1 shows the proliferation rate of T lymphocytes after 6 days of culture with or without encapsulation. [Figure 4]FIG. 1 is a comparative representation of cytokine expression profiles in T lymphocyte cultures after 6 days with or without encapsulation. Results are shown as normalized values ​​for GM-CSF and IFN-γ secretion. [Diagram 5] 13A-C are confocal microscopy images of microcompartments with polarized lymphocytes. [Figure 6] The proliferation rate of T lymphocytes after 14 days of culture with ("Capsule") or without ("Control") encapsulation is shown. [Figure 7a] Comparative representation of cytokine expression in NK lymphocyte cultures after 14 days of culture with or without encapsulation (for A supernatant donor diluted 10-fold). Results are shown as normalized values ​​for IFN-γ secretion. [Figure 7b] Comparative representation of granzyme B expression in NK lymphocyte cultures after 14 days of culture with or without encapsulation (for A supernatant donor diluted 100-fold). Results are shown as normalized values ​​for IFN-γ secretion. [Figure 8a] Comparative representation of cytokine expression in NK lymphocyte cultures after 11 days of culture with or without encapsulation (for B supernatant donors diluted 10-fold). Results are shown as normalized values ​​for IFN-γ secretion. [Figure 8b] Comparative representation of granzyme B expression in NK lymphocyte cultures after 11 days of culture with or without encapsulation (for B supernatant donors diluted 100-fold). Results are shown as normalized values ​​for IFN-γ secretion. [Figure 9] FIG. 1 shows comparative results of toxicity tests carried out on NK obtained without encapsulation and on NK obtained according to the invention (for donor B). [Figure 10] FIG. 2 shows the comparative results of the evolution of the viability during culture of NK without encapsulation and of NK encapsulated according to the invention (results for two donors A and B). [Figure 11a] A phase contrast microscope image at ×4 magnification of a set of microcompartments according to the invention containing NK on the day of encapsulation is shown in FIG. 11a. [Figure 11b]FIG. 10 is a phase contrast microscope image at ×10 magnification of a set of microcompartments according to the invention containing NK after 11 days of encapsulation culture. [Figure 12a] The proliferation rate of NK lymphocytes when cultured for 12 days with ("Capsule" encapsulation without activated beads) or without ("Control") encapsulation is shown. [Figure 12b] Comparative results are shown for the evolution of viability when culturing NK without encapsulation ("control") and NK encapsulated according to the invention (encapsulation without "capsule" activated beads) (results for two donors B and C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] definition For purposes of the present invention, "alginate" refers to a linear polysaccharide formed from β-D-mannuronate and α-L-guluronate, their salts, and derivatives.

[0040] For the purposes of the present invention, "hydrogel capsule" or "hydrogel microcompartment" refers to a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, preferably water.

[0041] For the purposes of the present invention, "human cells" refers to human cells or immunologically humanized non-human mammalian cells. Even if this is not specified, the cells, stem cells, progenitor cells, and tissues according to the invention consist of or are derived from human cells or from immunologically humanized non-human mammalian cells.

[0042] For purposes of the present invention, the term "mutated cell" refers to a cell that has at least one mutation.

[0043] The "Ferret diameter" of a microcompartment (part of a microcompartment) according to the present invention means the distance "d" between two tangents to the microcompartment (or part of the microcompartment), which are parallel, so that the entire projection of the microcompartment (or part of the microcompartment) lies between these two parallel tangents. The Feret diameter of an internal part of a microcompartment is measured between the two interfaces of the internal part of the microcompartment and the outer layer, i.e., the distance "d" between two tangents to the internal part, which are parallel, so that the entire projection of the internal part lies between these two parallel tangents.

[0044] For purposes of the present invention, "variable thickness" of a layer means that for a single microcompartment, the layer does not have the same thickness throughout.

[0045] For the purposes of the present invention, "microcompartment" or "capsule" means a partially or completely enclosed three-dimensional structure that contains several cells.

[0046] In the sense of the present invention, "medium" means an aqueous solution containing cells, suitable for the survival, development and / or metabolism of the cells. It may be a culture medium.

[0047] For purposes of the present invention, "convective culture medium" means a culture medium that is agitated by internal movement.

[0048] For the purposes of the present invention, the term "mutation" refers to a genetic or epigenetic mutation, preferentially a functional mutation, which may in particular involve a point modification of the gene sequence, a structural variant, an epigenetic modification or a modification of the mitochondrial DNA.

[0049] The term "functional mutation" in the sense of the present invention refers to a transmissible genetic or epigenetic modification that confers a potential gain or loss of function or a potential loss of function to the relevant mutant cells. It preferably involves a mutation that causes a modification of the phenotype of the affected mutant cells. Very preferentially, it involves a change in the genomic and / or epigenetic sequence that alters the therapeutic potential of the collection of cells or by increasing the risk associated with the therapy produced or by reducing the benefit provided by the therapy produced.

[0050] For the purposes of the present invention, the "smallest dimension" of a microcompartment or cell layer refers to the value of the smallest Feret diameter of said microcompartment. For the purposes of the present invention, the "lumen" refers to the volume of aqueous solution morphologically surrounded by the cells. Preferably, its contents are not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0051] The "minimum radius" of the inner part of the microcompartment according to the present invention means half the value of the minimum Feret diameter of the inner part of the microcompartment.

[0052] The "average radius" of the interior portion of a microcompartment according to the invention means the average of the radii of the smallest compartments, each radius corresponding to half the value of the Feret diameter of the interior portion of the microcompartment.

[0053] "Growth rate at day X" according to the present invention means a measurement of cell proliferation at time t=X. The proliferation rate is measured by taking the ratio of the number of cells counted at day X of culture divided by the number of cells at the start of culture (day of encapsulation or day placed in culture).

[0054] According to the present invention, "large-scale culture" means a cell culture method suitable for lymphopoiesis batches of liver microtissues that allow the treatment of at least one patient, preferably 10 patients, more preferably 100 patients, and even more preferably more than 1,000 patients.

[0055] A "functional phenotype" according to the present invention refers to lymphocytes that have the conventional membrane marker expression profile known to those skilled in the art (CD3+, CD4+ or CD8+ for T lymphocytes and CD3-, CD56+ for NK lymphocytes) and have the ability to secrete interleukins (IL2, IL6, IL10, IL12) or other factors of interest (Granzyme B, INFγ, GMCSF, and TNFα) upon induction of antigen presentation in vitro.

[0056] For the purposes of the present invention, "progenitor cells" refers to stem cells that are already committed to lymphoid cell differentiation but have not yet differentiated.

[0057] For the purposes of the present invention, "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4, NANOG, and SOX2, and surface markers such as SSEA3 / 4, Tra-1-60, and Tra-1-81. Embryonic stem cells used in the context of the present invention can be obtained without destroying the embryo from which they are derived, for example, using the techniques described in Chang et al. (Cell Stem Cell, 2008, 2(2):113-117).

[0058] Optionally, embryonic stem cells from humans can be excluded.

[0059] For the purposes of the present invention, "pluripotent stem cells" or "pluripotent cells" refers to cells that have the potential to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Human pluripotent stem cells may be referred to as hPSCs in this application. These may in particular be induced pluripotent stem cells (iPSCs, or in the case of human induced pluripotent stem cells, hiPSCs), embryonic stem cells, or MUSE cells (for "multi-lineage differentiation stress resistance").

[0060] For the purposes of the present invention, "induced pluripotent stem cells" refers to pluripotent stem cells that have been induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are positive for pluripotency markers, such as, inter alia, staining with alkaline phosphatase and expression of the proteins NANOG, SOX2, OCT4, and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by 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).

[0061] Cellular microcompartments An object of the present invention is therefore a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part comprising 500 to 5000 lymphocytes forming a grouped three-dimensional culture in a medium, and having a TNFα content of less than 100 ng / mL of medium.

[0062] Preferably, the inner part of the microcompartment contains a TNFα content of less than 50 ng / mL of medium, in particular less than 30 ng / mL of medium, more preferably less than 10 ng / mL of medium, even more preferably less than 3 ng / mL of medium. The microcompartment therefore contains at least lymphocytes which secrete little or no TNFα.

[0063] In the context of the present invention, the content of molecules in the medium inside the microcompartment (in particular cytokines such as TNF, granzyme B, perforin, etc.) can be measured directly in the capsule or, if the microcompartment is in the medium, in the medium outside the microcompartment. Indeed, molecules smaller than 150 KDa are in osmotic equilibrium between the inner part of the microcompartment and the medium in which the microcompartment is located.

[0064] According to a preferred embodiment, the inner part of the microcompartment contains a perforin content of less than 0.5 μg / mL of medium, preferably less than 0.1 μg / mL of medium, more preferably less than 50 ng / mL of medium, even more preferably less than 10 ng / mL of medium.

[0065] A further object of the present invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, having a smallest dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part comprising 500 to 5000 lymphocytes forming a grouped three-dimensional culture in a medium, and having a perforin content of less than 0.5 μg / mL of medium, preferably less than 0.1 μg / mL of medium, more preferably less than 50 ng / mL of medium, even more preferably less than 10 ng / mL of medium.

[0066] According to a preferred embodiment, the inner part of the microcompartment comprises a content of Granzyme B of less than 1 μg / mL of medium, in particular less than 0.5 μg / mL of medium, more preferably less than 0.1 μg / mL of medium, even more preferably less than 50 ng / mL of medium. The microcompartment therefore comprises at least lymphocytes which secrete little or no Granzyme B.

[0067] A further object of the present invention is therefore a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a smallest dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an internal part, said internal part comprising 500 to 5000 lymphocytes forming a grouped three-dimensional culture in a medium, with a granzyme B content of less than 1 μg / mL of medium. According to a preferred embodiment, when the lymphocytes are NK lymphocytes, the internal part of the microcompartment comprises a content of IL6 of less than 0.5 μg / mL of medium, preferably less than 0.1 μg / mL of medium, more preferably less than 50 ng / mL of medium, even more preferably less than 10 ng / mL of medium.

[0068] A further object of the present invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a smallest dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part comprising in a medium between 500 and 5000 NK lymphocytes forming a grouped three-dimensional culture, and having an IL6 content of less than 0.5 μg / mL of medium, preferably less than 0.1 μg / mL of medium, more preferably less than 50 ng / mL of medium, even more preferably less than 10 ng / mL of medium.

[0069] According to a preferred embodiment, when the lymphocytes are NK lymphocytes, the inner part of the microcompartment contains a GM-CSF content of less than 0.5 μg / mL of medium, preferably less than 0.1 μg / mL of medium, more preferably less than 50 ng / mL of medium, even more preferably less than 10 ng / mL of medium.

[0070] A further object of the present invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part comprising in a medium between 500 and 5000 NK lymphocytes forming a grouped three-dimensional culture, and a GM-CSF content of less than 0.5 μg / mL of medium, preferably less than 0.1 μg / mL of medium, more preferably less than 50 ng / mL of medium, even more preferably less than 10 ng / mL of medium.

[0071] According to one embodiment, the object of the present invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part forming a clustered three-dimensional culture in a medium, with a content of less than 100 ng / mL of medium, and - A perforin content of less than 0.5 μg / mL of medium, and / or - 500-5000 T lymphocytes with a granzyme B content of less than 1 µg / mL of medium.

[0072] According to one embodiment, the object of the present invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension between 200 and 400 μm, comprising an outer hydrogel layer defining an inner part, said inner part forming a clustered three-dimensional culture in a medium, a TNF-α content of less than 100 ng / mL of medium, and - A perforin content of less than 0.5 μg / mL of medium, and / or - A granzyme B content of less than 1 μg / mL of medium, and / or - a perforin-IL6 content of less than 0.5 μg / mL of medium, and / or - Contains 500-5000 NK lymphocytes with a GM-CSF content of less than 0.5 µg / mL of medium.

[0073] According to the invention, surprisingly, a TNFα content of the culture medium in the inner part of the microcompartment of less than 100 ng / mL, in particular less than 30 ng / mL, preferably less than 50 ng / mL, more preferably less than 10 μg / mL and even more preferably less than 3 ng / mL is a criterion that guarantees a functional phenotype of lymphocytes in the inner part of the microcompartment according to the invention.

[0074] Tumor necrosis factor (TNFα) is a pleiotropic cytokine involved in numerous physiological processes controlling inflammation, tumor response, and immune system homeostasis. TNFα is secreted by lymphocytes or other cells (especially activated macrophages) and can act directly on cells infected with certain pathogens or promote lymphocyte proliferation. Thus, TNFα can promote the activation and proliferation of naive and effector T cells, but can also induce apoptosis of activated effector T cells. TNFα is stored in intracellular secretory vesicles. These vesicles are typically released into the immediate environment of the target cells.

[0075] In conventional culture methods, lymphocytes are subjected to various physical constraints and / or stresses that tend to induce the release of interleukins, especially TNFα. This uncontrolled release of TNFα can change the immediate environment of lymphocytes and significantly reduce their viability by inducing apoptosis in some of them. On the other hand, the remaining lymphocytes no longer show a functional phenotype. Indeed, lymphocytes lose the ability to secrete interleukins under the induction of antigen presentation, thus affecting their ability to act on target antigens.

[0076] Advantageously, the microcompartments according to the invention make it possible to obtain lymphocytes with a functional phenotype.

[0077] Contrary to what is suggested in the prior art, a TNFα content of less than 100 ng / mL of medium, in particular less than 30 ng / mL of medium, preferably less than 50 ng / mL of medium, more preferably less than 10 μg / mL of medium, even more preferably less than 3 ng / mL of medium is particularly required in lymphocyte cultures.

[0078] TNFα concentrations above 100 ng / mL of medium and / or perforin concentrations above 0.5 μg / mL and / or granzyme B concentrations above 1 μg / mL indicate that an uncontrolled process of interleukin release is occurring within the microcompartments according to the invention, making them incompatible with large-scale culture of lymphocytes with a functional phenotype.

[0079] The same applies to NK lymphocytes with IL6 concentrations above 0.5 μg / mL and / or GM-CSF concentrations above 0.5 μg / mL. IL6 in particular is known to reduce the cytotoxicity of NK cells (Cifadi et al., "Inhibition of natural killer cell cytotoxicity by interleukin-6: implications for the pathogenesis of macrophage activation syndrome," Artritis Rheumatol, 2015 Nov; 67(II): 3037-46. doi: 10.1002 / art.39295.). Thus, reducing IL6 concentrations has the advantage of increasing NK efficacy.

[0080] Advantageously, the microcompartments according to the invention are suitable for large-scale cultivation.

[0081] The microcompartments according to the invention advantageously have at least a 20-fold increase in T lymphocyte proliferation rate after 3 days in culture and at least a 40-fold increase in NK lymphocyte proliferation rate after 14 days in culture.

[0082] The microcompartments according to the invention comprise an outer hydrogel layer. Preferably, the hydrogel used is biocompatible, i.e. non-toxic to the cells. The outer hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained within the microcompartments and allow them to survive. According to one embodiment, the outer hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be sodium alginate, composed of 80% α-L-guluronate and 20% α-D-mannuronate, with an average molecular weight of 100-400 kDa and a total concentration of 0.5-5% by weight. The outer hydrogel layer is cell-free.

[0083] The outer hydrogel layer in particular makes it possible to protect the cells from the mechanical stresses of the bioreactor and to limit the uncontrolled secretion by lymphocytes in the culture of cytokines that are potentially toxic if they accumulate in the medium.

[0084] The average outer layer thickness can be variable. It is preferably between 20 and 60 μm, more preferably between 30 and 40 μm. The ratio between the minimum radius of the inner part and this thickness is preferably between 2 and 10 μm.

[0085] The microcompartments according to the invention can be obtained after encapsulation of lymphocytes without the addition of any extracellular, natural or synthetic matrix.

[0086] According to one variant, the microcompartments according to the invention do not comprise in their internal part extracellular matrices such as Matrigel® and / or Geltrex®, and / or hydrogel-type matrices of plant or synthetic origin such as modified alginates, or copolymers of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) of the Mebiol® type.

[0087] According to one variant, the microcompartments according to the invention can be obtained after encapsulation of lymphocytes without the addition of extracellular matrix, which can be peptide or peptidomimetic sequences, mixtures of proteins, extracellular compounds or structural proteins, such as collagens, laminins, entactins, vitronectins, as well as growth factors or cytokines.

[0088] The absence of extracellular matrix elements in the interior part of the microcompartments according to the invention after encapsulation ensures freedom of structural organization of lymphocytes within the microcompartments according to the invention.

[0089] Advantageously, the absence of extracellular matrix elements improves lymphocyte proliferation rates.

[0090] Within the microcompartments, lymphocytes group together in three dimensions, preferably in clusters.

[0091] Preferably, the microcompartments according to the present invention contain lymphocytes that form grouped three-dimensional aluminal cultures.

[0092] The absence of a lumen advantageously limits autocrine and paracrine signaling of lymphocytes, making their phenotype particularly stable following encapsulation.

[0093] Advantageously, the absence of a lumen also makes it easier to control the size of the lymphocyte clusters present within the microcompartments.

[0094] The microcompartments according to the invention can be obtained after encapsulation of previously activated lymphocytes, thereby increasing their proliferation rate by stimulating their proliferation. Activation can be achieved by contacting the lymphocytes with artificial antigen presenting cells (aAPC), most preferably via CD3 / CD28 antibodies for T lymphocytes or via CD2 / NKp46 antibodies for NK lymphocytes.

[0095] If the lymphocytes have been previously activated, in order to maintain their activation, the inner part of the microcompartments according to the invention can contain a solution comprising a cytokine, in particular IL2 or a combination of IL7 and IL15 for T lymphocytes, and in particular IL2, IL12, IL18, IL21 for NK (preferably IL2 or IL12 or IL18 or IL21, or a combination of at least two cytokines selected from IL2, IL12, IL18 and IL21).

[0096] Cytokines in the inner part of the microcompartment promote lymphocyte proliferation.

[0097] In another embodiment, the microcompartments contain lymphocytes that are activated during and / or after encapsulation. In this embodiment, the lymphocytes may be co-encapsulated with artificial antigen presenting cells, such as activation beads coated with a suitable antibody (e.g., Cloudz Human NK Cell Expansion Kit (R&D Systems) or NK Cell Activation / Expansion Kit (Miltenyi Biotec)).

[0098] Preferably, in this embodiment, lymphocyte activation occurs within the microcompartment. Preferably, lymphocyte activation within the microcompartment is achieved at a lymphocyte / antigen mass ratio of greater than 8:1, preferably greater than 9:1, even more preferably greater than 10:1.

[0099] Advantageously, activation is performed in microcompartments by controlling the ratio of lymphocyte number to the amount of antigen required, so as to activate lymphocytes while limiting cell depletion.

[0100] The microcompartments of the present invention isolate lymphocytes and prevent cell mixing or convection. This mechanical isolation makes it possible to approximate the spatial and structural organization of lymphocytes in vivo.

[0101] Due to the mechanical isolation provided by the microcompartments, lymphocyte activation cannot be uniform or continuous, thus approximating the conditions of lymphocyte activation in vivo, favoring their polarization.

[0102] Preferably, the lymphocytes forming the clusters are polarized. The polarity of these lymphocytes within each cluster can be demonstrated by the uneven distribution of the nuclei within the cells. Polarized lymphocytes can also show anisotropy in the distribution of Dig, Scrib and Lgl proteins.

[0103] Polarization of lymphocytes in microcompartments can be an indicator of their functional phenotype: indeed, lymphocytes are polarized if they contain sufficient amounts of granules and vesicles that contain interleukins, especially TNFα.

[0104] Unexpectedly, the microcompartments of the invention preserve the clustered conformation of lymphocytes, allowing for better proliferation while maintaining a functional phenotype, which in turn allows for a reduction in the number of passagings and the culture time required to reach the required final number of lymphocytes.

[0105] The microcompartments can also contain cells in suspension within the microcompartments in addition to clusters.

[0106] The microcompartments according to the invention preferably contain naive and / or Tscm lymphocytes at a concentration of more than 20%, even more preferably more than 40%.

[0107] The concentration of naive and / or Tscm lymphocytes ensures a sustained effect during cell therapy. It is particularly important to obtain a naive and / or Tscm lymphocyte content of more than 20% in culture in order to obtain an optimal ratio between the duration of effect and the efficacy in vivo.

[0108] Naïve and / or Tscm lymphocyte concentrations below 20% indicate that lymphocyte life span is too short and do not guarantee a sustained effect.

[0109] Naïve and Tscm lymphocytes are immature lymphocytes that display the CD3+ CD45RO- CD45RA+ CCR7+ markers, which can be easily identified and quantified by detection methods well known to those skilled in the art, such as flow cytometry.

[0110] According to a particular embodiment, the present invention provides a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroidal or spherical shape, or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension of 200-400 μm, comprising an outer hydrogel layer defining an inner portion, said inner portion forming a clustered three-dimensional culture of lymphocytes in a medium, - a TNFα content of less than 100 ng / mL of medium, and / or - A granzyme B content of less than 1 μg / mL of medium, and / or -Has a perforin content of less than 0.5 µg / mL of medium.

[0111] In this embodiment, when the lymphocytes are T lymphocytes, the proportion of naive T lymphocytes and / or Tscm lymphocytes remains above 20%.

[0112] Preferably, the microcompartments are obtained after encapsulation of the lymphocytes without the addition of extracellular matrix and / or without the addition of extracellular matrix elements.

[0113] The lymphocytes present in the microcompartments according to the invention are preferably obtained after at least two cell division cycles after encapsulation of at least one lymphocyte in the outer hydrogel layer.

[0114] Preferably, the lymphocytes present in the microcompartments 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 cell division cycles after encapsulation in the outer hydrogel layer, preferably at least 5, even more preferably at least 6, in particular 5-100 lymphocytes per encapsulated lymphocyte. For example, the lymphocytes present in the microcompartments have been obtained after at least 6 cell division cycles after encapsulation of the cells in the outer hydrogel layer.

[0115] Preferably, the number of cell divisions for carrying out the method according to the invention is less than 100, even more preferably less than 30.

[0116] Preferably, the microcompartments are obtained after at least two passages after encapsulation, more preferably after at least 3, 4 or 5 passages, each passage lasting for example 2 to 10 days, in particular 2 to 4 days.

[0117] Preferably, the microcompartments are obtained after at least one reencapsulation, more preferably after 1 to 14 reencapsulations, in particular after 2 to 7 reencapsulations. Very preferably, the reencapsulation corresponds to a new passage, each encapsulation cycle corresponding to a passage.

[0118] Preferably, the microcompartments according to the invention are obtained less than 6 days after encapsulation, even more preferably less than 4 days after encapsulation of at least 5 lymphocytes in the internal portion defined by the outer hydrogel layer.

[0119] A microcompartment according to the invention may contain between 500 and 5000 lymphocytes, preferably between 1000 and 3000 lymphocytes.

[0120] Advantageously, the microcompartments according to the invention protect the lymphocytes from mechanical stresses and therefore make it possible to obtain proliferation rates that are particularly suitable for large-scale culture.

[0121] The microcompartments according to the invention may be of any three-dimensional form, i.e., they may have the shape of any object in space. They may have any form that is compatible with cell encapsulation. Preferably, the microcompartments according to the invention are spherical or elongated or substantially spherical or elongated in shape. The microcompartments according to the invention may have an ovoid, cylindrical, spheroidal or spherical shape or substantially this shape.

[0122] It is the outer layer of the microcompartment, i.e. the hydrogel layer, that gives the microcompartment according to the invention its size and shape. Preferably, the smallest dimension of the microcompartment according to the invention is between 200 μm and 400 μm, preferably between 200 μm and 350 μm, even more preferably between 200 μm and 300 μm, in particular between 200 μm and 250 μm.

[0123] Its maximum dimension is preferably greater than 10 μm, more preferably between 10 μm and 1 m, and even more preferably between 202 μm and 50 cm.

[0124] According to a particularly preferred embodiment, the lymphocytes encapsulated in the microcompartments according to the invention are T lymphocytes or NK lymphocytes.

[0125] Preferably, the lymphocytes contained in the microcompartments according to the invention express a chimeric antigen receptor.

[0126] Therefore, the microcompartments according to the invention are particularly suitable for large-scale lymphocyte culture for CAR-T or CAR-NK cell therapy.

[0127] According to one embodiment, the microcompartments according to the invention do not comprise and / or are not derived from human embryonic stem cells.

[0128] Microcompartments according to the invention may optionally be frozen for storage, after which they must preferably be thawed before use.

[0129] The present invention also relates to the assembly of multiple microcompartments.

[0130] The present invention also relates to an assembly or series of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one of the microcompartments is a microcompartment according to the invention.

[0131] Preferably, the series of microcompartments according to the invention are in a culture medium, in particular in an at least partially convective culture medium. Any culture medium suitable for lymphocyte culture can be used, such as, for example, "TexMACS (Miltenyi Biotec)" or "VIVO 15-20 (Lonza)" or "CTS™ OpTmizer™ (ThermoFisher)" or NKMACS (Miltenyi Biotec) or ExCellerate human NK cell growth medium (R&D Systems), provided that the concentration of dissolved salts is compatible with maintaining alginate cross-linking by divalent cations.

[0132] According to a particularly preferred embodiment, the subject of the present invention is a series of cell microcompartments as described above in a closed chamber, e.g. a bioreactor, preferentially in a culture medium in a closed chamber, e.g. a bioreactor. Thus, preferably, the microcompartments are arranged in a culture medium in a closed bioreactor.

[0133] The assembly or series of microcompartments according to the invention preferably comprises 2 to 10 16 It contains micro compartments.

[0134] The microcompartments according to the invention can be used for all applications, in particular as drugs in human or animal cell therapy.

[0135] Preferably, the microcompartments according to the invention may be for use in the prevention or treatment of autoimmune diseases, immune deficiency syndromes, cancer, viral diseases or inflammatory diseases.

[0136] Thus, the microcompartments according to the invention are suitable for large-scale culture and provide lymphocytes with a functional phenotype that ensures optimal efficacy and persistence in vivo.

[0137] Method for preparing microcompartments according to the invention The present invention also relates to a method for preparing the microcompartments according to the invention.

[0138] The microcompartments according to the invention may be obtained from lymphocytes or cells capable of differentiating into lymphocytes (stem cells, in particular induced pluripotent stem cells, hematopoietic stem cells including CD34+ cells, etc.).

[0139] In a first embodiment, the microcompartments according to the invention are obtained from lymphocytes.

[0140] The method for preparing a microcompartment or an assembly of microcompartments according to the invention comprises the following steps: -(a) incubating lymphocytes in a culture medium containing cytokines; - (b) encapsulating the lymphocytes washed in their culture medium in a hydrogel layer, preferably without the addition of extracellular matrix or without the addition of extracellular matrix elements, to form closed three-dimensional microcompartments of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension of 200 to 400 μm, so that each microcompartment contains at least 5 lymphocytes in its interior part, and - (c) culturing the microcompartments obtained in step (b) for 2 to 6 days in a culture medium containing cytokines.

[0141] The incubation step (a) of the method according to the invention comprises between 500,000 and 2,000,000 lymphocytes per mL of culture medium, preferably 1,000,000 lymphocytes per mL of culture medium.

[0142] The lymphocytes in step (a) are preferably T lymphocytes and / or NK lymphocytes.

[0143] Preferably, the preparation method according to the invention comprises, prior to lymphocyte incubation, a step consisting of activating said lymphocytes using an artificial activation system (aAPC) for 3 days prior to encapsulation.

[0144] In a particular embodiment, lymphocyte activation is carried out using an artificial activation system (aAPC) in the microcompartments obtained from step (b). Any culture medium suitable for culturing lymphocytes, in particular T lymphocytes or NK lymphocytes, can be used, such as, for example, "TexMACS (Miltenyi Biotec)" or "VIVO 15-20 (Lonza)" or "CTS™ OpTmizer™ (ThermoFisher)" medium. Each microcompartment obtained in step (b) comprises at least 500,000 cells per mL of the volume of the inner part of the microcompartment, more preferably at least 1,000,000 cells / mL, in particular at least 2,000,000 cells / mL, even more preferably 5,000,000 cells / mL.

[0145] Preferably, each microcompartment obtained in step (b) comprises in its interior portion at least 5 lymphocytes, preferably at least 8 lymphocytes.

[0146] The cytokines included in the culture medium may be one or more cytokines, such as, for example, IL-2, or a mixture of IL-7 and IL-15, or IL-17 and / or IL-18 and / or IL-21, or other cytokines known to those skilled in the art to stimulate lymphocyte proliferation.

[0147] Preferably, the culture medium used in the preparation method of the present invention for T lymphocytes is supplemented with 100 to 1000 units / mL of IL2, or a combination of 300 to 600 units / mL of IL7 and 50 to 100 units / mL of IL15.

[0148] In the method according to the invention, all of the lymphocytes initially encapsulated in step (b) occupy 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.

[0149] The encapsulated lymphocytes are suspended in the form of single cells and / or clusters or assemblies of at least two cells ("clusters" / groups). Preferably, when at least 15 lymphocytes are present, single cells represent less than 50% of the total number of cells initially encapsulated in step (b).

[0150] Preferably, each lymphocyte cluster initially encapsulated in step (b) has a larger dimension that is less than 20% and even more preferentially less than 10% of the maximum dimension of the microcompartment in which it is encapsulated. In fact, the lymphocyte clusters must not have a size that is too large compared to the size of the microcompartment, because the dimensions of these initial cell clusters that are too large may lead to premature cell confluence in the capsule during cell division. This premature confluence of all or part of the capsule may cause an increase in intracellular pressure, resulting in cell stress that affects not only cell proliferation but also chromosome segregation.

[0151] The encapsulation step (b) is carried out according to techniques known to those skilled in the art. In fact, any method of producing hydrogel capsules and cellular microcompartments containing cells inside can be used to carry out the preparation method according to the present invention. In particular, it is possible to prepare the microcompartments by adapting the method and microfluidic device described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells hNSC", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604) according to the steps described below.

[0152] Preferably, step (b) is carried out without the addition of any extracellular matrix, either natural or synthetic. According to one variant, step (b) is carried out without the addition of extracellular matrix elements.

[0153] Preferably, step (b) is carried out in a device capable of producing hydrogel capsules using a microfluidic chip. For example, the device may include syringe pumps for several solutions that are injected concentrically by a microfluidic injector, making it possible to form a jet that breaks into droplets, which are then collected in a calcium bath. According to a particularly preferred embodiment, the following two or three solutions are loaded into two or three syringe pumps: - hydrogel solutions, e.g. alginate, Optionally, an isotonic intermediate solution, preferably one that does not contain divalent cations such as Ca2+ Mg2+, e.g. a sorbitol solution, to avoid excessively premature crosslinking of the hydrogel in the syringe; - the solution from step (a) comprising lymphocytes, culture medium.

[0154] The three solutions are co-injected concentrically by a microfluidic injector or microfluidic chip that allows forming a jet that breaks into droplets, the outer layer of which is the hydrogel solution and the core of which is the solution from step (a) containing the lymphocytes; these droplets are collected in a calcium bath that crosslinks and / or gels the alginate solution to form a shell.

[0155] To improve the monodispersity of the cell microcompartments, the hydrogel solution is preferably charged with direct current (1-10 kV). A ring to ground may be optionally placed at a distance of 1 mm to 20 cm from the tip, preferably 3 mm to 10 cm, even more preferably 1 cm to 5 cm from the tip, in a plane perpendicular to the axis of the jet emerging from the microfluidic injector (coextrusion tip) to generate an electric field.

[0156] According to the invention, it is necessary to produce capsules whose inner part has an average radius of at least 100 μm or less. To produce capsules with such dimensions using a coextrusion tip (microfluidic injector or microfluidic chip), the invention proposes in particular to modify the flow rate of the coextrusion solution and the final opening of the coextrusion tip. "Flow rate" means the flow rate of each solution that reaches the injector. "Final opening of the coextrusion tip" means the inner opening of the exit channel of the tip. - Regarding the flow rates: the method preferably goes from values ​​of 20-40 mL / h up to values ​​of 45-150 mL / h, in the variants of the invention preferably from 45-110 mL per hour, for each coextrusion solution of standard capsule sizes known from the prior art. - for the final opening diameter of the coextrusion tip (microfluidic injector), for standard capsule sizes known from the prior art, preferably from values ​​of 50-120 μm up to diameter values ​​of 150-300 μm, preferably from diameter values ​​of 180-240 μm.

[0157] Thus, according to a particular embodiment, the encapsulation step (c) is carried out using a microfluidic injector, the final opening diameter of which is between 150 and 300 μm, preferably between 180 and 240 μm, and the flow rate of each of the three solutions is between 45 and 150 mL / h, preferably between 45 and 110 mL / h.

[0158] According to one embodiment, steps (a) and / or (c) are carried out with continuous or sequential stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of diffusion gradients. For example, it allows uniform control of the cellular oxygenation level, thus avoiding the phenomenon of necrosis associated with hypoxia or oxidative stress associated with hyperoxia.

[0159] The method according to the invention is preferably carried out in a closed chamber, such as a closed bioreactor.

[0160] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of lymphocytes after step (c), i.e. at least two encapsulation cycles.Preferably, each encapsulation cycle corresponds to a passage.In this variant of the method (at least one re-encapsulation of cells after step (e)), the number of cell divisions throughout the method (for all passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0161] In the method according to the invention there may be several reencapsulations, preferably 1 to 100, in particular 1 to 10 reencapsulations.

[0162] According to a particular embodiment, a lymphocyte activation step using an artificial activation system (aAPC) can be carried out in the microcompartments after at least one reencapsulation.

[0163] Each reencapsulation is - a step consisting of dissociating the microcompartment or a series of microcompartments in order to obtain a suspension of lymphocytes or a suspension of lymphocyte clusters. The outer hydrogel layer can be removed in particular by hydrolysis, dissolution, perforation and / or destruction by any biocompatible means, i.e. means that are not toxic to cells. For example, removal can be achieved using phosphate buffered saline, divalent ion chelators, enzymes such as alginate lyase if the hydrogel comprises alginate, and / or laser microdissection, and - a lymphocyte activation step using artificial activation systems (aAPC), preferably using CD3 / CD28 antibodies; This can be achieved using a process of reencapsulation of all or part of the lymphocytes or lymphocyte clusters into hydrogel capsules, which is a suitable means to increase the cell expansion resulting from the pluripotency process and to reduce the risk of mutations.

[0164] Reencapsulation involves removing the outer layer of the hydrogel by resuspending the cells in a partially or fully dissociated manner, preferably in the form of cysts within the microcompartments, and resuming the steps of the method.

[0165] According to one embodiment, the reencapsulation comprises the following steps: -(i) removing the outer hydrogel layer; -(ii) resuspending the lymphocytes contained within the microcompartments to obtain single cells and / or at least one assembly or cluster of lymphocytes in a culture medium containing cytokines; - (iii) encapsulating the solution of lymphocytes in a hydrogel layer to form ovoid, cylindrical, spheroidal or spherical, or substantially ovoid, cylindrical, spheroidal or spherical microcompartments, comprising an outer hydrogel layer defining an interior portion, the minimum or average radius of the interior portion being at least 100 μm; - (iv) culturing the obtained microcompartments in a cytokine-containing culture medium; - (vi) preferably rinsing the microcompartments to remove cytokines; - (vi) culturing the microcompartments in a cytokine-free culture medium for at least one cell division cycle; -(vii) optionally, recovering the resulting cell micro-compartments.

[0166] Compartmentalization into microcompartments allows the elimination of microcompartments that contain more mutant cells than other capsules. Even if mutant cells grow rapidly, they reach intracapsular confluence, limiting their increase. Compartmentalization also allows not to contaminate the entire cell population and allows the elimination of capsules that contain mutant cells at any time, in particular before the reencapsulation step. This selection can be done, for example, by in-line analysis or by eliminating filled capsules more quickly than other capsules.

[0167] In one embodiment, at least one of the steps (preferably all steps) is carried out at a temperature suitable for cell survival, including between 4 and 42° C. The temperature during cell growth should preferably be between 32 and 37° C. to avoid inducing mutations by reducing the performance of repair enzymes. Similarly, the temperature should preferably be low (ideally around 4° C.) to manage stress on the cells in step (c).

[0168] At any point, the method according to the invention may include a step consisting of verifying the phenotype of the cells contained in the microcompartments, which can be carried out by identifying the expression by at least some of the cells contained in the microcompartments of at least four genes specific for the desired phenotype selected from CD45RARO, CCR7, CD62L, CD3, CD4, CD8, CD56, CD16.

[0169] The cell microcompartments obtained according to the method of the invention can then be frozen before any use. Freezing is preferably performed at a temperature between -190°C and -80°C. Thawing can be performed by immersing the sealed freezing container (screwable ampoule or plastic pouch) in a warm water bath (preferably at 37°C), so that the cells are thawed very quickly. The microcompartments according to the invention before use may be kept at a temperature higher than 4°C, preferentially between 4°C and 38°C, for a limited time before use.

[0170] By carrying out the method according to the invention it is possible to obtain microcompartments which contain at least 80, preferably at least 500, at least 800, at least 1,000 and in particular at least 3000 lymphocytes.

[0171] The present invention specifically promotes expansion at high proliferation rates, thereby reducing culture times and the number of divisions required to obtain large numbers of functional lymphocytes.

[0172] According to a second embodiment, the microcompartments according to the invention are obtained from cells capable of differentiating into lymphocytes and comprise at least the implementation of a method for differentiating cells capable of differentiating into lymphocytes (such as stem cells, in particular induced pluripotent stem cells, hematopoietic stem cells including CD34+ cells) into lymphocytes.

[0173] The cells capable of differentiating into lymphocytes are preferably chosen from: * lymphocytes, at least T lymphocytes or NK lymphocytes, preferably embryonic stem cells or induced pluripotent stem cells, or hematopoietic stem cells comprising CD34+ cells, very preferably stem cells capable of differentiating into induced pluripotent stem cells, and / or * or progenitor cells capable of differentiating into lymphocytes, at least T lymphocytes or NK lymphocytes; * or differentiated cells that can undergo reprogramming to become induced pluripotent stem cells capable of differentiating into lymphocytes, at least T lymphocytes or NK lymphocytes.

[0174] In this variant, the method may comprise carrying out at least the following steps: -Inside the hydrogel capsule, * Preferably at least an extracellular matrix element or a natural or synthetic extracellular matrix, * generating cellular microcompartments comprising cells capable of differentiating into lymphocytes, preferably induced pluripotent stem (IPS) cells; Inducing lymphocyte differentiation in the cell microcompartments to obtain at least lymphocytes. EXAMPLES

[0175] Example 1: Method according to the invention (T lymphocytes) Reagents used: - Solution 1: Culture medium used: TexMACS (Miltenyi Biotec) supplemented with 1000 units / mL of recombinant IL-2 (Miltenyi Biotec) - Solution 2: CD3 / CD28 polyantigen activation reagent (Transact™ Miltenyi Biotec) - Solution 3: Capsule rinse medium: DMEM / F-12 + Glutamax - Solution 4: Solution for dissolving alginate capsules: Non-enzymatic cell detachment buffer (RelesR™ Stem Cell Technology).

[0176] Step 1: Cell thawing (day 0) Ampoules of T lymphocytes sorted from healthy donor peripheral blood were thawed according to the supplier's recommendations (Hu PB Pan-T, Stem Cell Technologies, #70024).

[0177] Step 2: T cell activation (day 0) T lymphocytes were counted on a NucleoCounter NC2000 (standard 1 cassette protocol) and cultured in solution 1 at a density of 1,000,000 cells per mL in culture flasks.

[0178] Solution 2 was added 1:100 to the cell suspension thus prepared.

[0179] The treated cells were cultured in a 37°C, 5% CO2 incubator for 3 days.

[0180] Step 3: Medium change and encapsulation (Day 3): To remove Transcart™ from the medium, the cells were centrifuged at 300 g for 10 minutes and the supernatant was aspirated off.

[0181] The cells were rinsed once with solution 1.

[0182] A portion of the cell pellet was taken up in solution 1 at a cell density of 5,000,000 cells / mL for encapsulation.

[0183] The remainder of the cell pellet was taken up in solution 1 at a cell density of 5,000,000 cells per mL (control).

[0184] Step 4: Encapsulation (Day 3) The encapsulation device is prepared as described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604).

[0185] The three required solutions are loaded into three syringe pumps: i) alginate solution (2 wt% PRONOOVA® SLG100 in distilled water), ii) intermediate solution (300 mM sorbitol), and iii) cell solution (prepared in the previous step). The three solutions are co-injected concentrically using a microfluidic injector to form a jet that splits into droplets with the alginate solution as the outer layer and the cell solution as the core. These droplets are collected in a calcium bath (100 mM) where the alginate solution hardens to form a shell.

[0186] The invention in particular promotes high proliferation rates, thereby reducing the culture time and number of divisions required to obtain a large number of functional lymphocytes, thus limiting new mutations.

[0187] A phase contrast microscope image at ×20 magnification of the assembly after 3 days of capsule culture is shown in Figure 1a. A phase contrast microscope image at ×10 magnification after 3 days of capsule culture is shown in Figure 1b.

[0188] Step 5: Post-encapsulation treatment (Day 3) The capsules were collected using a 40 μm cell sieve and then rinsed with solution 3 (1×) before being separated into 75 cm 2 Stock in flasks with solution 1 at a cell density of 100,000 cells per mL (condition 1) or 200,000 cells per mL (condition 2).

[0189] The flasks are kept in an incubator at 37° C. and 5% CO 2 for 3 days.

[0190] The non-encapsulated cells prepared in step 3 are grown in parallel under the same atmospheric conditions for 3 days.

[0191] Step 6: Capsule dissolution and counting (Day 6) -(a): Group 1: Capsule -Capsule suspension was collected and allowed to settle for 5 minutes in a 50 mL Falcon tube. - The supernatant was carefully removed and discarded. The capsules were then treated with solution 4 for 5 minutes at 37°C. - The cells released from the capsules were centrifuged at 300g for 10 minutes to remove solution 4. - Finally, cells were counted in a NC2000 (Standard Protocol 1 cassette). -(b): Group 2: Suspension control - The cell suspension was collected and centrifuged at 300g for 10 minutes. - The supernatant was aspirated. -The cells were then counted in a NC2000 (Standard Protocol 1 cassette).

[0192] The results of T lymphocyte encapsulation using the described protocol show better cell expansion in capsules than in conventional suspension culture. Thus, we obtain a 20-fold expansion rate in capsule culture over 3 days, compared to a 2-fold expansion rate for conventional culture.

[0193] Example 2: Measurement of cytokine secretion from encapsulated and non-encapsulated T lymphocytes The detection kit used was the MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi Biotec, 130-125-800).

[0194] The various reagents and solutions were prepared in MACSPlex Filter Plates according to the supplier's recommendations.

[0195] Culture supernatants from both encapsulated and non-encapsulated cells were harvested after 6 days, undiluted, incubated in duplicate, and incubated in plates prepared in step 2 of Example 1. Incubations were performed according to the supplier's recommendations. Data acquisition was performed on a MACSQuant 10 cytometer (Miltenyi Biotec) using the dedicated Express Mode (Miltenyi Biotec).

[0196] Results are expressed in Table 1 and FIG. 4 as mean fluorescence per 1,000,000 cells (MFI / e6 cells) (results are normalized to GM-CSF and IFNg secretion).

[0197] [Table 1]

[0198] In particular, these results confirm that lymphocytes encapsulated in microcompartments according to the invention have lower concentrations of extracellular granzyme B, perforin and TNFα than lymphocytes in suspension.

[0199] Example 3: Polarization of microcompartments according to the invention Microcompartments according to the invention containing T lymphocytes were fixed in PFA 4% diluted in PBS containing calcium and stored at 4°C.

[0200] Staining protocol: Phalloidin and Lipilight (Membright) staining was performed at the dilutions recommended by the supplier for 72 h under agitation at room temperature, protected from light. Image acquisition (Figure 5) was performed using a confocal microscope.

[0201] It can be seen that the microcompartments according to the invention contain lymphocytes with eccentric nuclei, such that the lymphocytes are polarized within the microcompartment and display a functional phenotype.

[0202] Example 4: Method according to the invention (NK lymphocytes) Reagents used: - Solution 1: basal culture medium for NK expansion (ExCellerate Human NK Cell Expansion Medium (R&D Systems)) supplemented with 27 ng / mL recombinant IL-2, 10 ng / mL recombinant IL-12, 10 ng / mL recombinant IL-18 and 10 ng / mL recombinant IL-21. - Solution 2: Solution 1 containing CD2 / NKp46 multi-antigen activation beads (e.g., Cloudz Human NK Cell Expansion Kit (R&D Systems)) - Solution 3: Capsule rinse medium: DMEM / F-12 + Glutamax - Solution 4: Solution for dissolving alginate capsules: 2.5 mM EDTA solution

[0203] Step 1: Cell thawing (day 0) NK ampoules sorted from peripheral blood of healthy donors (donor A and donor B) were thawed according to the supplier's recommendations (Hu PB NK, Stem Cell Technologies, #70036).

[0204] NK were counted on a NucleoCounter NC2000 (standard 1 cassette protocol).

[0205] The cells were centrifuged at 300 g for 10 min and the supernatant was aspirated.

[0206] A portion of the cell pellet was taken up in solution 2 at a cell density of 15 million cells per mL for encapsulation.

[0207] The other part of the cell pellet was taken up in solution 2 at a cell density of 200,000 to 1,000,000 cells per mL (control).

[0208] Step 2: Encapsulation (Day 0) The encapsulation device is prepared as described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604).

[0209] The three required solutions are loaded into three syringe pumps: i) alginate solution (2 wt% PRONOOVA® SLG100 in distilled water), ii) intermediate solution (300 mM sorbitol), and iii) cell solution (prepared in the previous step). The three solutions are co-injected concentrically using a microfluidic injector to form a jet that splits into droplets with the alginate solution as the outer layer and the cell solution as the core. These droplets are collected in a calcium bath (100 mM) where the alginate solution hardens to form a shell.

[0210] The invention in particular promotes high proliferation rates, thereby reducing the culture time and number of divisions required to obtain a large number of functional lymphocytes, thus limiting new mutations.

[0211] Step 3: Post-encapsulation treatment (day 0) Capsules were collected using a 40 μm cell sieve and then rinsed with solution 3 (1×) before being soaked in 75 cm 2 PBS at a cell density of 200,000–1,000,000 cells per mL. 2 (Donor A) or in a 30 mL mini-bioreactor with solution 1 (Donor B).

[0212] Incubate the 30 mL flasks or mini-bioreactors at 37 °C and 5% CO2 for 11-14 days. Agitate the mini-bioreactor capsules at 100 RPM. Refresh the culture medium according to the supplier's recommendations.

[0213] Grow the non-encapsulated cells prepared in step 3 in parallel under the same atmospheric conditions for 11-14 days.

[0214] Step 6: Capsule dissolution and counting (day 11 or 14, depending on capsule confluence). -(a): Group 1: Capsule -Capsule suspension was collected and allowed to settle for 5 minutes in a 50 mL Falcon tube. - The supernatant was carefully removed and discarded. The capsules were then treated with solution 4 for 5 minutes at 37°C. - The cells released from the capsules were centrifuged at 300g for 10 minutes to remove solution 4. - Activated beads are disposed of according to the supplier's recommendations. - Finally, cells were counted in a NC2000 (Standard Protocol 1 cassette). -(b): Group 2: Suspension control - The cell suspension was collected and centrifuged at 300g for 10 minutes. - The supernatant was aspirated. - Activated beads are disposed of according to the supplier's recommendations. -The cells were then counted in a NC2000 (Standard Protocol 1 cassette).

[0215] The results of NK encapsulation according to the protocol illustrate a better cell expansion in capsules compared to conventional suspension culture, as shown in Figure 6. Thus, for donor A, we obtain a 40-fold expansion rate in encapsulated culture for 14 days, compared to a 3-fold expansion rate for conventional culture. For donor B, we obtain a 110-fold expansion rate in encapsulated culture for 11 days, compared to a 70-fold expansion rate for conventional culture.

[0216] Figure 10 shows that the viability of NK obtained according to the present invention is better than that of NK cultured in 2D. For donor A, the viability at day 14 was 74% for encapsulated cultures versus 64% for suspension cultures. For donor B, the viability at day 11 was 88% in encapsulated cultures versus 62% in suspension.

[0217] Example 5: Measurement of cytokine secretion from encapsulated and non-encapsulated NK lymphocytes The detection kit used was the MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi Biotec, 130-125-800).

[0218] The various reagents and solutions were prepared in MACSPlex Filter Plates according to the supplier's recommendations.

[0219] Culture supernatants from encapsulated or non-encapsulated cells were collected after 14 days, diluted 10-fold or 100-fold, and incubated in duplicate in plates prepared in step 2 of Example 4. Incubations were performed according to the supplier's recommendations. Data acquisition was performed on a MACSQuant 10 cytometer (Miltenyi Biotec) using the dedicated Express Mode (Miltenyi Biotec).

[0220] Results are expressed as mean fluorescence per 1,000,000 cells (MFI / e6 cells) in Tables 2 and 3 and Figures 7a, 7b (donor A) and 8a, 8b (donor B).

[0221] [Table 2]

[0222] [Table 3]

[0223] In particular, these results confirm that NK lymphocytes encapsulated in microcompartments according to the invention have lower concentrations of granzyme B, perforin, GM-CSF and IL-6 than NK lymphocytes in suspension.

[0224] Example 6: Measurement of the functionality of encapsulated and non-encapsulated NK lymphocytes Measurement of K562 by flow cytometry was performed according to the following protocol.

[0225] After decapsulation, NK (B donor NK obtained according to the procedure of Example 4, capsule group in suspension and control group) are stained with PKH-26 probe, then NK are incubated with K562 at 37°C, 5% CO2 for 4 hours at different E:T ratios (effector to target): 12.5:1 / 6.25:1 / 3.125:1 / 1.5:1. Annexin V and 7-AAD staining. Cytometry data analysis strategy was performed as follows. Separation of NK and K562 populations using PKH-26 probe (K562 = negative population): - Viable K562 are 7aad- / Annexin V-negative. - Early apoptotic K562 is 7AAD- / Annexin V+. -Late apoptotic K562 is 7AAD+ / Annexin V+. -Necrotic or dead K562 is 7AAD+ / Annexin V-.

[0226] The percentage of cytotoxicity was calculated as follows: % Cytotoxicity = (Early apoptosis + Late apoptosis + Necrosis) / Total K562

[0227] The results obtained are shown in Figure 9. The NK cytotoxicity obtained according to the invention is similar to that obtained with 2D.

[0228] Example 7 Method according to the invention (NK lymphocytes) Reagents used: - Solution 1: basal culture medium for NK expansion (ExCellerate Human NK Cell Expansion Medium (R&D Systems)) supplemented with 27 ng / mL recombinant IL-2, 10 ng / mL recombinant IL-12, 10 ng / mL recombinant IL-18 and 10 ng / mL recombinant IL-21. - Solution 2: Capsule rinse medium: DMEM / F-12 + Glutamax - Solution 3: Solution for dissolving alginate capsules: 2.5mM EDTA solution

[0229] Step 1: Cell thawing (day 0) NK ampoules sorted from peripheral blood of healthy donors (donor B and donor C) were thawed according to the supplier's recommendations (Hu PB NK, Stem Cell Technologies, #70036).

[0230] NK were counted on a NucleoCounter NC2000 (standard 1 cassette protocol).

[0231] The cells were centrifuged at 300 g for 10 min and the supernatant was aspirated.

[0232] A portion of the cell pellet was taken up in solution 1 at a cell density of 5-15 million cells per mL for encapsulation.

[0233] The remainder of the cell pellet was taken up in solution 1 at a cell density of 200,000 to 1,000,000 cells per mL (control).

[0234] Step 2: Encapsulation (Day 0) The encapsulation device is prepared as described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604).

[0235] The three required solutions are loaded into three syringe pumps: i) alginate solution (2 wt% PRONOOVA® SLG100 in distilled water), ii) intermediate solution (300 mM sorbitol), and iii) cell solution (prepared in the previous step). The three solutions are co-injected concentrically using a microfluidic injector to form a jet that splits into droplets with the alginate solution as the outer layer and the cell solution as the core. These droplets are collected in a calcium bath (100 mM) where the alginate solution hardens to form a shell.

[0236] The invention in particular promotes high proliferation rates, thereby reducing the culture time and number of divisions required to obtain a large number of functional lymphocytes, thus limiting new mutations.

[0237] Step 3: Post-encapsulation treatment (day 0) The capsules are collected using a 40 μm cell sieve and then after rinsing with solution 2 (1×), they are stored in a 30 mL mini-bioreactor with solution 1 at a cell density of 200,000 cells per mL.

[0238] The 30 mL mini-bioreactors are incubated at 37° C. and 5% CO2 for 11 days. The mini-bioreactor capsules are agitated at 100 RPM. The culture medium is refreshed according to the supplier's recommendations.

[0239] The non-encapsulated cells prepared in step 14 are grown in parallel under the same atmospheric conditions for 11 days.

[0240] Step 6: Capsule dissolution and counting (Day 11) -(a): Group 1: Capsule -Capsule suspension was collected and allowed to settle for 5 minutes in a 50 mL Falcon tube. - The supernatant was carefully removed and discarded. The capsules were then treated with solution 3 for 5 minutes at 37°C. - The cells released from the capsules were centrifuged at 300g for 10 minutes to remove solution 3. - Finally, cells were counted in a NC2000 (Standard Protocol 1 cassette). -(b): Group 2: Suspension control - The cell suspension was collected and centrifuged at 300g for 10 minutes. - The supernatant was aspirated. -The cells were then counted (Standard Protocol 1 Cassette).

[0241] A phase contrast microscope image at ×4 magnification of the assembly on the day of encapsulation is shown in Figure 11a, and a phase contrast microscope image at ×10 magnification after 11 days of capsule culture is shown in Figure 11b.

[0242] The results of NK encapsulation according to the protocol demonstrate better cell expansion in capsules compared to conventional suspension culture, as shown in Figure 12a. Thus, for donor B, the expansion rate is 135-fold in capsule culture over 11 days compared to 72-fold for conventional culture. For donor C, the expansion rate is 41-fold in capsule culture over 11 days compared to 28-fold for conventional culture.

[0243] Figure 12b shows that the viability of NK obtained according to the present invention is better than that of NK cultured in 2D. For donor B, the viability at day 11 is 82% for encapsulated cultures versus 69% for suspension cultures. For donor C, the viability at day 11 is 73% in encapsulated cultures versus 51% in suspension.

Claims

1. 1. A closed three-dimensional cellular microcompartment having an ovoid, cylindrical, spheroidal or spherical shape, or substantially ovoid, cylindrical, spheroidal or spherical shape, the closed three-dimensional cellular microcompartment having a minimum dimension of 200-400 μm and comprising an outer hydrogel layer defining an interior portion, the interior portion comprising 500-5000 lymphocytes forming a grouped three-dimensional culture in medium, the TNFα content being less than 100 ng / mL of medium.

2. 2. The microcompartment of claim 1, wherein the inner portion contains lymphocytes that form a grouped three-dimensional culture without a lumen.

3. 3. Microcompartments according to claim 1 or 2, characterized in that they are obtained after encapsulation of lymphocytes without the addition of extracellular matrix or without the addition of extracellular matrix elements.

4. Microcompartments according to any one of claims 1 to 3, characterized in that they are obtained after encapsulation of preactivated lymphocytes.

5. Microcompartments according to any one of claims 1 to 4, characterized in that the thickness of said outer layer is variable and is between 20 and 60 μm.

6. Microcompartments according to any one of claims 1 to 5, characterized in that the lymphocytes are polarized.

7. Microcompartments according to any one of claims 1 to 6, characterized in that the number of lymphocytes forming the grouped three-dimensional culture is between 500 and 5000.

8. Microcompartments according to any one of claims 1 to 7, characterized in that the number of lymphocytes forming the grouped three-dimensional culture is between 1000 and 3000.

9. Microcompartments according to any one of claims 1 to 8, characterized in that the lymphocytes are T lymphocytes or NK lymphocytes.

10. The microcompartment according to any one of claims 1 to 9, characterized in that said lymphocytes are lymphocytes expressing a chimeric antigen receptor.

11. the cell is a T lymphocyte, - a perforin content of less than 0.5 μg / mL of medium, and / or Microcompartments according to any one of claims 1 to 10, characterized in that they have a Granzyme B content of less than -1 μg / mL of medium.

12. the lymphocytes are NK lymphocytes, - a perforin content of less than 0.5 μg / mL of medium, and / or - a granzyme B content of less than 1 μg / mL of medium, and / or - a perforin IL6 content of less than 0.5 μg / mL of medium, and / or Microcompartments according to any one of claims 1 to 10, characterized in that they have a GM-CSF content of less than -0.5 μg / mL of medium.

13. Microcompartment according to any one of claims 1 to 12, characterized in that the outer layer comprises alginate.

14. 14. The microcompartment according to any one of claims 1 to 13, characterized in that the internal part contains a solution comprising IL2, or a combination of IL7 and IL15, or IL12, IL18, IL21, or a combination of at least two cytokines selected from IL2, IL12, IL18 and IL21.

15. Microcompartments according to any one of claims 1 to 14, characterized in that they contain T lymphocytes and that the proportion of naive and / or Tscm T lymphocytes remains above 20%.

16. An assembly of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to any one of claims 1 to 15.

17. Assembly of micro-compartments according to any one of claims 1 to 16, characterized in that said micro-compartments are placed in a culture medium in a bioreactor.

18. A microcompartment according to any one of claims 1 to 15 for use as a medicament.

19. The microcompartment according to any one of claims 1 to 15 for use in the prevention or treatment of an autoimmune disease, an immune deficiency syndrome, cancer, a viral disease or an inflammatory disease.

20. The following steps: -(a) incubating lymphocytes in a culture medium containing cytokines; - (b) encapsulating lymphocytes in their culture medium in a hydrogel layer, preferably without the addition of extracellular matrix, to form closed three-dimensional microcompartments of ovoid, cylindrical, spheroidal or spherical shape or substantially ovoid, cylindrical, spheroidal or spherical shape, with a minimum dimension between 200 and 400 μm, so that each microcompartment contains at least 5 lymphocytes in its interior portion; - (c) culturing the microcompartments obtained in step (b) for 2 to 6 days in a culture medium containing at least cytokines.

21. A method for preparing microcompartments according to any one of claims 1 to 20, characterized in that it comprises, prior to the incubation of the lymphocytes, a step consisting of activating said lymphocytes.

22. 22. Method for preparing microcompartments according to claim 20 or 21, characterized in that the culture medium contains 100-1000 units / ml of IL2 or a combination of 300-600 units / ml of IL7 and 50-100 units / ml of IL15.

23. A method for preparing microcompartments according to any one of claims 20 to 22, characterized in that in step (a) between 500,000 and 2,000,000 lymphocytes are incubated per ml of culture medium.

24. Step b) comprises mixing two solutions: - hydrogel solutions, Method according to any one of claims 20 to 23, characterized in that the inner part of each droplet is performed by co-injection of a solution from step a) comprising lymphocytes and culture medium, concentrically via a microfluidic injector, said microfluidic injector making it possible to form at the outlet of said injector a jet consisting of a mixture of the two solutions, said jet breaking up into droplets, said droplets being collected in a calcium bath that hardens the hydrogel solution forming the outer layer of each micro-compartment, the inner part of each droplet being performed by co-injection of a solution consisting of the solution from step (a) comprising lymphocytes and culture medium.

25. 25. The method of claim 24, wherein the microfluidic injector has a final orifice diameter of 150 to 300 μm.

26. 26. The method according to any one of claims 20 to 25, characterized in that the flow rate of each of the two solutions is between 45 and 150 mL / h.

27. 27. The method according to any one of claims 20 to 26, characterized in that all of the cells initially encapsulated in step (b) occupy a volume less than 50% of the volume of the microcompartment in which they are encapsulated.

28. 28. The method according to any one of claims 20 to 27, characterized in that steps (a) and / or (c) are carried out under continuous or sequential stirring.

29. The method according to any one of claims 20 to 28, characterized in that the method comprises at least one reencapsulation of the cells after step (c).

30. Method according to any one of claims 1 to 29, characterized in that each reencapsulation consists of removing the outer hydrogel layer, preferably resuspending the lymphocytes, partially or completely lysed in the microcompartments, in a partially or completely dissociated manner, and reperforming the steps of the method.

31. A method for preparing micro-compartments according to any one of claims 20 to 30, characterized in that it comprises, after step (c), a step consisting of freezing said micro-compartments.

32. A method for preparing micro-compartments according to any one of claims 20 to 31, characterized in that step (c) is carried out in a bioreactor.

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