Cell microcompartment comprising lymphocytes forming 3D aggregated cultures and having low granzyme b content

Abstract

The present invention relates to an oval, cylindrical, spheroid or spherical shape or substantially oval, cylindrical, spheroid or spherical shape three-dimensional cell microcell or assembly of three-dimensional cell microcells, the minimum size of which is between 200 [mu] m and 400 [mu] m, the three-dimensional cell microcell comprising an outer hydrogel layer defining an inner portion, the inner portion has a granzyme B content of less than 1 [mu] g / ml medium and comprises between 500 and 5000 lymphocytes forming a three-dimensionally aggregated culture. The 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 lymphocyte cell culture, and in particular to a specific cell microcompartment, a preparation method thereof and a use thereof. Background Art

[0002] The progress achieved in recent decades has highlighted the benefits of cell therapy in treating a wide range of pathologies.

[0003] Cell therapy is defined as the administration of living cells to a patient with the goal of preventing, curing or alleviating a disease. The goal is to achieve long-lasting effects from infusions of therapeutic cells.

[0004] A variety of cell therapy approaches are envisioned, such as:

[0005] -Autologous approach, in which the patient is their own donor.

[0006] - Allogeneic approach, where the donor is different from the patient.

[0007] Cell therapy covers a broad range of therapeutic areas and is applicable to a variety of cell types from pluripotent cells to specialized adult cells with metabolic functions (e.g. hepatocytes) or mechanical functions (e.g. myoblasts, chondrocytes) as well as the hematopoietic lineage such as mononuclear blood cells (e.g. T lymphocytes or NK lymphocytes).

[0008] One of the most promising therapies to date is CAR (chimeric antigen receptor) cell therapy. CAR-T cells are T lymphocytes expressing chimeric antigen receptors, which are artificial receptors designed to recognize antigens present on the surface of tumor cells. CAR-T cells guide the immune system to target and eradicate cancer cells. This method is considered a major advance in immunotherapy, and many CAR-T cells are currently being developed to expand their uses. Autologous methods are usually used to produce CAR-T cells. However, it is also possible to form allogeneic CAR-T cells.

[0009] There is also an alternative format using NK (Natural Killer) lymphocytes. NK lymphocytes are highly potent cytotoxic effector cells with proven anti-tumor effects.

[0010] In order to exploit the potential of cell-based therapies using lymphocytes, in particular CAR-T or CAR-NK therapies, it is necessary to be able to rely on large-scale lymphocyte cell culture technologies.

[0011] The in vitro production (especially large-scale production) of immune cells (such as T or NK lymphocytes) is particularly complex. In fact, the in vitro culture methods developed so far are too stressful and rapidly cause cell exhaustion, 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). Since the bioreactor environment is full of non-physiological shear stress, the amplification rate obtained is usually still insufficient compared to the in vivo amplification capacity.

[0012] Currently, there is no satisfactory solution for the rapid large-scale production of functional lymphocytes, especially CAR-T or CAR-NK cells.

[0013] The following culture methods are mainly used for CAR cell expansion: static flask culture, static gas permeable bag culture and rocking motion bioreactor culture (Vormittag P, Gunn R, Ghorashian S, Veraitch FS. Curr Opin Biotechnol. 2018), Platform (Ludwig et al. "Methods and Process Optimization for Large-Scale CAR T Expansion Using the G-Rex Cell Culture Platform" Methods MolBiol 2020; 2086:165-177).

[0014] However, these culture methods are not suitable for large-scale lymphocyte production due to exposure of cells to multiple handling steps increasing the risk of contamination, exposure of cells to excessive stress and / or development and capacity constraints of the platforms and methods used. In addition, the work involved in culture and storage would be too demanding for large-scale production.

[0015] In order to solve this problem, the prior art proposes to activate lymphocytes before culturing them so as to achieve rapid lymphocyte proliferation.

[0016] However, early lymphocyte activation triggers the physiological process of cell exhaustion. Exhaustion corresponds to the early maturation of naive T cells into effector T cell subsets, limiting the persistence of the graft in vivo.

[0017] Another solution proposed by the prior art is to use hydrogels and alginate microtubes suspended in cell culture medium. This method reported a lymphocyte expansion rate of 320 at 14 days in culture (Lin et al. Adv. Healthcare Mater. 2018, 1701297).

[0018] However, although this method provides satisfactory expansion rates, it is not suitable for large-scale culture. This method would require the development of a dedicated culture system and would be too complex to be applicable on a large scale.

[0019] On the other hand, this approach does not guarantee a functional phenotype of the lymphocytes and therefore does not guarantee that the effects of the lymphocytes are long-lasting.

[0020] The complexity of differentiation and function of each lymphocyte subset means that analysis of phenotype during proliferation is essential for the success of any lymphocyte-based therapy, particularly those using CAR-T or CAR-NK cells.

[0021] Less differentiated T-cell subsets such as naive T lymphocytes and Tscm (stem cell-like memory T cells) lymphocytes appear to be particularly important in ensuring long-lasting effects in patients (Vita Golubovskaya and Lijun Wu (Cancers 2016, 8, 36). Moreover, memory-like NK subsets enable the persistence of clinical responses (Romee et al. "Cytokine activation induces human memory-like NK cells" Blood 2012 Dec 6; 120(24): 4751-60).

[0022] On the other hand, we also know that effector T lymphocyte subsets are essential to ensure a significant effect on target cells.

[0023] Therefore, it seems necessary to control cell subpopulations during large-scale lymphocyte culture to obtain the optimal ratio between persistence and in vivo efficacy.

[0024] Current production methods involving lymphocytes are very expensive and difficult to scale up, resulting in lymphocytes of mixed quality and reduced sustainability.

[0025] Therefore, a solution that enables large-scale production of lymphocytes with a functional phenotype is urgently needed to meet the urgent need for lymphocyte-based cell therapies.

[0026] It is therefore an object of the present invention to meet all of these needs and to overcome the shortcomings and limitations of the prior art. Summary of the invention

[0027] To meet this objective, the present invention proposes the use of a specific microcompartment to obtain a large-scale production of lymphocytes suitable for use in cell therapy.

[0028] To this end, the object of the present invention is a closed three-dimensional cell microcompartment of ovoid, cylindrical, spheroid or spherical shape or of substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, comprising an outer hydrogel layer delimiting an inner part, the inner part containing between 500 and 5000 lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a content of granzyme B of less than 1 μg / mL of culture medium. Preferably, the inner part of the microcompartment comprises a content of perforin of less than 0.5 μg / mL of culture medium, and / or a content of endogenous tumor necrosis factor (TNFα) of less than 100 ng / mL of culture medium, and / or a content of IL6 of less than 0.5 μg / mL of culture medium, and / or a content of GM-CSF of less than 0.5 μg / mL of culture medium.

[0029] Advantageously, the microcompartments according to the invention enable the production of lymphocytes with a functional phenotype while ensuring rapid large-scale production.

[0030] According to a particularly suitable embodiment, the microcompartments according to the invention contain lymphocytes forming aggregated three-dimensional cultures without a lumen.

[0031] The microcompartments according to the invention can be obtained after encapsulation of lymphocytes with or without addition of an extracellular matrix and, depending on the variant, with or without addition of extracellular matrix elements.

[0032] Advantageously, the absence of added extracellular matrix or matrix elements during encapsulation of lymphocytes improved their expansion rate after 3 days in culture.

[0033] According to a preferred object of the present invention, the microcompartment according to the present invention has been obtained after encapsulating previously activated lymphocytes. Lymphocyte activation advantageously stimulates lymphocyte proliferation in the inner part of the microcompartment according to the present invention. According to a variant, lymphocyte activation can be performed in the microcompartment after encapsulation.

[0034] According to another particularly suitable embodiment, the microcompartments according to the invention contain lymphocytes expressing a chimeric antigen receptor.

[0035] When the microcompartment according to the invention contains T lymphocytes, it preferably contains a concentration of naive lymphocytes and / or Tscm lymphocytes greater than 20%, even more preferably greater than 40%.

[0036] Advantageously, such high concentrations of naive lymphocytes and / or Tscm lymphocytes ensure a long-lasting effect and avoid the phenomenon of early lymphocyte depletion.

[0037] According to a preferred object of the present invention, the present invention relates to a set of microcompartments.

[0038] According to another aspect, the present invention relates to a method for preparing a microcompartment or a set of microcompartments, the method comprising carrying out at least the following steps: incubation, encapsulation and cultivation steps.

[0039] Finally, a subject of the present invention is a microcompartment according to the invention for use as a medicament, preferably for the prevention or treatment of autoimmune diseases, immunodeficiency syndromes, cancer, viral diseases or inflammatory diseases.

[0040] Other features and advantages will become apparent from the detailed description of the invention and from the examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1a is a set of phase contrast microscopy images at x20 magnification of the microcompartments containing T lymphocytes according to the present invention after 3 days of encapsulation culture.

[0042] Figure 1b is a set of phase contrast microscopy images of microcompartments containing T lymphocytes according to the present invention at a magnification of x10 after 3 days of encapsulation culture.

[0043] Figure 2 is a representation of the results of tests carried out on the expansion of lymphocytes in microcompartments according to the invention.

[0044] Figure 3 Shown are the expansion factors of T lymphocytes after 6 days of culture with or without encapsulation.

[0045] Figure 4 Comparative representation of cytokine expression profiles during T lymphocyte culture after 6 days with or without encapsulation. Results are expressed as values ​​normalized to GM-CSF and IFN-γ secretion. Figure 5 is a confocal microscopy image of a microcompartment with polarized lymphocytes.

[0046] Figure 6 Shown are the expansion factors of T lymphocytes after 14 days of culture with encapsulation ("Capsule") or without encapsulation ("Control").

[0047] Figure 7a Comparative representation of cytokine expression during NK lymphocyte culture after 14 days of culture with or without encapsulation (for A supernatant donor diluted 10-fold). Results are expressed as values ​​normalized to IFN-γ secretion.

[0048] Figure 7bComparative representation of granzyme B expression during NK lymphocyte culture after 14 days of culture with or without encapsulation (for A supernatant donor diluted 100-fold). Results are expressed as values ​​normalized to IFN-γ secretion.

[0049] Figure 8a Comparative representation of cytokine expression during NK lymphocyte culture after 11 days of culture with or without encapsulation (for 10-fold dilution of B supernatant donor). Results are expressed as values ​​normalized to IFN-γ secretion.

[0050] Figure 8b Comparative representation of granzyme B expression during NK lymphocyte culture after 11 days of culture with or without encapsulation (for B supernatant donor diluted 100-fold). Results are expressed as values ​​normalized to IFN-γ secretion.

[0051] Fig. 9 Comparative results of toxicity tests performed on NK obtained without encapsulation and on NK obtained according to the present invention are shown (for donor B).

[0052] Fig.10 Comparative results of the evolution of the survival rate during culture of NKs without encapsulation and NKs encapsulated according to the present invention are shown (results for two donors A and B).

[0053] Fig.11a yes Fig.11a A set of phase contrast microscopy images of NK-containing microcompartments according to the present invention at x4 magnification on the day of encapsulation are shown in FIG.

[0054] Fig.11b is a set of phase contrast microscopy images of NK-containing microcompartments according to the present invention at x10 magnification after 11 days of encapsulation culture.

[0055] Fig.12a Shown are the expansion factors of NK lymphocytes when cultured for up to 12 days with encapsulation ("capsule" encapsulation without activation beads) or without encapsulation ("control").

[0056] Figure 12b Comparative results are shown for the evolution of the viability when NKs were cultured without encapsulation ("control") and NKs encapsulated according to the invention ("capsule" encapsulation without activation beads) (results for two donors B and C). DETAILED DESCRIPTION

[0057] definition

[0058] For the purposes of the present invention, "alginate" means a linear polysaccharide formed from β-D-mannuronic acid and α-L-guluronic acid, their salts and derivatives.

[0059] For the purposes of the present invention, "hydrogel capsule" or "hydrogel microcompartment" means a three-dimensional structure formed by a matrix of polymer chains swollen with a liquid, preferentially water.

[0060] For the purposes of the present 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 present invention consist of or are obtained from human cells or immunologically humanized non-human mammalian cells.

[0061] For the purposes of the present invention, the term "mutant cell" refers to a cell that carries at least one mutation.

[0062] The "Feret's diameter" of a microcompartment (or a part of a microcompartment) according to the invention means the distance "d" between two tangents of the microcompartment (or the part), which are parallel so that the entire projection of the microcompartment (or the part) is between the two parallel tangents. The Feret's diameter of the inner part of the microcompartment is measured between the two interfaces of the inner part of the microcompartment and the outer layer, i.e. the distance "d" between the two tangents of the inner part, which are parallel so that the entire projection of the inner part is between the two parallel tangents.

[0063] For the purposes of the present invention, "variable thickness" of a layer means that the layer does not have the same thickness everywhere for a single microcompartment.

[0064] For the purposes of the present invention, "microcompartment" or "capsule" means a partially or fully enclosed three-dimensional structure containing several cells.

[0065] Within the meaning of the present invention, "culture medium" means an aqueous solution containing cells that is compatible with the survival, development and / or metabolism of the cells. It can be a culture medium.

[0066] For the purposes of the present invention, "convective medium" means a medium that is agitated by internal motion.

[0067] For the purposes of the present invention, the term "mutation" means a genetic or epigenetic mutation, preferably a functional mutation. In particular, it may involve point modifications to the genetic sequence, structural variants, epigenetic modifications or modifications to mitochondrial DNA.

[0068] Within the meaning of the present invention, the term "functional mutation" refers to a transmissible genetic or epigenetic modification that confers a potential gain or loss of function or loss of potential function to the relevant mutant cell. It preferably relates to a mutation that causes a modification of the phenotype of the affected mutant cell. Very preferentially, it relates to a change in the genomic and / or epigenomic sequence that alters the therapeutic potential of the cell population either by increasing the risk associated with the resulting therapy or by reducing the benefit provided by the resulting therapy.

[0069] For the purposes of the present invention, the "minimum dimension" of a microcompartment or cell layer means the value of the minimum Feret's diameter of the microcompartment. For the purposes of the present invention, "lumen" means a volume of aqueous solution topologically surrounded by cells. Preferably, its contents are not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0070] The "smallest radius" of the inner part of a microcell according to the present invention means half the value of the smallest Feret's diameter of the inner part of the microcell.

[0071] The "average radius" of the inner part of a microcompartment according to the invention means the average value of the radii of the smallest compartments, each radius corresponding to half the value of the Feret's diameter of the inner part of a microcompartment.

[0072] According to the present invention, "expansion rate at day X" means the measurement of cell proliferation at time t = X. The rate of expansion is measured by taking the ratio of the number of cells counted on day X of culture divided by the number of cells at the beginning of culture (the day of packaging or the day of placement in culture).

[0073] According to the present invention, "large-scale culture" means a cell culture method suitable for production batches of lymphocytes from liver microtissues, which makes it possible to treat at least 1 patient, preferably 10 patients, more preferably 100 patients, even more preferably more than 1,000 patients.

[0074] According to the present invention, "functional phenotype" means lymphocytes having a 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 the ability to secrete interleukins (IL2, IL6, IL10, IL12) or other factors of interest (granzyme B, INFγ, GMCSF and TNFα) under the induction of antigen presentation in vitro.

[0075] For the purposes of the present invention, "progenitor cell" means a stem cell that has participated in lymphocyte differentiation but has not yet differentiated.

[0076] For the purposes of the present invention, "embryonic stem cells" means pluripotent stem cells derived from the cells of the internal cell mass of the 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). The embryonic stem cells used in the context of the present invention are obtained without destroying the embryos from which they originate, for example using the techniques described in Chang et al. (Cell Stem Cell, 2008, 2 (2): 113-117). Optionally, embryonic stem cells from humans can be excluded.

[0077] For the purposes of the present invention, "pluripotent stem cells" or "multipotent cells" means cells that have the ability to form all tissues present in the entire source organism, but cannot form the entire organism itself. Human pluripotent stem cells may be referred to as hPSCs in this application. These cells may be particularly induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells, or MUSE cells (for "multi-lineage differentiation stress persistence").

[0078] For the purposes of the present invention, "induced pluripotent stem cells" means pluripotent stem cells induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are particularly positive for pluripotency markers (such as staining with alkaline phosphatase and expression of proteins NANOG, SOX2, OCT4 and SSEA3 / 4). Examples of methods for obtaining induced pluripotent stem cells are described in articles such as 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).

[0079] Cell Microcompartment

[0080] The object of the present invention is therefore a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or of substantially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, comprising an outer hydrogel layer delimiting an inner part, said inner part containing between 500 and 5000 lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a content of granzyme B of less than 1 μg / mL culture medium. Preferably, the inner part of the microcompartment contains a content of granzyme B of less than 0.5 μg / mL culture medium, more preferably less than 0.1 μg / mL, even more preferably less than 50 ng / mL. Thus, the microcompartment contains at least lymphocytes that secrete little or no granzyme B.

[0081] In the context of the present invention, the content of molecules in the culture medium inside the microcompartment (especially cytokines, such as TNF, granzyme B, perforin, etc.) can be measured directly in the capsule, or in the culture medium outside the microcompartment when the microcompartment is in the culture medium. In fact, molecules smaller than 150KDa are in osmotic equilibrium between the inner part of the microcompartment and the culture medium in which the microcompartment is located.

[0082] According to a preferred embodiment, the inner part of the microcompartment comprises a perforin content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0083] Another object of the present invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or essentially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, which closed three-dimensional cell microcompartment comprises an outer hydrogel layer defining an inner part, the inner part containing between 500 and 5000 lymphocytes forming an aggregated three-dimensional culture in culture medium and having a perforin content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0084] According to a preferred embodiment, the inner part of the microcompartment comprises a TNFα content of less than 100 ng / mL, particularly less than 50 ng / mL culture medium, particularly less than 30 ng / mL, more preferably less than 10 ng / mL, even more preferably less than 3 ng / mL.

[0085] Another object of the invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or substantially oval, cylindrical, spheroid or spherical shape, the minimum dimension of which is between 200 μm and 400 μm, the closed three-dimensional cell microcompartment comprising an outer hydrogel layer defining an inner portion, the inner portion containing between 500 and 5000 lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a TNFα content of less than 100 ng / mL, and in particular less than 50 ng / mL culture medium, especially less than 30 ng / mL, more preferably less than 10 ng / mL, even more preferably less than 3 ng / mL. According to a preferred embodiment, when the lymphocytes are NK lymphocytes, the inner portion of the microcompartment contains an IL6 content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0086] Another object of the present invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or essentially oval, cylindrical, spheroid or spherical shape, wherein the minimum dimension of the closed three-dimensional cell microcompartment is between 200 μm and 400 μm, the closed three-dimensional cell microcompartment comprising an outer hydrogel layer defining an inner part, the inner part containing between 500 and 5000 NK lymphocytes forming an aggregated three-dimensional culture in a culture medium and having an IL6 content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0087] 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 medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0088] Another object of the present invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or essentially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, which comprises an outer hydrogel layer defining an inner part, the inner part containing between 500 and 5000 NK lymphocytes forming an aggregated three-dimensional culture in culture medium and having a GM-CSF content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0089] According to one embodiment, the object of the invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or of substantially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, comprising an outer hydrogel layer delimiting an inner part, the inner part containing between 500 and 5000 T lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a granzyme B content of less than 1 μg / mL of culture medium, and:

[0090] - less than 0.5 μg / mL perforin content in culture medium, and / or

[0091] - TNF-α content less than 100 ng / mL of culture medium.

[0092] According to one embodiment, the object of the invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or of substantially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, comprising an outer hydrogel layer defining an inner part, the inner part containing between 500 and 5000 NK lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a granzyme B content of less than 1 μg / mL of culture medium, and:

[0093] - less than 0.5 μg / mL perforin content in culture medium, and / or

[0094] - TNF-α content less than 100 ng / mL of culture medium, and / or

[0095] - IL6 content less than 0.5 μg / mL of culture medium, and / or

[0096] -GM-CSF content below 0.5 μg / mL culture medium.

[0097] According to one embodiment, the object of the invention is a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or of substantially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, comprising an outer hydrogel layer defining an inner part, the inner part containing between 500 and 5000 NK lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a granzyme B content of less than 1 μg / mL of culture medium, and:

[0098] - less than 0.5 μg / mL perforin content in culture medium, and / or

[0099] - IL6 content less than 0.5 μg / mL of culture medium, and / or

[0100] -GM-CSF content below 0.5 μg / mL culture medium.

[0101] According to the present invention, surprisingly, a granzyme B content of less than 1 μg / mL culture medium, preferably less than 0.5 μg / mL, more preferably less than 0.1 μg / mL, even more preferably less than 50 ng / mL, and / or a perforin content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL in the inner part of the microcompartment is a criterion for ensuring the functional phenotype of the lymphocytes within the inner part of the microcompartment according to the present invention.

[0102] Granzymes belong to the family of serine proteases and are known to be the means by which cytotoxic immune cells, notably T lymphocytes and NK lymphocytes, lyse their targets. Granzymes are stored in cytotoxic granules that also contain perforin. These granules are usually released into the immediate environment of the target cell and then penetrate the cytoplasm through the action of perforin. Among these granzymes is granzyme B, which is known to activate apoptosis through a signaling pathway involving caspases.

[0103] In conventional culture methods, lymphocytes are subjected to various physical constraints and / or stresses that tend to cause the release of interleukins and granules containing granzymes (particularly granzyme B). This release leads to a significant reduction in lymphocyte survival by inducing apoptosis in some lymphocytes. On the other hand, the remaining lymphocytes no longer display a functional phenotype. In fact, lymphocytes have lost their ability to secrete interleukins under the induction of antigen presentation, thus affecting their ability to act on target antigens.

[0104] Tumor necrosis factor (TNFα) is a pleiotropic cytokine that participates in many physiological processes that control inflammation, tumor response, and immune system homeostasis. TNFα can be secreted by lymphocytes or other cells (notably activated macrophages) to act directly on cells infected by certain pathogens or to 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 usually released into the direct environment of target cells.

[0105] In conventional culture methods, lymphocytes are subjected to various physical constraints and / or stresses that tend to trigger the release of interleukins (particularly TNFα). This uncontrolled release of TNFα changes the immediate environment of lymphocytes, and can significantly reduce their survival rate by inducing apoptosis of some lymphocytes in them. On the other hand, the remaining lymphocytes no longer show functional phenotypes. In fact, lymphocytes have lost their ability to secrete interleukins under the induction of antigen presentation, thus affecting their ability to act on target antigens.

[0106] Advantageously, the microcompartment according to the invention makes it possible to obtain lymphocytes with a functional phenotype.

[0107] Contrary to what is suggested in the prior art, a granzyme B content of less than 1 μg / mL culture medium, preferably less than 0.5 μg / mL, more preferably less than 0.1 μg / mL, even more preferably less than 50 ng / mL, and / or a perforin content of less than 0.5 μg / mL culture medium, preferably less than 0.1 μg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL is particularly desirable in lymphocyte cultures.

[0108] A granzyme B concentration of greater than 1 μg / mL of culture medium and / or a perforin content of greater than 0.5 μg / mL of culture medium and / or a TNFα concentration of greater than 100 ng / mL would indicate that an uncontrolled interleukin release process is occurring in the microcompartment according to the invention, making it incompatible with large-scale culture of lymphocytes with a functional phenotype.

[0109] This also applies to NK lymphocytes with an IL6 concentration higher than 0.5 μg / mL and / or a GM-CSF concentration higher than 0.5 μg / mL for a culture medium of NK lymphocytes. In particular, it is known that IL6 reduces NK cytotoxic activity (Cifadi et al., "Inhibition of natural killer cell cytotoxicity by interleukin-6: implications for the pathogenesis of macrophage activation syndrome" Artritis Rheumatol, November 2015; 67 (ll): 3037-46.doi: 10.1002 / art.39295.). Therefore, a reduction in IL6 concentration has the advantage of increasing NK efficacy.

[0110] Advantageously, the microcompartments according to the present invention are suitable for large-scale culture.

[0111] The microcompartment according to the invention advantageously has a T lymphocyte expansion rate of at least 20 times after 3 days of culture and a NK lymphocyte expansion rate of at least 40 times after 14 days of culture.

[0112] The microcompartment according to the present invention comprises an outer hydrogel layer. Preferentially, the hydrogel used is biocompatible, that is to say it is non-toxic to cells. The outer hydrogel layer must allow diffusion of oxygen and nutrients to supply the cells contained in the microcompartment and enable them to survive. According to one embodiment, the outer hydrogel layer comprises at least alginate. It can be composed exclusively of alginate. Alginate can be sodium alginate in particular, which is composed of 80% α-L-guluronic acid and 20% α-D-mannuronic acid, with an average molecular weight of 100kDa to 400kDa and a total concentration between 0.5% and 5% by weight. The outer hydrogel layer does not have cells.

[0113] The outer hydrogel layer makes it possible in particular to protect the cells from the mechanical stresses of the bioreactor, in order to limit the uncontrolled secretion of cytokines by lymphocytes in culture, which lymphocytes could be toxic when accumulated in the culture medium.

[0114] The average outer layer thickness may be variable. It is preferably between 20 μm and 60 μm, more preferably between 30 μm and 40 μm. The ratio between the smallest radius of the inner part and this thickness is preferably between 2 μm and 10 μm.

[0115] The microcompartments according to the invention can be obtained after encapsulation of lymphocytes without addition of an extracellular natural or synthetic matrix.

[0116] According to a variant, the microcompartment according to the invention does not contain in its internal part an extracellular matrix (such as and / or ) and / or a hydrogel-type matrix of plant origin (such as modified alginate) or a hydrogel-type matrix of synthetic origin, or Type of copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG).

[0117] According to a variant, the microcompartments according to the invention may be obtained after encapsulation of lymphocytes without the addition of an extracellular matrix. The extracellular matrix elements may be peptide or peptidomimetic sequences, mixtures of proteins, extracellular compounds or structural proteins (such as collagen, laminin, entactin, vitronectin) and growth factors or cytokines.

[0118] The absence of extracellular matrix elements in the inner part of the microcompartments according to the invention after encapsulation ensures a free organization of the lymphocyte structures within the microcompartments according to the invention.

[0119] Advantageously, the absence of extracellular matrix elements improves the rate of lymphocyte expansion.

[0120] Within the microcompartments, lymphocytes aggregate together in three dimensions, preferably into clusters.

[0121] Preferably, the microcompartments according to the invention contain lymphocytes forming aggregated three-dimensional cultures without a lumen.

[0122] The absence of a lumen advantageously limits autocrine and paracrine signaling of lymphocytes, rendering their phenotype particularly stable from the outset of encapsulation.

[0123] Advantageously, the absence of an inner lumen also makes it easier to control the size of the lymphocyte clusters present in the microcompartment.

[0124] The microcompartments according to the invention can be obtained after encapsulating previously activated lymphocytes, thereby increasing their rate of expansion by stimulating their proliferation. Activation can be achieved by contacting lymphocytes with artificial antigen presenting cells (aAPCs), most preferably via CD3 / CD28 antibodies for T lymphocytes, or via CD2 / NKp46 antibodies for NK lymphocytes.

[0125] When the lymphocytes have been previously activated and in order to maintain their activation, the internal part of the microcompartment according to the present invention may contain a solution comprising cytokines, notably IL2, or a combination of IL7 and IL15 for T lymphocytes, notably 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).

[0126] Cytokines in the inner part of the microcompartment accelerate lymphocyte proliferation.

[0127] In another embodiment, the microcompartment contains lymphocytes activated during and / or after encapsulation. In this embodiment, lymphocytes can be co-encapsulated with artificial antigen presenting cells (e.g., activated beads coated with suitable antibodies (e.g., Cloudz Human NK Cell Expansion Kit (R&D Systems) or NK Cell Activation / Expansion Kit (Miltenyi Biotec))).

[0128] Preferably, in this embodiment, lymphocyte activation occurs within the microcompartment. Preferably, lymphocyte activation within the microcompartment is achieved at a lymphocyte / antigen amount ratio of greater than 8:1, preferably greater than 9:1, even more preferably greater than 10:1.

[0129] Advantageously, activation is performed in microcompartments by controlling the ratio of lymphocyte numbers to the amount of antigen required in order to activate lymphocytes while limiting cell depletion.

[0130] The microcompartments of the present invention isolate lymphocytes, preventing cell mixing or convection. This mechanical isolation makes it possible to access the spatial and structural organization of lymphocytes in vivo.

[0131] Due to the mechanical isolation provided by the microcompartments, lymphocyte activation cannot be uniform or continuous. Thus, the activation approaches the conditions of lymphocyte activation in vivo, favoring their polarization.

[0132] Preferably, the lymphocytes forming the clusters are polarized. The polarity of these lymphocytes within each cluster can be demonstrated by the eccentric positioning of the nucleus within the cell. Polarized lymphocytes can also exhibit anisotropy in the distribution of Dig, Scrib and Lgl proteins.

[0133] Polarization of lymphocytes in microcompartments can be an indicative marker for a functional phenotype. In fact, lymphocytes are polarized if they contain a sufficient amount of granules containing granzymes, notably granzyme B.

[0134] Unexpectedly, the microcompartments of the present invention retain the aggregated conformation of lymphocytes, enabling better proliferation while maintaining a functional phenotype. Thus, this makes it possible to reduce the number of passages and the culture time necessary to reach the desired final lymphocyte number.

[0135] In addition to clusters, microcompartments may also contain cells suspended in the microcompartments.

[0136] The microcompartments according to the invention preferably contain a concentration of naive lymphocytes and / or Tscm lymphocytes greater than 20%, even more preferably greater than 40%.

[0137] The concentration of naive lymphocytes and / or Tscm lymphocytes ensures a long-lasting effect during cell therapy. It is particularly important to obtain a naive lymphocyte and / or Tscm lymphocyte content of more than 20% during the culture period in order to obtain an optimal ratio between the duration of the effect and the in vivo efficacy.

[0138] A naive lymphocyte and / or Tscm lymphocyte concentration of less than 20% would indicate that the lymphocyte life cycle is too short to ensure a lasting effect.

[0139] Naive lymphocytes and Tscm lymphocytes are immature lymphocytes showing 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.

[0140] According to a specific embodiment, the present invention relates to a closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or substantially oval, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 μm and 400 μm, the closed three-dimensional cell microcompartment comprising an outer hydrogel layer defining an inner part, the inner part forming an aggregated three-dimensional culture of lymphocytes in culture medium, the inner part having:

[0141] - Granzyme B content less than 1 μg / mL culture medium; and

[0142] - a TNFα content of less than 100 ng / mL of culture medium; and / or

[0143] -Perforin content less than 0.5 μg / mL culture medium.

[0144] In this embodiment, when the lymphocytes are T lymphocytes, the proportion of naive T lymphocytes and / or Tscm lymphocytes is preferably kept above 20%.

[0145] Preferably, the microcompartments are obtained after encapsulation of the lymphocytes without addition of an extracellular matrix and / or without addition of extracellular matrix elements. 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.

[0146] Preferably, the lymphocytes present in the microcompartments according to the invention are obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 (preferably at least 5, even more preferably at least 6) cell division cycles after encapsulation in the outer hydrogel layer, in order in particular to obtain between 5 and 100 lymphocytes according to the encapsulated lymphocytes. For example, the lymphocytes present in the microcompartments are obtained after at least six cell division cycles after encapsulation of the cells in the outer hydrogel layer.

[0147] Preferably, the number of cell divisions for a particular implementation of the method according to the invention is less than 100, even more preferably less than 30.

[0148] Preferably, the microcompartments are obtained after at least 2, more preferably at least 3, 4 or 5 passes after packaging. Each pass may for example last between 2 and 10 days, in particular between 2 and 4 days.

[0149] Preferably, the microcompartments are obtained after at least one repackaging, more preferably between 1 and 14 repackagings, in particular between 2 and 7 repackagings. Very preferentially, the repackaging corresponds to a new pass, and each packaging cycle corresponds to a pass.

[0150] Preferably, the microcompartments according to the invention are obtained within less than 6 days after encapsulation of at least 5 lymphocytes in the inner portion delimited by the outer hydrogel layer, even more preferably within less than 4 days after encapsulation.

[0151] The microcompartment according to the invention may contain between 500 and 5000 lymphocytes, preferably between 1000 and 3000 lymphocytes.

[0152] Advantageously, the microcompartments according to the invention protect the lymphocytes from mechanical stresses, thus making it possible to obtain expansion rates particularly suitable for large-scale cultivation.

[0153] The microcompartment according to the present invention may be in any three-dimensional form, i.e., it may have the shape of any object in space. The microcompartment may have any form compatible with cell encapsulation. Preferentially, the microcompartment according to the present invention is spherical or elongated or substantially spherical or elongated. It may have the shape of an ovoid, a cylinder, a spheroid or a sphere or substantially this shape.

[0154] 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 minimum 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 200 μm and 300 μm, in particular between 200 μm and 250 μm.

[0155] Its largest dimension is preferentially greater than 10 μm, more preferentially between 10 μm and 1 m, even more preferentially between 10 μm and 50 cm.

[0156] According to a particularly preferred embodiment, the lymphocytes encapsulated in the microcompartments according to the invention are T lymphocytes or NK lymphocytes.

[0157] Preferably, the lymphocytes contained in the microcompartment according to the present invention express a chimeric antigen receptor.

[0158] Therefore, the microcompartment according to the present invention is particularly suitable for large-scale lymphocyte culture for CAR-T or CAR-NK cell therapy.

[0159] According to one embodiment, the microcompartment according to the invention does not contain human embryonic stem cells and / or is not obtained from human embryonic stem cells.

[0160] The microcompartments according to the invention can optionally be frozen for storage. They should then preferably be thawed before use.

[0161] The invention also relates to a plurality of microcompartments together.

[0162] The invention also relates to a microcompartment assembly comprising at least two three-dimensional cell microcompartments or a series of microcompartments comprising at least two three-dimensional cell microcompartments, characterized in that at least one microcompartment is a microcompartment according to the invention.

[0163] 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 the culture of lymphocytes can be used, such as, for example, "TexMACS (Miltenyi Biotec)", or "VIVO 15-20 (Lonza)", or "CTS TM OpTmizer TM (ThermoFisher), or NKMACS (Miltenyi Biotec), or ExCellerate Human NK Cell Expansion Media (R&D Systems), provided that the concentration of dissolved salts is compatible with maintaining alginate cross-linking by divalent cations.

[0164] According to a particularly suitable embodiment, the present invention targets a series of cell microcompartments as described above in a culture medium in a closed chamber such as a bioreactor, preferentially in a closed chamber such as a bioreactor.

[0165] Thus, preferentially, the microcompartments are arranged in the culture medium in a closed bioreactor.

[0166] The microcompartment assembly or the series of microcompartments according to the invention preferably comprises between 2 and 10 16 microcompartments between them.

[0167] The microcompartments according to the invention can be used for all applications, in particular as drugs in cell therapy in humans or animals.

[0168] Preferably, the microcompartments according to the present invention can be used to prevent or treat autoimmune diseases, immunodeficiency syndromes, cancer, viral diseases or inflammatory diseases.

[0169] Thus, the microcompartments according to the present invention are suitable for large-scale culture, providing lymphocytes with a functional phenotype that ensures optimal efficacy and persistence in vivo.

[0170] Method for preparing the microcompartments according to the invention

[0171] The invention also relates to a method for producing the microcompartments according to the invention.

[0172] The microcompartment according to the present invention can be obtained from lymphocytes or cells capable of differentiating into lymphocytes (particularly, stem cells, in particular induced pluripotent stem cells, hematopoietic stem cells (including CD34+ cells), etc.).

[0173] In a first embodiment, the microcompartments according to the invention are obtained from lymphocytes.

[0174] The method for preparing a microcompartment or microcompartment assembly according to the present invention may comprise the following steps: - (a) culturing lymphocytes in a culture medium comprising cytokines - (b) encapsulating the washed lymphocytes in a hydrogel layer in the culture medium to form a closed three-dimensional microcompartment of oval, cylindrical, spheroid or spherical shape or substantially oval, cylindrical, spheroid or spherical shape, preferably without adding an extracellular matrix or without adding extracellular matrix elements, wherein the minimum dimension of the closed three-dimensional microcompartment is between 200 μm and 400 μm, so that each microcompartment contains at least 5 lymphocytes in its inner part.

[0175] - (c) culturing the microcompartments obtained in step (b) in a culture medium containing cytokines for 2 to 6 days.

[0176] The incubation step (a) of the method according to the invention comprises between 500,000 and 2 million lymphocytes per mL of culture medium, preferably 1 million lymphocytes per mL of culture medium.

[0177] The lymphocytes in step (a) are preferably T lymphocytes and / or NK lymphocytes.

[0178] Preferably, the preparation method according to the invention comprises a step prior to lymphocyte cultivation consisting in activating said lymphocytes for 3 days using an artificial activation system (aAPC) before encapsulation.

[0179] In a specific embodiment, lymphocyte activation is performed using an artificial activation system (aAPC) in the microcompartment caused by step (b). Any culture medium suitable for culturing lymphocytes (particularly T lymphocytes or NK lymphocytes) can be used, for example, such as "TexMACS (Miltenyi Biotec)", or "VIVO 15-20 (Lonza)", or "CTS TM OpTmizer TM (ThermoFisher)" medium. Each microcompartment obtained in step (b) contains at least 500,000 cells per mL of the microcompartment inner partial volume, more preferably at least 1 million cells / mL, in particular at least 2 million cells / mL, even more preferably 5 million cells / mL.

[0180] Preferably, each microcompartment obtained in step (b) contains at least 5 lymphocytes, preferably at least 8 lymphocytes in its inner part.

[0181] The cytokines contained in the culture medium may be, for example, one or more cytokines, such as a mixture of IL-7 and IL-15, or IL-2, or IL-17 and / or IL-18 and / or IL-21, or other cytokines known to those skilled in the art that stimulate lymphocyte proliferation.

[0182] Preferably, the culture medium used in the production method of the present invention for T lymphocytes is supplemented with a combination of 300 to 600 units / mL IL7 and 50 to 100 units / mL IL15, or 100 to 1000 units / mL IL2.

[0183] In the method according to the present invention, all of the lymphocytes initially encapsulated in step (b) preferably exhibit a volume that is less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0184] The encapsulated lymphocytes are suspended in the form of single cells and / or clusters or assemblies ("clusters" / groups) of at least two cells. Preferably, when there are at least 15 lymphocytes, single cells account for less than 50% of the number of all cells initially encapsulated in step (b).

[0185] Preferably, each lymphocyte cluster initially encapsulated in step (b) has a larger dimension that is less than 20%, even more preferably less than 10%, of the maximum dimension of the microcompartment in which it is encapsulated. In fact, the lymphocyte population cannot be too large in size compared to the size of the microcompartment, because too large a size of these initial cell clusters can cause premature cell confluence in the capsule during cell division; such premature confluence of all or part of the capsules in the capsule can cause an increase in intracellular pressure and cause cell stress, thereby affecting cell growth, and also affecting chromosome segregation.

[0186] The encapsulation step (b) is implemented according to techniques known to those skilled in the art. In fact, any method for producing cell microcompartments containing hydrogel capsules and cells inside can be used to implement the preparation method according to the present invention. In particular, it is possible to prepare 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.

[0187] Preferably, step (b) is performed without addition of natural or synthetic extracellular matrix.According to one variant, step (b) is performed without addition of extracellular matrix elements.

[0188] Preferably, step (b) is implemented in a device capable of using a microfluidic chip to generate hydrogel capsules. For example, the device may include a syringe pump for concentrically injecting several solutions by means of a microfluidic syringe, which makes it possible to form a jet that breaks up into droplets that are then collected in a calcium bath. According to a particularly suitable embodiment, two or three solutions are loaded on two or three syringe pumps:

[0189] - hydrogel solutions, such as alginate,

[0190] - optionally an isotonic intermediate solution, preferentially an isotonic solution free of divalent cations (such as Ca2+Mg2+) to avoid excessive premature crosslinking of the hydrogel in the syringe, such as, for example, a sorbitol solution,

[0191] - The solution from step (a) comprising lymphocytes and culture medium.

[0192] The three solutions are co-injected (simultaneously injected) concentrically by means of a microfluidic syringe or a microfluidic chip, which makes it possible to form a jet that breaks up into droplets whose outer layer is the hydrogel solution and whose core is the solution containing lymphocytes from step (a); these droplets are collected in a calcium bath that crosslinks and / or gels the alginate solution to form a shell.

[0193] To improve the monodispersity of the cell microcompartments, the hydrogel solution is preferentially charged with a direct current at (between 1 kV and 10 kV). A grounded ring may optionally be arranged at a distance between 1 mm and 20 cm from the tip, preferentially 3 mm to 10 cm, even more preferentially 1 cm to 5 cm, in a plane perpendicular to the axis of the jet leaving the microfluidic injector (coextrusion chip) to generate an electric field.

[0194] According to the present invention, it is necessary to generate capsules whose inner part has an average radius of at least 100 μm or a smaller radius. In order to generate capsules with such sizes with a co-extruded chip (microfluidic syringe or microfluidic chip), the present invention particularly proposes to modify the flow rate of the co-extruded solution and the final opening of the co-extruded chip. "Flow rate" means the flow rate of each solution reaching the syringe. "Final opening of the co-extruded chip" means the inner opening of the outlet channel of the chip.

[0195] - for the flow rate: the process preferably goes from a value between 20 and 40 mL / h to a value between 45 and 150 mL / h for each coextruded solution for standard capsule sizes known from the prior art, preferably between 45 and 110 mL per hour for the variants of the invention,

[0196] - The final opening diameter for the coextruded chip (microfluidic syringe) changes from values ​​preferentially between 50 and 120 μm for standard capsule sizes known from the prior art to diameter values ​​between 150 and 300 μm, preferentially between 180 and 240 μm.

[0197] Therefore, according to a specific embodiment, the encapsulation step (c) is carried out using a microfluidic syringe (the final opening diameter of which is between 150 μm and 300 μm, preferably between 180 μm and 240 μm) and at a flow rate of between 45 mL / h and 150 mL / h, preferably between 45 mL / h and 110 mL / h for each of the three solutions.

[0198] According to one embodiment, steps (a) and / or (c) are achieved with constant or sequential stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusion gradients. For example, it allows a uniform control of the cellular oxygenation level; thus avoiding the phenomena of hypoxia-related necrosis or hyperoxia-related oxidative stress.

[0199] The method according to the invention is preferably carried out in a closed chamber, such as a closed bioreactor.

[0200] In a preferred variant, the method according to the invention comprises at least one lymphocyte repackaging after step (c), i.e. at least two packaging cycles. Preferably, each packaging cycle corresponds to one pass. In this variant of the method (at least one cell repackaging after step (e)), the number of cell divisions of the entire method (for all passes) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0201] In the method according to the invention, there may be several repackagings, preferentially between 1 and 100, in particular between 1 and 10 repackagings.

[0202] According to a specific embodiment, a lymphocyte activation step using an artificial activation system (aAPC) can be performed within the microcompartments after at least one recapsulation.

[0203] Each repackaging may include:

[0204] a step consisting of dissociating the microcompartment or the series of microcompartments so as to obtain a suspension of lymphocytes or a suspension of lymphocyte clusters; the outer hydrogel layer may be eliminated in particular by hydrolysis, dissolution, puncture and / or decomposition by any biocompatible means (that is to say, non-toxic means for the cells). For example, this elimination may be achieved using phosphate-buffered saline, divalent ion chelators, enzymes such as alginate lyase (if the hydrogel comprises alginate) and / or laser microdissection, and

[0205] - a lymphocyte activation step using an artificial activation system (aAPC), preferably using CD3 / CD28 antibodies;

[0206] - A step of re-encapsulating all or part of the lymphocytes or lymphocyte clusters in a hydrogel capsule. Re-encapsulation is a means suitable for increasing the expansion of cells resulting from the pluripotency step and reducing the risk of mutation.

[0207] Reencapsulation consists in removing the outer hydrogel layer, preferentially in resuspending the cells in the form of cysts in the microcompartments in a partially or completely dissociated manner, and in re-implementing this step of the method.

[0208] According to one embodiment, repackaging comprises the following steps:

[0209] - (i) removing the outer hydrogel layer,

[0210] - (ii) resuspending the lymphocytes contained in the microcompartments in order to obtain single cells and / or at least one assembly or cluster of lymphocytes in a medium containing cytokines;

[0211] - (iii) encapsulating the solution of lymphocytes in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the microcompartment comprising an outer hydrogel layer defining an inner portion, the inner portion having a minimum radius or an average radius of at least 100 μm;

[0212] - (iv) culturing the resulting microcompartments in a medium containing cytokines,

[0213] - (vi) preferentially flushing the microcompartments to eliminate cytokines;

[0214] - (vi) culturing the microcompartments in a medium without cytokines for at least one cell division cycle, and

[0215] - (vii) optionally recovering the resulting cell microcompartments.

[0216] Compartmentalization in microcompartments makes it possible to eliminate microcompartments containing even more mutant cells than other capsules. Even if mutant cells grow rapidly, they will reach capsule confluence, which will limit their proliferation. Compartmentalization also makes it possible not to contaminate the entire cell population at any time, especially before the repackaging step, and also to eliminate capsules containing mutant cells. For example, the sorting can be done by in-line analysis or by eliminating the filled capsules faster than the other capsules.

[0217] In one embodiment, at least one of the steps (preferably all steps) is performed at a temperature suitable for cell survival (including between 4°C and 42°C). The temperature during cell proliferation must preferably be between 32°C and 37°C to avoid triggering mutations by reducing the performance of repair enzymes. Likewise, preferably, the temperature must be low (ideally about 4°C) to manage stress on the cells in step (c).

[0218] At any time, the method according to the invention may comprise a step consisting of: verifying the phenotype of the cells contained in the microcompartment. The verification may be performed by identifying the expression of at least 4 genes specific for the desired phenotype by at least some of the cells contained in the microcompartment, these genes being selected from the group consisting of CD45 RA RO, CCR7, CD62L, CD3, CD4, CD8, CD56, CD16.

[0219] The cell microcompartments obtained according to the method of the present invention can then be frozen before any use. Freezing is preferably carried out at a temperature between -190°C and -80°C. Thawing can be carried out by immersing the sealed freezing container (screwable ampoule or plastic bag) in a warm water bath (preferably 37 degrees), so that the cells are thawed quite quickly. The microcompartment according to the present invention can be kept for a limited time before it is used at more than 4°C (preferably between 4°C and 38°C) before it is used.

[0220] Implementing the method according to the invention makes it possible to obtain microcompartments comprising at least 80, preferably at least 500, at least 800, at least 1,000, in particular at least 3,000 lymphocytes.

[0221] The present invention in particular facilitates expansion with a high expansion rate, which thus reduces the culture time and the number of divisions for obtaining very large numbers of functional lymphocytes.

[0222] According to a second embodiment, the microcompartment according to the present invention is obtained from cells capable of differentiating into lymphocytes, and includes at least a method for differentiating cells capable of differentiating into lymphocytes (stem cells, in particular induced pluripotent stem cells, hematopoietic stem cells (including CD34+ cells), etc.) into lymphocytes.

[0223] The cells capable of differentiating into lymphocytes are preferably selected from the following:

[0224] * Stem cells capable of differentiating into lymphocytes, at least into T lymphocytes or NK lymphocytes, preferably embryonic stem cells or induced pluripotent stem cells or hematopoietic stem cells (including CD34+ cells), very preferably induced pluripotent stem cells, and / or

[0225] * or a progenitor cell capable of differentiating into a lymphocyte, at least a T lymphocyte or a NK lymphocyte,

[0226] * or are capable of undergoing reprogramming such that they become differentiated cells of induced pluripotent stem cells capable of differentiating into lymphocytes, at least into T lymphocytes or NK lymphocytes.

[0227] In this variation, the method may include implementing at least the following steps:

[0228] - Producing cellular microcompartments that contain inside the hydrogel capsules:

[0229] *preferably at least extracellular matrix elements or natural or synthetic extracellular matrix,

[0230] * cells capable of differentiating into lymphocytes, preferably induced pluripotent stem (IPS) cells,

[0231] - Inducing differentiation of cells into lymphocytes within the cell microcompartments, so as to obtain at least lymphocytes.

[0232] Example

[0233] Example 1: Method according to the invention (T lymphocytes)

[0234] Reagents used

[0235] -Solution 1: Medium used: TexMACS (Miltenyi Biotec) supplemented with 1000 units / mL recombinant IL-2 (Miltenyi Biotec)

[0236] -Solution 2: CD3 / CD28 polyantigen activation reagent (Transact TM Miltenyi Biotec

[0237] -Solution 3: Capsule flushing medium: DMEM / F-12+Glutamax

[0238] -Solution 4: Solution for dissolving alginate capsules: Non-enzymatic cell detachment buffer (RelesR TM Stem Cell Technology).

[0239] Step 1: Thawing cells (D0)

[0240] Ampoules of T lymphocytes sorted from peripheral blood of healthy donors were thawed according to the supplier's recommendations (Hu PB Pan-T, Stem Cell Technologies, #70024).

[0241] Step 2: T cell activation (D0)

[0242] T lymphocytes were counted on a NucleoCounter NC2000 (standard 1 box protocol) and cultured in solution 1 at a density of 1 million cells per mL in culture flasks.

[0243] Add solution 2 at 1:100 to the cell suspension thus prepared.

[0244] The treated cells were cultured in an incubator at 37°C and 5% CO2 for up to 3 days.

[0245] Step 3: Change of culture medium and packaging (D3)

[0246] To remove Transcart from the culture medium TM The cells were centrifuged at 300 g for 10 min and the supernatant was aspirated.

[0247] Rinse cells once in Solution 1.

[0248] A portion of the cell pellet was taken up in Solution 1 at a cell density of 5 million cells per mL for encapsulation.

[0249] The remaining cell pellet was taken up in solution 1 at a cell density of 5 million cells per mL (control).

[0250] Step 4: Packaging (D3)

[0251] The encapsulation device was 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).

[0252] The three required solutions were loaded onto three syringe pumps: i) alginate solution (2% by weight in distilled water) SLG100), ii) intermediate solution (sorbitol at 300 mM), iii) cell solution (prepared in the previous step). The three solutions are co-injected concentrically using a microfluidic syringe to form a jet that breaks up into droplets with alginate solution as the outer layer and cell solution as the core. These droplets are collected in a calcium bath (at 100 mM), which hardens the alginate solution to form a shell.

[0253] The present invention promotes in particular a high expansion rate, which thus reduces the culture time and the number of divisions for obtaining very large numbers of functional lymphocytes and thus limits new mutagenesis.

[0254] Phase contrast microscopy images of the assembly at x20 magnification after 3 days of capsule culture are shown in FIG. 1 a. Figure 1b Phase contrast microscopy images at x10 magnification after 3 days of capsule culture are shown in .

[0255] Step 5: Post-packaging processing (D3)

[0256] The capsules were collected using a 40 μm cell sieve and then, after rinsing (1×) with Solution 3, stored in a 75 cm flask with Solution 1 at a cell density of 100,000 cells per mL (Condition 1) or 200,000 cells per mL (Condition 2). 2 The flasks were kept in an incubator at 37°C and 5% CO2 for up to 3 days.

[0257] Unencapsulated cells as prepared in step 3 were grown in parallel under the same atmospheric conditions for 3 days.

[0258] Step 6: Capsule dissolution and counting (D6)

[0259] -(a): Arm 1: Capsule

[0260] - The capsule suspension was collected and allowed to settle in a 50 mL Falcon tube for 5 minutes.

[0261] - Carefully remove the supernatant and discard.

[0262] - The capsules were then treated with solution 4 for 5 min at 37°C.

[0263] - Cells released from the capsules were centrifuged at 300 g for 10 minutes to remove Solution 4.

[0264] -Finally, count the cells using NC2000 (Standard Protocol 1 Kit)

[0265] -(b): Arm 2: Suspension control

[0266] - The cell suspension was collected and centrifuged at 300 g for 10 min.

[0267] - Aspirate the supernatant.

[0268] - Next, the cells were counted using NC2000 (standard protocol 1 box).

[0269] Results of T lymphocyte encapsulation using the described protocol showed better cell expansion in capsules than in conventional suspension culture. Thus, we obtained a 20-fold expansion factor in 3 days of capsule culture compared to a factor of 2 for conventional culture.

[0270] Example 2: Measurement of cytokine secretion from encapsulated and non-encapsulated T lymphocytes

[0271] The detection kit used was MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi Biotec, 130-125-800)

[0272] Prepare various reagents and solutions in the MACSPlex Filter Plate according to the supplier's recommendations.

[0273] The culture supernatants from the encapsulated and non-encapsulated cells were obtained on the 6th day thereafter, without dilution, and cultured in duplicate and in the plates prepared in step 2 of Example 1;

[0274] Cultivate according to the supplier's recommendations;

[0275] Data acquisition was performed on a MACSQuant 10 cytometer (Miltenyi Biotec) using the dedicated Express Mode (Miltenyi Biotec).

[0276] The results are shown in Table 1 and Figure 4 Figures are expressed as mean fluorescence per million cells (MFI / e6 cells) (results were normalized to GMCSF and IFNg secretion).

[0277] Table 1

[0278]

[0279] In particular, these results demonstrate that lymphocytes encapsulated in microcompartments according to the invention have lower concentrations of extracellular granzyme B, perforin and TNFα than lymphocytes in suspension.

[0280] Example 3: Polarization of microcompartments according to the invention

[0281] The microcompartments according to the invention containing T lymphocytes were fixed in PFA 4% diluted in PBS with calcium and stored at 4°C.

[0282] Staining protocol:

[0283] Phalloidin and Lipilight (Membright) staining was performed at the dilution recommended by the supplier at room temperature with agitation and protected from light for 72 h. Images were acquired using a confocal microscope ( Figure 5 ).

[0284] It can be seen that the microcompartments according to the invention contain lymphocytes with eccentric nuclei. Thus, the lymphocytes are polarized within the microcompartments and display a functional phenotype.

[0285] Example 4: Method according to the present invention (NK lymphocytes)

[0286] Reagents used:

[0287] - Solution 1: Basal medium for NK expansion (ExCellerate Human NK Cell Expansion Media (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.

[0288] -Solution 2: Solution 1 supplemented with CD2 / NKp46 multi-antigenic activation beads (e.g. Cloudz Human NK Cell Expansion Kit (RD Systems))

[0289] -Solution 3: Capsule flushing medium: DMEM / F-12+Glutamax

[0290] -Solution 4: Solution for dissolving alginate capsules: 2.5 mM EDTA solution

[0291] Step 1: Thawing cells (D0)

[0292] Ampules of NK isolated 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).

[0293] NKs were counted on a NucleoCounter NC2000 (standard 1 box protocol).

[0294] The cells were centrifuged at 300 g for 10 min and the supernatant was aspirated.

[0295] A portion of the cell pellet was taken up in Solution 2 at a cell density of 15 million cells per mL for encapsulation.

[0296] The remaining cell pellet was taken up in Solution 2 at a cell density of 0.2 million to 1 million cells per mL (control).

[0297] Step 2: Packaging (D0)

[0298] The encapsulation device was prepared as described in Alessandri et al., 2016 (“A3D 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).

[0299] The three required solutions were loaded onto three syringe pumps: i) alginate solution (2% by weight in distilled water) SLG100), ii) intermediate solution (sorbitol at 300 mM), iii) cell solution (prepared in the previous step). The three solutions are co-injected concentrically using a microfluidic syringe to form a jet that breaks up into droplets with alginate solution as the outer layer and cell solution as the core. These droplets are collected in a calcium bath (at 100 mM), which hardens the alginate solution to form a shell.

[0300] The present invention promotes in particular a high expansion rate, which thus reduces the culture time and the number of divisions for obtaining very large numbers of functional lymphocytes and thus limits new mutagenesis.

[0301] Step 3: Post-packaging processing (D0)

[0302] The capsules were collected using a 40 μm cell sieve and then, after rinsing (1×) with Solution 3, stored in a 75 cm column of Solution 1 with a cell density between 200,000 and 1,000,000 cells per mL. 2 flask (donor A) or a 30 mL microbioreactor (donor B).

[0303] Incubate the 30 mL flask or microbioreactor at 37°C and 5% CO2 for 11 to 14 days. Agitate the microbioreactor capsule at 100 RPM. Refresh the culture medium according to the supplier's recommendations.

[0304] Unencapsulated cells as prepared in step 3 were grown in parallel under the same atmospheric conditions for 11 to 14 days.

[0305] Step 6: Capsule lysis and counting (D11 or D14, depending on capsule confluence)

[0306] -(a): Arm 1: Capsule

[0307] - The capsule suspension was collected and allowed to settle in a 50 mL Falcon tube for 5 minutes.

[0308] - Carefully remove the supernatant and discard.

[0309] - The capsules were then treated with solution 4 for 5 min at 37°C.

[0310] - Cells released from the capsules were centrifuged at 300 g for 10 minutes to remove Solution 4.

[0311] -Discard activated beads according to the supplier's recommendations.

[0312] -Finally, count the cells using NC2000 (Standard Protocol 1 Kit)

[0313] -(b): Arm 2: Suspension control

[0314] - The cell suspension was collected and centrifuged at 300 g for 10 min.

[0315] - Aspirate the supernatant.

[0316] -Discard activated beads according to the supplier's recommendations.

[0317] - Next, the cells were counted using NC2000 (standard protocol 1 box).

[0318] like Figure 6 As shown, the results of NK encapsulation according to this protocol depict a better cell expansion in capsules compared to conventional suspension culture. Thus, for donor A, we obtained a 40-fold expansion factor in 14 days of encapsulation culture compared to a factor of 3 for conventional culture. For donor B, we obtained a 110-fold expansion factor in 11 days of encapsulation culture compared to a factor of 70 for conventional culture.

[0319] Fig.10 It was shown that the survival rate of NK obtained according to the present invention was better than that of NK cultured in 2D. For donor A, at D14, the survival rate for encapsulated culture was 74%, compared to 64% for suspension culture. For donor B, at D11, the survival rate in encapsulated culture was 88%, compared to 62% in suspension.

[0320] Example 5: Measurement of cytokine secretion from encapsulated and non-encapsulated NK lymphocytes

[0321] The detection kit used was MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi Biotec, 130-125-800)

[0322] Prepare various reagents and solutions in the MACSPlex Filter Plate according to the supplier's recommendations.

[0323] 14 days later, the culture supernatant from the encapsulated or non-encapsulated cells was collected, diluted 10-fold or 100-fold, and cultured in duplicate in the plate prepared in step 2 of Example 4;

[0324] Cultivate according to the supplier's recommendations;

[0325] Data acquisition was performed on a MACSQuant 10 cytometer (Miltenyi Biotec) using the dedicated Express Mode (Miltenyi Biotec).

[0326] The results are shown in Tables 2 and 3 and Figure 7a , Figure 7b (Donor A) and Figure 8a , Figure 8b (Donor B) is expressed as mean fluorescence per million cells (MFI / e6 cells).

[0327] Table 2

[0328]

[0329] Table 3

[0330]

[0331] In particular, these results demonstrate that NK lymphocytes encapsulated in the microcompartments according to the present invention have lower concentrations of granzyme B, perforin, GM-CSF and IL-6 than NK lymphocytes in suspension.

[0332] Example 6: Measurement of functionality of encapsulated and non-encapsulated NK lymphocytes

[0333] Measurements were performed by flow cytometry on K562 according to the following protocol.

[0334] After decapsulation, NK (B donor NK obtained according to the process in Example 4, capsule arm and control arm in suspension) were stained with PKH-26 probe, and then incubated with K562 at 37°C 5% CO2 for 4h 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.

[0335] The cytometry data analysis strategy was performed as follows: PKH-26 probe was used to separate NK and K562 populations (K562 = negative population):

[0336] - Live K562 is negative for 7aad- / Annexin V

[0337] - Early apoptotic cells K562 are 7AAD- / Annexin V+

[0338] - Late apoptosis K562 is 7AAD+ / Annexin V+

[0339] - Necrotic or dead K562 are 7AAD+ / Annexin V-,

[0340] The percentage of cytotoxicity was calculated as follows:

[0341] % cytotoxicity = (early apoptosis + late apoptosis + necrosis) / total K562

[0342] The results obtained are shown in Fig. 9 The cytotoxicity of NK obtained according to the present invention is similar to that obtained with 2D.

[0343] Example 7 Method according to the present invention (NK lymphocytes)

[0344] Reagents used

[0345] - Solution 1: Basal medium for NK expansion (ExCellerate Human NK Cell Expansion Media (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.

[0346] -Solution 2: Capsule flushing medium: DMEM / F-12+Glutamax

[0347] -Solution 3: Solution for dissolving alginate capsules: 2.5 mM EDTA solution

[0348] Step 1: Thawing cells (D0)

[0349] Ampules of NK isolated 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).

[0350] NKs were counted on a NucleoCounter NC2000 (standard 1 box protocol).

[0351] The cells were centrifuged at 300 g for 10 min and the supernatant was aspirated.

[0352] A portion of the cell pellet was taken up in Solution 1 at a cell density of 5 to 15 million cells per mL for encapsulation.

[0353] The remaining cell pellet was taken up in Solution 1 at a cell density of 0.2 million to 1 million cells per mL (control).

[0354] Step 2: Packaging (D0)

[0355] The encapsulation device was 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).

[0356] The three required solutions were loaded onto three syringe pumps: i) alginate solution (2% by weight in distilled water) SLG100), ii) intermediate solution (sorbitol at 300 mM), iii) cell solution (prepared in the previous step). The three solutions are co-injected concentrically using a microfluidic syringe to form a jet that breaks up into droplets with alginate solution as the outer layer and cell solution as the core. These droplets are collected in a calcium bath (at 100 mM), which hardens the alginate solution to form a shell.

[0357] The present invention promotes in particular a high expansion rate, which thus reduces the culture time and the number of divisions for obtaining very large numbers of functional lymphocytes and thus limits new mutagenesis.

[0358] Step 3: Post-packaging processing (D0)

[0359] Capsules were collected using a 40 μm cell sieve and then stored in a 30 mL microbioreactor with solution 1 at a cell density of 200,000 cells per mL after rinsing with solution 2 (1×).

[0360] The 30 mL micro-bioreactor was incubated at 37° C. and 5% CO 2 for up to 11 days. The micro-bioreactor capsule was agitated at 100 RPM. The culture medium was refreshed according to the supplier's recommendations.

[0361] Unencapsulated cells as prepared in step 3 were grown in parallel under the same atmospheric conditions for 11 days.

[0362] Step 6: Capsule dissolution and counting (D11)

[0363] -(a): Arm 1: Capsule

[0364] - The capsule suspension was collected and allowed to settle in a 50 mL Falcon tube for 5 minutes.

[0365] - Carefully remove the supernatant and discard.

[0366] - The capsules were then treated with solution 3 for 5 min at 37°C.

[0367] - Cells released from capsules were centrifuged at 300 g for 10 min to remove Solution 3.

[0368] -Finally, count the cells using NC2000 (Standard Protocol 1 Kit)

[0369] -(b): Arm 2: Suspension control

[0370] - The cell suspension was collected and centrifuged at 300 g for 10 min.

[0371] - Aspirate the supernatant.

[0372] - Next, count the cells using (Standard Protocol 1 Box).

[0373] Fig.11a A phase contrast microscope image of the component at x4 magnification on the day of encapsulation is shown in FIG. Fig.11b Phase contrast microscopy images at x10 magnification after 11 days of capsule culture are shown in FIG.

[0374] like Fig.12a As shown, the results of NK encapsulation according to this protocol depict a better cell expansion in capsules compared to conventional suspension culture. Thus, for donor B, the expansion factor was 135-fold in 11 days of capsule culture compared to a factor of 72 for conventional culture. For donor C, the expansion factor was 41-fold in 11 days of capsule culture compared to a factor of 28 for conventional culture.

[0375] Figure 12b It was shown that the survival rate of NK obtained according to the present invention was better than that of NK cultured in 2D. For donor B, at D11, the survival rate for encapsulated culture was 82%, compared to 69% for suspension culture. For donor C, at D11, the survival rate for encapsulated culture was 73%, compared to 51% in suspension.

Claims

1. A closed three-dimensional cell microcompartment of oval, cylindrical, spheroid or spherical shape or substantially oval, cylindrical, spheroid or spherical shape, the smallest dimension of the closed three-dimensional cell microcompartment being between 200 μm and 400 μm, the closed three-dimensional cell microcompartment comprising an outer hydrogel layer defining an interior portion, the interior portion containing between 500 and 5000 lymphocytes forming an aggregated three-dimensional culture in a culture medium and having a granzyme B content of less than 1 μg / mL of the culture medium.

2. Microcompartment according to the preceding claim, characterized in that The inner portion comprises a perforin content of less than 0.5 μg / mL of culture medium.

3. Microcompartment according to one of the preceding claims, characterized in that The inner portion contains lymphocytes that form an aggregated three-dimensional culture without a lumen.

4. Microcompartment according to one of the preceding claims, characterized in that Said microcompartments have been obtained after encapsulation of lymphocytes without addition of an extracellular matrix or without addition of extracellular matrix elements.

5. Microcompartment according to one of the preceding claims, characterized in that Said microcompartments have been obtained after encapsulation of previously activated lymphocytes.

6. Microcompartment according to one of the preceding claims, characterized in that The thickness of the outer layer is variable and lies between 20 and 60 μm.

7. Microcompartment according to one of the preceding claims, characterized in that The lymphocytes are polarized.

8. Microcompartment according to one of the preceding claims, characterized in that The number of lymphocytes forming aggregated three-dimensional cultures ranged between 500 and 5,000.

9. Microcompartment according to one of the preceding claims, characterized in that The number of lymphocytes forming aggregated three-dimensional cultures ranged between 1000 and 3000.

10. Microcompartment according to one of the preceding claims, characterized in that The lymphocytes are T lymphocytes or NK lymphocytes.

11. Microcompartment according to one of the preceding claims, characterized in that The lymphocytes are lymphocytes expressing chimeric antigen receptors.

12. Microcompartment according to one of the preceding claims, characterized in that The cells are T lymphocytes, and the microcompartments have: - less than 0.5 μg / mL perforin content in culture medium, and / or - TNF-α content less than 100 ng / mL of culture medium.

13. Microcompartment according to one of claims 1 to 11, characterized in that The lymphocytes are NK lymphocytes, and the microcompartment has: - less than 0.5 μg / mL perforin content in culture medium, and / or - TNF-α content less than 100 ng / mL of culture medium, and / or - IL6 content less than 0.5 μg / mL of culture medium, and / or -GM-CSF content below 0.5 μg / mL culture medium.

14. Microcompartment according to one of the preceding claims, characterized in that The outer layer comprises alginate.

15. Microcompartment according to one of the preceding claims, characterized in that The inner portion comprises 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.

16. Microcompartment according to one of the preceding claims, characterized in that The microcompartments contain T lymphocytes, and the proportion of naive T lymphocytes and / or Tscm lymphocytes remains greater than 20%.

17. A microcompartment assembly comprising at least two three-dimensional cell microcompartments, characterized in that: At least one microcompartment is a microcompartment according to one of claims 1 to 16.

18. Microcompartment assembly according to the preceding claim, characterized in that The microcompartments are arranged in a culture medium in a bioreactor.

19. Microcompartment according to one of claims 1 to 16, for use as a medicine.

20. The microcompartment according to one of claims 1 to 16, for use in preventing or treating autoimmune diseases, immunodeficiency syndromes, cancer, viral diseases or inflammatory diseases.

21. A method for preparing a microcompartment according to one of claims 1 to 16 or a cell microcompartment assembly according to one of claims 17 to 18, the method comprising the following steps: (a) culturing lymphocytes in a medium containing cytokines, (b) encapsulating lymphocytes in their culture medium in a hydrogel layer to form closed three-dimensional microcompartments of oval, cylindrical, spheroid or spherical shape or substantially oval, cylindrical, spheroid or spherical shape, preferably without adding an extracellular matrix, wherein each microcompartment contains at least 5 lymphocytes in its inner part, (c) culturing the microcompartments obtained in step (b) in a culture medium comprising at least cytokines for 2 to 6 days.

22. Method for producing microcompartments according to the preceding claim, characterized in that The method comprises a step prior to the cultivation of lymphocytes, the step consisting in activating the lymphocytes.

23. Method for producing microcompartments according to one of claims 21 or 22, characterized in that The culture medium contains 300 to 600 units / mL of IL7 in combination with 50 to 100 units / mL of IL15, or 100 to 1000 units / mL of IL2.

24. Method for producing microcompartments according to one of claims 21 to 23, characterized in that In step (a), between 500,000 and 2,000,000 lymphocytes are cultured per mL of culture medium.

25. The method according to one of claims 21 to 24, characterized in that Step b) is performed by concentrically co-injecting via a microfluidic syringe at least two solutions: - hydrogel solution, - a solution containing lymphocytes and culture medium from step a), said microfluidic syringe making it possible to form, at the outlet of said syringe, a jet consisting of a mixture of said two solutions, said jet breaking up into droplets which are collected in a calcium bath which hardens said hydrogel solution to form the outer layer of each microcompartment, the inner part of each droplet consisting of the solution containing lymphocytes and culture medium from step (a).

26. The method according to claim 25, characterized in that The microfluidic syringe has a final opening diameter between 150 μm and 300 μm.

27. The method according to claim 25 or 26, characterized in that The flow rate of each of the two solutions was between 45 mL / h and 150 mL / h.

28. The method according to one of claims 25 to 27, characterized in that All of the cells initially encapsulated in step (b) exhibit a volume that is less than 50% of the volume of the microcompartment in which they are encapsulated.

29. The method according to one of claims 25 to 28, characterized in that Step (a) and / or step (c) are carried out under constant or sequential stirring.

30. The method according to one of claims 25 to 29, characterized in that The method comprises re-encapsulating the cells at least once after step (c).

31. The method according to the preceding claim, characterized in that Each re-encapsulation consists in removing said outer hydrogel layer, preferentially in resuspending said lymphocytes partially or completely dissolved in said microcompartments in a partially or completely dissociated manner, and in re-implementing said steps of said method.

32. Method for producing microcompartments according to one of claims 21 to 31, characterized in that The method comprises, after step (c), a step consisting in freezing the microcompartments.

33. The method according to one of claims 21 to 32, characterized in that The method is implemented in a bioreactor.

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