Cellular microcompartments comprising lymphocytes forming a grouped 3D culture and having a low TNF alpha content

Abstract

The invention relates to a three-dimensional cellular microcompartment or an assembly of three-dimensional cellular microcompartments of ovoid, cylindrical, spheroid or spherical shape or of substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 µm, comprising an outer hydrogel layer that delimits an internal part, said internal part comprising, in a medium, between 500 and 5000 lymphocytes forming a grouped three-dimensional culture and having a TNF alpha content of less than 100 ng / ml of medium. The invention also relates to a method for producing such a microcompartment or microcompartment assembly.

Description

[0001] CELLULAR MICROCOMPARTMENTS COMPRISING LYMPHOCYTES FORMING A 3D CLUSTERED CULTURE AND HAVING A LOW CONTENT OF TNF ALPHA

[0002] Technical field

[0003] The present invention relates to the field of large-scale lymphocyte cell culture. The invention relates in particular to a particular cellular microcompartment, its preparation method and its uses.

[0004] Prior art

[0005] The progress made over the past few decades has highlighted the benefits of cell therapy for treating many pathologies.

[0006] Cell therapy is defined as the administration of living cells to a patient for the purpose of preventing, treating, or alleviating a disease. The goal is to achieve a lasting effect through the injection of therapeutic cells.

[0007] Many cell therapy approaches can be considered such as:

[0008] - the so-called autologous approach in which the patient is his own donor.

[0009] - the so-called allogeneic approach in which the donor is different from the patient.

[0010] Cell therapy covers broad therapeutic areas and applies to many cell types ranging from pluripotent cells to specialized adult cells with metabolic function (e.g. hepatocytes) or mechanical function (e.g. myoblasts, chondrocytes), including hematopoietic lineages such as blood mononuclear cells (e.g. T lymphocytes or NK lymphocytes).

[0011] 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 an antigen present on the surface of tumor cells. CAR-T cells guide the immune system to target and eradicate cancer cells. This approach is considered a major advancement in immunotherapy, and many CAR-T cells are being developed to expand their uses. CAR-T cells are generally produced using the autologous approach. However, it is also possible to create allogeneic CAR-T cells. Another alternative is natural killer (NK) cells. NK cells are highly potent cytotoxic effector cells that have demonstrated antitumor effects.

[0012] In order to develop the potential of cell 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.

[0013] The ex vivo production of immune cells such as T or NK lymphocytes is particularly complex, especially on a large scale. Indeed, the in vitro culture methods developed to date are too stressful and quickly lead to cell exhaustion, making it impossible to produce lymphocytes on a large scale. In vivo, immune cell proliferation occurs mainly in mechanically stable environments such as solid tissues. Since the environment of a bioreactor is filled with non-physiological shear stresses, the expansion rates achieved are generally still insufficient compared to in vivo expansion capacities.

[0014] Currently, there is no satisfactory solution for rapidly producing functional lymphocytes, particularly CAR-T or CAR-NK cells, on a large scale.

[0015] Several culture methods are mainly used for CAR cell expansion: 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).

[0016] However, these culture methods are not suitable for large-scale lymphocyte production due to the exposure of cells to numerous handling steps increasing the risk of contamination, the exposure of cells to excessive stress, and / or the development and capacity constraints of the platforms and methods used. Furthermore, the work of maintaining culture and storage would be too demanding for large-scale production.

[0017] To address this problem, the prior art proposes activating the lymphocytes before culturing them in order to obtain rapid proliferation of the lymphocytes.

[0018] However, early lymphocyte activation leads to a physiological process of cellular exhaustion. Exhaustion corresponds to an early maturation of naive T cells towards a subpopulation of effector T lymphocytes limiting graft persistence in vivo.

[0019] Another solution proposed by the prior art is the use of hydrogel and alginate microtubes suspended in a cell culture medium. This method reports a lymphocyte expansion rate of 320 at 14 days of culture (Lin et al. Adv. Healthcare Mater. 2018, 1701297)

[0020] However, although this method provides a satisfactory expansion rate, it is not suitable for large-scale cultivation. This method would require the development of a dedicated cultivation system and would be too complicated to adapt on a large scale.

[0021] Furthermore, this method does not guarantee the functional phenotype of the lymphocytes and therefore that the action of the lymphocytes is sustainable.

[0022] The complexity of the differentiation and function of each lymphocyte subpopulation makes the analysis of the phenotype during proliferation an essential piece of data for carrying out any lymphocyte-based therapy, in particular therapies using CAR-T or CAR-NK cells.

[0023] The least differentiated T lymphocyte subpopulations such as naive T lymphocytes and Tscm lymphocytes (memory stem cells) appear to be particularly important to ensure a lasting effect in patients (Vita Golubovskaya and Lijun Wu (Cancers 2016, 8, 36). In addition, “memory-like” NK subpopulations allow for persistence of the clinical response (Romee et al. “Cytokine activation induces human memory-like NK cells” Blood 2012 Dec 6;120(24):4751-60).

[0024] On the other hand, it is also known that the effector T lymphocyte subpopulation is essential to ensure a significant effect on the target cell(s).

[0025] It therefore appears necessary to control cell subpopulations during large-scale lymphocyte culture to obtain an optimal ratio between persistence and efficacy in vivo.

[0026] Current production processes involving lymphocytes are very expensive and difficult to scale up, resulting in lymphocytes of mixed quality and reduced persistence.

[0027] There is therefore a significant need for a solution enabling large-scale production of lymphocytes with a functional phenotype, to meet an essential demand for lymphocyte-based cell therapy.

[0028] The objective of the invention is therefore to meet all of these needs and to overcome the drawbacks and limitations of the prior art.

[0029] Summary of the invention

[0030] To meet this objective, the invention proposes to use a particular microcompartment to obtain large-scale production of lymphocytes, suitable for use in cell therapy.

[0031] For this purpose, the subject of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 lymphocytes forming a three-dimensional grouped culture having an endogenous tumor necrosis factor (TNF alpha) content of less than 100 ng / mL of the medium.

[0032] Preferably, the internal part of the microcompartment comprises a perforin content of less than 0.5 pg / mL of the medium and / or a granzyme B content of less than 1 pg / mL of the medium and / or an IL6 content of less than 0.5 pg / mL of the medium and / or a GM-CSF content of less than 0.5 pg / mL of the medium.

[0033] Advantageously, the microcompartment according to the invention makes it possible to produce lymphocytes presenting a functional phenotype while guaranteeing rapid production on a large scale.

[0034] According to a particularly suitable embodiment, the microcompartment according to the invention comprises lymphocytes forming a three-dimensional grouped culture without lumen.

[0035] The microcompartment according to the invention can be obtained after encapsulation of lymphocytes with or without the addition of extracellular matrix, and according to a variant with or without the addition of an extracellular matrix element.

[0036] Advantageously, the absence of addition of extracellular matrix or matrix element at the time of encapsulation of lymphocytes makes it possible to improve their expansion rate after 3 days of culture.

[0037] According to a preferred object of the invention, the microcompartment according to the invention was obtained after encapsulation of previously activated lymphocytes. The activation of the lymphocytes advantageously makes it possible to stimulate the proliferation of the lymphocytes in the internal part of the microcompartment according to the invention. According to a variant, the activation of the lymphocytes can be carried out in the microcompartment after encapsulation.

[0038] According to another particularly suitable embodiment, the microcompartment according to the invention comprises lymphocytes expressing a chimeric antigenic receptor. When the microcompartment according to the invention comprises T lymphocytes, it preferably comprises a concentration of naive and / or Tscm lymphocytes greater than 20%, even more preferably greater than 40%.

[0039] Advantageously, such a concentration of naive and / or Tscm lymphocytes makes it possible to guarantee a lasting effect and to avoid the phenomenon of early lymphocyte exhaustion.

[0040] According to a preferred object of the invention, the invention relates to a set of microcompartments.

[0041] According to another aspect, the invention relates to a method for preparing a microcompartment or a set of microcompartments comprising at least the implementation of the following steps, an incubation, an encapsulation and a culture step. Finally, the invention relates to a microcompartment according to the invention for its use as a medicament, preferably in the prevention or treatment of autoimmune diseases, immunodeficiency syndromes, cancers, viral diseases or inflammatory diseases.

[0042] Other features and advantages will emerge from the detailed description of the invention and the examples which follow.

[0043] Brief description of the figures

[0044] Figure 1a is a phase contrast microscopy image at 20x magnification of a set of microcompartments according to the invention comprising T lymphocytes after 3 days of capsule cultures.

[0045] Figure 1b is a phase contrast microscopy image at 10x magnification of a set of microcompartments according to the invention comprising T lymphocytes after 3 days of capsule cultures.

[0046] Figure 2 is a representation of the results of tests on the amplification of lymphocytes in microcompartments according to the invention.

[0047] Figure 3 is a representation of T cell expansion factor after 6 days of culture with or without encapsulation.

[0048] Figure 4 is a comparative representation of the cytokine expression profile in T lymphocyte culture after 6 days of culture with or without encapsulation. The results are presented with values ​​normalized to GM-CSF and IFN-g secretion. Figure 5 is a confocal microscopy image of a microcompartment showing polarized lymphocytes.

[0049] Figure 6 is a representation of NK cell expansion factor after 14 days of culture with encapsulation (“Capsules”) or without encapsulation (“Control”).

[0050] Figure 7a is a comparative representation of cytokine expression in NK lymphocyte culture after 14 days of culture with or without encapsulation (for donor A-supernatant diluted to 10). The results are presented with values ​​normalized to IFN-g secretion.

[0051] Figure 7b is a comparative representation of granzyme B expression in NK lymphocyte culture after 14 days of culture with or without encapsulation (for donor A-supernatant diluted to 100). The results are presented with values ​​normalized to IFN-g secretion.

[0052] Figure 8a is a comparative representation of cytokine expression in NK lymphocyte culture after 11 days of culture with or without encapsulation (for donor B - supernatant diluted to 10). The results are presented with values ​​normalized to IFN-g secretion.

[0053] Figure 8b is a comparative representation of granzyme B expression in NK lymphocyte culture after 11 days of culture with or without encapsulation (for a 100-fold diluted donor B-supernatant). The results are presented with values ​​normalized to IFN-g secretion.

[0054] Figure 9 represents the comparative results of a toxicity test carried out on NK obtained without encapsulation and on NK obtained according to the invention (for donor B).

[0055] Figure 10 represents the comparative results of the evolution of viability during the culture of NK without encapsulation and NK encapsulated according to the invention (results for two donors A and B).

[0056] Figure 11a is a phase contrast microscopy image at x4 magnification of a set of microcompartments according to the invention containing NKs on the day of encapsulation is presented in Figure 11a.

[0057] Figure 11b is a phase contrast microscopy image at 10x magnification of a set of microcompartments according to the invention containing NK after 11 days of capsule cultures.

[0058] Figure 12a is a representation of NK cell expansion factor when cultured for 12 days of culture with encapsulation (“Capsules” encapsulation without activation bead) or without encapsulation (“Control”).

[0059] Figure 12b represents the comparative results of the evolution of viability during the culture of NK without encapsulation (“control”) and NK encapsulated according to the invention (“capsules” encapsulation without activation bead) (results for two donors B and C).

[0060] Detailed description of the invention

[0061] Definitions

[0062] For the purposes of the invention, the term "alginate" means linear polysaccharides formed from pD-mannuronate and aL-guluronate, salts and derivatives thereof.

[0063] For the purposes of the invention, the term “hydrogel capsule” or “hydrogel microcompartment” means a three-dimensional structure formed from a matrix of polymer chains, swollen by a liquid and preferably water.

[0064] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention consist of or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0065] For the purposes of the invention, the term “mutant cell” means a cell carrying at least one mutation.

[0066] By "Feret diameter" of a microcompartment (or of a part of a microcompartment) according to the invention, we mean the distance "d" between two tangents to said microcompartment (or to said part), these two tangents being parallel, such that the entire projection of said microcompartment (or of said part) is between these two parallel tangents. A Ferret diameter of the internal part of the microcompartment is measured between two interfaces of the internal part and the external layer of the microcompartment, i.e. the distance "d" between two tangents to said internal part, these two tangents being parallel, such that the entire projection of said internal part is between these two parallel tangents.

[0067] For the purposes of the invention, the term "variable thickness" of a layer means that the layer for the same microcompartment does not have the same thickness everywhere.

[0068] For the purposes of the invention, the term “microcompartment” or “capsule” means a partially or totally closed three-dimensional structure containing several cells.

[0069] For the purposes of the invention, the term "medium" means an aqueous solution containing cells, compatible with the survival, development and / or metabolism of the cells. It may be a culture medium.

[0070] For the purposes of the invention, the term “convective culture medium” means a culture medium driven by internal movements.

[0071] For the purposes of the invention, the term "mutation" means a genetic or epigenetic mutation, preferably a functional mutation. In particular, it may be a specific modification of the genetic sequence, a structural variant, an epigenetic modification, or a modification of mitochondrial DNA.

[0072] For the purposes of the invention, the term "functional mutation" means a transmissible genetic or epigenetic modification which confers a gain or loss of function or potential loss of function to the mutant cell concerned. This is preferably a mutation resulting in a modification of the phenotype of the mutant cell concerned. Very preferably, it is a change in the sequence of the genome and / or the epigenome which alters the therapeutic potential of a population of cells, either by increasing the risk associated with the therapy produced or by reducing the benefit provided by the therapy produced.

[0073] By "smallest dimension" of a microcompartment or a layer of cells within the meaning of the invention, we mean the value of the smallest Feret diameter of said microcompartment. By "light" or "lumen" within the meaning of the invention, we mean a volume of aqueous solution topologically surrounded by cells. Preferably, its contents are not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment.

[0074] By "smallest radius" of the internal part of a microcompartment according to the invention, we mean half the value of the smallest Ferret diameter of the internal part of the microcompartment.

[0075] By "average radius" of the internal part of a microcompartment according to the invention, we mean the average of the radii of the smallest compartment, each radius corresponding to half the value of a Ferret diameter of the internal part of the microcompartment.

[0076] By "expansion rate at X days" according to the invention, we mean a measurement of cell proliferation at time t=X. The expansion rate is measured by taking the ratio of the number of cells counted on day X of the culture divided by the number of cells at the start of the culture (day of encapsulation or day of culturing).

[0077] By "large-scale culture" according to the invention, we mean a cell culture method suitable for a production batch of lymphocytes making it possible to treat at least 1 patient, preferably 10 patients, more preferably 100 patients, even more preferably more than 1000 patients.

[0078] By "functional phenotype" according to the invention, we mean lymphocytes having a classical membrane marker expression profile (CD3+, CD4+ or CD8+ for T lymphocytes and CD3-, CD56+ for NK lymphocytes), known to those skilled in the art and a capacity to secrete interleukins (IL2, IL6, IL10, IL12) or other factors of interest (granzyme B, INF gamma, GMCSF, and TNF alpha) under the induction of the presentation of an antigen in vitro.

[0079] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in cellular differentiation into lymphocytes but not yet differentiated.

[0080] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, embryonic stem cells of human beings can be excluded.

[0081] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the present application. In particular, they may be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0082] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106).

[0083] Cellular microcompartment

[0084] The subject of the invention is therefore a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 pm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 lymphocytes forming a three-dimensional grouped culture and having a TNF alpha content of less than 100 ng / mL of medium.

[0085] Preferably, the internal part of the microcompartment comprises a TNF alpha content of less than 50 ng / mL of medium, in particular less than 30 ng / mL, more preferably less than 10 ng / mL, even more preferably less than 3 ng / mL. The microcompartment according to the invention therefore comprises at least lymphocytes which do not secrete or secrete little TNF alpha.

[0086] In the context of the invention, the content of molecule(s) (in particular cytokines such as TNF, such as granzyme B, perforin, etc.) in the medium internal to the microcompartment can be measured directly in the capsule, or in the medium external to the microcompartment when the latter is in a medium. Indeed, molecules lower than 150KDa are in osmotic equilibrium between the internal part of the microcompartment and the medium in which the microcompartment is included.

[0087] According to a preferred embodiment, the internal part of the microcompartment comprises a perforin content of less than 0.5 pg / ml of medium, preferably less than 0.1 pg / ml, more preferably less than 50 ng / ml, even more preferably less than 10 ng / ml.

[0088] The invention also relates to a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 lymphocytes forming a three-dimensional grouped culture and having a perforin content of less than 0.5 pg / ml of medium, preferably less than 0.1 pg / ml, more preferably less than 50 ng / ml, even more preferably less than 10 ng / ml.

[0089] According to a preferred embodiment, the internal part of the microcompartment comprises a granzyme B content of less than 1 pg / mL of medium, in particular less than 0.5 pg / mL of medium, more preferably less than 0.1 pg / mL, even more preferably less than 50 ng / mL. The microcompartment according to the invention therefore comprises at least lymphocytes which do not secrete or secrete little granzyme B.

[0090] The invention therefore also relates to a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 lymphocytes forming a three-dimensional grouped culture and having a granzyme B content of less than 1 pg / mL of medium. According to one embodiment, when the lymphocytes are NK lymphocytes, the internal part of the microcompartment comprises an IL6 content of less than 0.5 pg / ml of medium, preferably less than 0.1 pg / mL, more preferably less than 50 ng / mL, even more preferably less than 10 ng / mL.

[0091] The invention also relates to a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 NK lymphocytes forming a three-dimensional grouped culture and having an IL6 content of less than 0.5 pg / ml of medium, preferably less than 0.1 pg / ml, more preferably less than 50 ng / ml, even more preferably less than 10 ng / ml.

[0092] According to one embodiment, when the lymphocytes are NK lymphocytes, the internal part of the microcompartment comprises a GM-CSF content of less than 0.5 pg / ml of medium, preferably less than 0.1 pg / ml, more preferably less than 50 ng / ml, even more preferably less than 10 ng / ml.

[0093] The invention also relates to a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 NK lymphocytes forming a three-dimensional grouped culture and having a GM-CSF content of less than 0.5 pg / ml of medium, preferably less than 0.1 pg / ml, more preferably less than 50 ng / ml, even more preferably less than 10 ng / ml.

[0094] According to one embodiment, the subject of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 T lymphocytes forming a three-dimensional grouped culture and having a content of less than 100 ng / mL of medium and:

[0095] - a perforin content of less than 0.5 pg / ml of medium, and / or

[0096] - a granzyme B content of less than 1 pg / mL of medium.

[0097] According to one embodiment, the subject of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 pm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 NK lymphocytes forming a three-dimensional grouped culture and having a TNF-α content of less than 100 ng / mL of medium and:

[0098] - a perforin content of less than 0.5 pg / ml of medium, and / or

[0099] - a granzyme B content of less than 1 pg / mL of medium, and / or

[0100] - an IL6 content of less than 0.5pg / ml of medium and / or

[0101] - a GM-CSF content of less than 0.5pg / ml of medium.

[0102] According to the invention, surprisingly, a TNF alpha content of less than 100ng / mL of medium in the internal part of the microcompartment, in particular less than 30ng / mL, preferably less than 50ng / mL, more preferably less than 100g / mL, even more preferably less than 3ng / mL is a criterion guaranteeing the functional phenotype of the lymphocytes within the internal part of the microcompartment according to the invention.

[0103] Tumor necrosis factor (TNF alpha) is a pleiotropic cytokine involved in numerous physiological processes that control inflammation, tumor response, and immune system homeostasis. TNF alpha can be secreted by lymphocytes or other cells (including activated macrophages) to act directly on cells infected by certain pathogens or to promote lymphocyte proliferation. TNF alpha can thus promote the activation and proliferation of naive and effector T cells, but can also induce apoptosis of activated effector T cells. TNF alpha is stored in intracellular secretory vesicles. These vesicles are classically released into the immediate environment of target cells.

[0104] In conventional culture methods, lymphocytes are subjected to various physical constraints and / or stresses that tend to cause a release of interleukins, particularly TNF alpha. This uncontrolled release of TNF alpha will modify the direct environment of the lymphocytes and can cause a significant decrease in lymphocyte viability by causing apoptosis of some of them. On the other hand, the remaining lymphocytes no longer present a functional phenotype. Indeed, the lymphocytes have lost their ability to secrete interleukins under the induction of antigen presentation, which thus affects their ability to act on the target antigen.

[0105] Advantageously, the microcompartment according to the invention makes it possible to obtain lymphocytes exhibiting a functional phenotype.

[0106] Contrary to what is suggested in the prior art, a TNF alpha content of less than 100ng / mL of medium, in particular less than 30ng / mL, preferably less than 50ng / mL, more preferably less than 100g / mL, even more preferably less than 3ng / mL is particularly sought in the context of lymphocyte culture.

[0107] A TNF alpha concentration greater than 100ng / mL of medium and / or a perforin concentration greater than 0.5pg / ml and / or a granzyme B concentration greater than 1pg / ml would indicate that an anarchic process of interleukin release is taking place in the microcompartment according to the invention and would make it incompatible with a large-scale culture of lymphocytes exhibiting a functional phenotype.

[0108] The same is true for a concentration higher than 0.5pg / ml IL6 and / or a concentration higher than 0.5pg / ml GM-CSF for NK lymphocytes. IL6 in particular is known to decrease the cytotoxic activity of NK (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(ll):3037-46. doi: 10.1002 / art.39295.). Thus, a decrease in the concentration of IL6 advantageously increases the effectiveness of NK.

[0109] Advantageously, the microcompartment according to the invention is suitable for large-scale cultivation.

[0110] 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.

[0111] The microcompartment according to the invention comprises an outer hydrogel layer. Preferably, the hydrogel used is biocompatible, i.e., it is not toxic to the cells. The outer hydrogel layer must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival. According to one embodiment, the outer hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be a sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular mass of 100 to 400 kDa and a total concentration of between 0.5 and 5% by mass. The outer hydrogel layer is devoid of cells.

[0112] The external hydrogel layer helps protect cells from the mechanical stress of bioreactors and limits the anarchic secretion by lymphocytes in culture of potentially toxic cytokines when accumulated in the medium.

[0113] The average thickness of the outer layer can be variable. It is preferably between 20 and 60 um, more preferably between 30 and 40 um. The ratio between the smallest radius of the inner part and this thickness is preferably between 2 and 10 um.

[0114] The microcompartment according to the invention can be obtained after encapsulation of lymphocytes without adding extracellular matrix, natural or synthetic.

[0115] According to a variant, the microcompartment according to the invention does not comprise in its internal part an extracellular matrix such as Matrigel® and / or Geltrex® and / or a hydrogel type matrix of plant origin such as modified alginates or of synthetic origin or a copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) type Mebiol®.

[0116] Alternatively, the microcompartment according to the invention can be obtained after encapsulation of lymphocytes without adding any extracellular matrix element. The extracellular matrix elements can be peptide or peptidomimetic sequences, protein mixtures, extracellular compounds or structural proteins, such as collagen, laminins, entactin, vitronectin, as well as growth factors or cytokines.

[0117] The absence of extracellular matrix element in the internal part of the microcompartment according to the invention after encapsulation guarantees the lymphocytes freedom of structural organization within the microcompartment according to the invention.

[0118] Advantageously, the absence of extracellular matrix element improves lymphocyte expansion rates.

[0119] Within the microcompartment, the lymphocytes group together in three dimensions, preferably in clusters. Preferably, the microcompartment according to the invention comprises lymphocytes forming a three-dimensional grouped culture without a lumen.

[0120] The absence of lumen advantageously limits autocrine and paracrine signals from lymphocytes, making their phenotype particularly stable from their encapsulation.

[0121] Advantageously, the absence of a lumen also allows for better control of the size of lymphocyte groupings present in the form of clusters or clusters in the microcompartment.

[0122] The microcompartment according to the invention can be obtained after encapsulation of previously activated lymphocytes, thus increasing their expansion rate by stimulating their proliferation. Activation can be achieved by bringing the lymphocytes into contact with Artificial antigen presenting cells called aAPC (artificial antigen presenting cells), very preferably via CD3 / CD28 antibodies for T lymphocytes, via CD2 / NKp46 for NK lymphocytes.

[0123] When the lymphocytes have been previously activated and to maintain their activation, the internal part of the microcompartment according to the invention may comprise a solution comprising cytokines, in particular IL2 or the combination of IL7 and IL15 for T lymphocytes, in particular IL2, IL12, IL18, IL21 (preferably IL2 or IL12 or IL18 or IL21 or the combination of at least two cytokines chosen from IL2, IL12, IL18 and IL21) for NK cells.

[0124] The cytokines that can be contained in the internal part of the microcompartment thus make it possible to accelerate the proliferation of lymphocytes.

[0125] According to another embodiment, the microcompartment according to the invention comprises lymphocytes activated during and / or after encapsulation. In this embodiment, the lymphocytes may be co-encapsulated with artificial antigen-presenting cells, for example activation beads coated with one or more suitable antibodies (for example Cloudz Human NK Cell Expansion Kit (R&D Systems) or NK Cell Activation / Expansion Kit (Miltenyi Biotec)).

[0126] Preferably, in this embodiment, the activation of the lymphocytes is carried out within the microcompartment. Preferably, the activation of the lymphocytes within the microcompartment is carried out in a ratio of number of lymphocytes / quantity of antigen greater than 8 / 1, preferably greater than 9 / 1, even more preferably greater than 10 / 1.

[0127] Advantageously, activation is carried out in the microcompartment by controlling the ratio of the number of lymphocytes / quantity of antigen required in order to activate the lymphocytes while limiting cellular exhaustion.

[0128] The microcompartment according to the invention makes it possible to isolate lymphocytes, preventing mixing or convection of cells. This mechanical isolation makes it possible to approximate the spatial and structural organization of lymphocytes existing in vivo.

[0129] Due to the mechanical isolation provided by the microcompartment, lymphocyte activation cannot occur in a homogeneous or continuous manner. In this way, activation approaches the conditions of in vivo lymphocyte activation, which promotes their polarization.

[0130] Preferably, lymphocytes forming clusters or clusters are polarized. The polarity of these lymphocytes within each cluster or cluster can be demonstrated by the eccentric positioning of the nucleus within the cell. Furthermore, polarized lymphocytes may exhibit anisotropy in the distribution of Dig, Scrib, and Lgl proteins.

[0131] Lymphocyte polarization in the microcompartment can be an indicator of functional phenotype. Indeed, if a lymphocyte contains a sufficient amount of granules and vesicles containing interleukins, including TNF alpha, then the lymphocytes are polarized.

[0132] Unexpectedly, the microcompartment according to the invention maintains the conformation in the form of clusters or clusters of lymphocytes, thus allowing for better proliferation while maintaining a functional phenotype. Consequently, this makes it possible to reduce the number of passages and to reduce the culture time required to reach the final number of lymphocytes required.

[0133] The microcompartment may also include, in addition to the clusters or clusters, cells suspended in the microcompartment.

[0134] The microcompartment according to the invention preferably comprises a concentration of naive lymphocytes and / or Tscm greater than 20%, even more preferably greater than 40%.

[0135] The concentration of naive and / or Tscm lymphocytes ensures a lasting effect during cell therapy. It is particularly important during culture to obtain a naive and / or Tscm lymphocyte content greater than 20% in order to obtain an optimal ratio between persistence of the effect and in vivo efficacy.

[0136] A concentration of naive and / or Tscm lymphocytes below 20% would be indicative of a lymphocyte lifespan that is too short, not guaranteeing a persistent effect.

[0137] Naive and Tscm lymphocytes are immature lymphocytes presenting the markers CD3+ CD45RO- CD45RA+ CCR7+ easily identifiable and quantifiable by detection methods well known to those skilled in the art such as flow cytometry.

[0138] According to a particular embodiment, the invention relates to a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 pm, comprising an external hydrogel layer defining an internal part, said internal part forming in a medium a three-dimensional grouped culture of lymphocytes having:

[0139] - a TNF alpha content of less than 100ng / mL of medium; and

[0140] - a granzyme B content of less than 1 pg / mL of medium; and / or

[0141] - a perforin content of less than 0.5 pg / mL of medium.

[0142] In this embodiment, when the lymphocytes are T lymphocytes, the proportion of naive T lymphocytes and / or Tscm remains greater than 20%.

[0143] Preferably, the microcompartment is obtained after encapsulation of lymphocytes without addition of extracellular matrix and / or without addition of extracellular matrix element.

[0144] The lymphocytes present in the microcompartment according to the invention were preferentially obtained after at least two cycles of cell division after encapsulation in an external layer of hydrogel of at least one lymphocyte.

[0145] Preferably, the lymphocytes present in the microcompartment according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division after encapsulation in an external hydrogel layer, preferably at least 5, even more preferably at least 6, to obtain in particular between 5 and 100 lymphocytes per encapsulated lymphocyte. For example, the lymphocytes present in the microcompartment have been obtained after at least six cycles of cell division after encapsulation of the cells in the external hydrogel layer. Preferably the number of cell divisions for implementing the method according to the invention is less than 100, even more preferably less than 30.

[0146] Preferably the microcompartment is obtained after at least 2 passes after encapsulation, more preferably at least 3, 4 or 5 passes. Each pass can last for example between 2 and 10 days, in particular between 2 and 4 days.

[0147] Preferably, the microcompartment is obtained after at least one re-encapsulation, more preferably between 1 and 14 re-encapsulations, in particular between 2 and 7 re-encapsulations. Very preferably, one re-encapsulation corresponds to a new passage and each encapsulation cycle corresponds to one passage.

[0148] Preferably, the microcompartment according to the invention was obtained in less than 6 days after encapsulation, even more preferably in less than 4 days after encapsulation of at least 5 lymphocytes in the internal part defined by the external hydrogel layer.

[0149] The microcompartment according to the invention can contain between 500 and 5000 lymphocytes, preferably between 1000 and 3000 lymphocytes.

[0150] Advantageously, the microcompartment according to the invention protects the lymphocytes from mechanical stress, thus making it possible to obtain an expansion rate particularly suited to large-scale culture.

[0151] The microcompartment according to the invention can be in any three-dimensional form, that is to say it can have the shape of any object in space. The microcompartment can have any shape compatible with the encapsulation of cells. Preferably, the microcompartment according to the invention is in a spherical or elongated or substantially spherical or elongated shape. It can have the shape of an ovoid, a cylinder, a spheroid or a sphere or substantially this shape.

[0152] It is the outer layer of the microcompartment, i.e. the hydrogel layer, which gives the microcompartment according to the invention its size and shape. Preferably, the smallest dimension of the microcompartment according to the invention is between 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.

[0153] Its largest dimension is preferably greater than 10 pm, more preferably between 10 pm and 1 m, even more preferably between 10 pm and 50 cm.

[0154] According to a particularly preferred embodiment, the lymphocytes encapsulated in the microcompartment according to the invention are T lymphocytes or NK lymphocytes.

[0155] Preferably, the lymphocytes included in the microcompartment according to the invention express a chimeric antigenic receptor.

[0156] The microcompartment according to the invention is thus particularly suitable for large-scale cultures of lymphocytes for CAR-T or CAR-NK type cell therapy.

[0157] According to one embodiment, the microcompartment according to the invention does not comprise human embryonic stem cells and / or is not obtained from human embryonic stem cells.

[0158] The microcompartment according to the invention can optionally be frozen for storage. It should then preferably be defrosted before use.

[0159] The invention also relates to several microcompartments together.

[0160] The invention also relates to a set or series of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to the invention.

[0161] Preferably, the series of microcompartments according to the invention is 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 the medium “TexMACS (Miltenyi Biotec)” or “VIVO 15-20 (Lonza)” or “CTS™ OpTmizer™ (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 the maintenance of the crosslinking of the alginate by the divalent cations.

[0162] According to a particularly suitable embodiment, the invention relates to a series of cellular microcompartments in a closed enclosure, such as a bioreactor, preferably in a culture medium in a closed enclosure, such as a bioreactor. Thus, preferably, the microcompartments are arranged in a culture medium in a closed bioreactor.

[0163] The set or series of microcompartments according to the invention preferably comprises between 2 and 10 16 microcompartments.

[0164] The microcompartments according to the invention can be used for all applications, in particular as a drug in cell therapy in humans or animals.

[0165] Preferably, the microcompartments according to the invention can be used in the prevention or treatment of autoimmune diseases, immunodeficiency syndromes, cancers, viral diseases or inflammatory diseases.

[0166] Thus, the microcompartment according to the invention is suitable for large-scale culture by providing lymphocytes with a functional phenotype guaranteeing optimal efficiency and persistence in vivo.

[0167] Method for preparing microcompartments according to the invention

[0168] The invention also relates to a method for preparing microcompartments according to the invention.

[0169] The microcompartment according to the invention can 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.).

[0170] According to a first embodiment, the microcompartment according to the invention is obtained from lymphocytes.

[0171] The method for preparing a microcompartment or a set of microcompartments according to the invention may comprise the following steps: -(a) incubating the lymphocytes in a culture medium comprising cytokines

[0172] -(b) encapsulating the washed lymphocytes in a culture medium in a hydrogel layer, preferably without adding extracellular matrix or without adding any extracellular matrix element, to form a closed three-dimensional microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 pm, so that each microcompartment comprises at least 5 lymphocytes in its internal part. -(c) culturing the microcompartments obtained in step (b) in a culture medium comprising cytokines, for 2 to 6 days.

[0173] Incubation step (a) of the method according to the invention comprises between 0.5 and 2 million lymphocytes per ml of culture medium, preferably 1 million lymphocytes per ml of culture medium.

[0174] The lymphocytes in step (a) are preferentially T lymphocytes and / or NK lymphocytes.

[0175] Preferably, the preparation method according to the invention comprises a step prior to the incubation of the lymphocytes, which consists of activating said lymphocytes using an artificial activation system (aAPC) for 3 days pre-encapsulation.

[0176] According to a particular embodiment, the activation of the lymphocytes is carried out using an artificial activation system (aAPC) within the microcompartment resulting from step (b). Any culture medium suitable for the culture of lymphocytes, in particular T lymphocytes or NK lymphocytes, may be used, such as the medium “TexMACS (Miltenyi Biotec)” or “VIVO 15-20 (Lonza)” or “CTS™ OpTmizer™ (ThermoFisher)” for example. Each microcompartment obtained in step (b) comprises at least 0.5 million cells per mL of volume of the internal part of the microcompartment, more preferably at least 1 million cells / mL, in particular at least 2 million cells / mL, even more preferably 5 million cells per mL.

[0177] Preferably, each microcompartment obtained in step (b) comprises at least 5 lymphocytes, preferably at least 8 lymphocytes in its internal part.

[0178] The cytokines contained in the culture medium may be, for example, one or more cytokines such as 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 for stimulating lymphocyte proliferation.

[0179] Preferably, the culture media used in the preparation method of the invention for T lymphocytes are supplemented with 100 to 1000 units / ml of IL2 or the combination of 300 to 600 units / ml of IL7 and 50 to 100 units / ml of IL15.

[0180] In the method according to the invention, all of the lymphocytes initially encapsulated in step (b) preferably represent a volume less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0181] The encapsulated lymphocytes are suspended in the form of single cells and / or clusters or group(s) of at least two cells ("cluster(s)"). Preferably, when there are at least 15 lymphocytes, the single cell(s) represent less than 50% in number of all the cells initially encapsulated in step (b).

[0182] Preferably, each cluster of lymphocytes initially encapsulated in step (b) has a largest dimension less than 20% of the largest dimension of a microcompartment in which it is encapsulated, even more preferably less than 10%. Indeed, the lymphocyte clusters must not be too large in size compared to the size of the microcompartment because too large a dimension of these initial cell clusters could lead, during cell divisions, to earlier cell confluence in the capsule; this too early confluence of all or part of the capsules could lead to an increase in intracellular pressures and cause cellular stress, impacting cell growth but also chromosome segregation.

[0183] The encapsulation step (b) is carried out according to techniques known to those skilled in the art. Indeed, any method for producing cellular microcompartments containing cells and a hydrogel capsule can be used for implementing the preparation method according to the invention. In particular, it is possible to prepare microcompartments by adapting the method and the 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), in accordance with the steps described below.

[0184] Preferably, step (b) is carried out without adding any extracellular matrix, natural or synthetic. According to a variant, step (b) is carried out without adding any extracellular matrix element.

[0185] Preferably, step (b) is implemented in a device capable of generating hydrogel capsules using a microfluidic chip. For example, the device may comprise syringe pumps for several solutions injected concentrically using a microfluidic injector which makes it possible to form a jet which splits into drops then collected in a calcium bath. According to a particularly suitable embodiment, two or three solutions loaded onto two or three syringe pumps:

[0186] - a hydrogel solution, for example alginate,

[0187] - optionally an isotonic intermediate solution, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ to avoid crosslinking of the hydrogel too early in the injector, such as for example a sorbitol solution,

[0188] - the solution from step (a) comprising lymphocytes and culture medium.

[0189] The three solutions are co-injected (injected simultaneously) concentrically using a microfluidic injector or microfluidic chip which allows the formation of a jet which splits into drops whose outer layer is the hydrogel solution and the core the solution from step (a) comprising the lymphocytes; These drops are collected in a calcium bath which crosslinks and / or gels the alginate solution to form the shell.

[0190] To improve the mono-dispersity of the cellular microcompartments, the hydrogel solution is preferably charged with a direct current at (between 1 and 10kV). A grounded ring can optionally be placed at a distance from the tip of between 1mm and 20cm, preferably 3mm to 10cm, even more preferably 1cm to 5cm, from the tip in the plane perpendicular to the axis of the jet exiting the microfluidic injector (coextrusion chip) to generate the electric field.

[0191] According to the invention, it is necessary to generate capsules whose internal part has a mean radius or smaller radius of at least 100 pm. To generate capsules with such dimensions with a coextrusion chip (microfluidic injector or microfluidic chip) the invention proposes in particular to modify the flow rate of the coextruded solutions and the final opening of the coextrusion chip. By flow rate is meant the flow rate of each solution that arrives at the injector. By final opening of the coextrusion chip is meant the internal opening of the outlet channel of the chip.

[0192] - for the flow rate: we preferably go from a value between 20 and 40 ml / h for each coextruded solution for the standard sizes of capsules known from the prior art, to a value between 45 and 150 mL / h, preferably between 45 and 110 mL per hour for a variant of the invention,

[0193] - for the diameter of the final opening of the coextrusion chip (microfluidic injector) which goes from a value preferably between 50 and 120 pm for the standard sizes of capsules known from the prior art, to a diameter value between 150 and 300 pm, preferably between 180 and 240 pm.

[0194] Thus, according to a particular embodiment, the encapsulation step (b) is carried out using a microfluidic injector whose final opening diameter is between 150 and 300 pm, preferably between 180 and 240 pm, and with the flow rate of each of the 3 solutions between 45 and 150 mL / h, preferably between 45 and 110 mL / h.

[0195] 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 any diffusive gradient. For example, it allows homogeneous control of the level of cellular oxygenation; thus avoiding the phenomena of necrosis linked to hypoxia, or oxidative stress linked to hyperoxia.

[0196] The method according to the invention is preferably implemented in a closed enclosure such as a closed bioreactor.

[0197] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the lymphocytes after step (c), i.e. at least two encapsulation cycles. Preferably, each encapsulation cycle corresponds to one passage. In this variant of the method (at least one re-encapsulation of the cells after step (c)) the number of cell divisions of the entire method (for all the passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0198] In a method according to the invention there may be several re-encapsulations, preferably between 1 and 100, in particular between 1 and 10 re-encapsulation(s).

[0199] According to a particular embodiment, a step of activating lymphocytes using an artificial activation system (aAPC) can be carried out within the microcompartment after at least one re-encapsulation.

[0200] Each re-encapsulation may include:

[0201] - a step of dissociating the microcompartment or series of microcompartments to obtain a suspension of lymphocytes or a suspension of lymphocyte clusters; the removal of the external hydrogel layer may be carried out in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, the removal may be carried out using a saline phosphate buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection, and

[0202] - a step of activating lymphocytes using an artificial activation system (aAPC) preferentially using CD3 / CD28 antibodies;

[0203] - a step of re-encapsulation of all or part of the lymphocytes or lymphocyte clusters in a hydrogel capsule. Re-encapsulation is a suitable means for increasing the cellular amplification obtained from the pluripotent stage, and reducing the risks of mutation.

[0204] Re-encapsulation consists of removing the outer layer of hydrogel, preferably resuspending the cells that were in the form of cysts in the microcompartments in a partially or totally dissociated manner and repeating the process steps.

[0205] According to one embodiment, the re-encapsulation comprises the following steps:

[0206] - (i) remove the outer hydrogel layer,

[0207] - (ii) resuspending the lymphocytes which were contained in the microcompartment so as to obtain single cells and / or at least one set or cluster of lymphocytes in a culture medium containing cytokines

[0208] - (iii) encapsulating the lymphocyte solution 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, comprising an outer hydrogel layer defining an inner portion, the smallest radius or the average radius of said inner portion being at least 100 pm;

[0209] - (iv) culturing the microcompartments obtained in a culture medium containing cytokines,

[0210] - (vi) preferentially rinse the microcompartments, so as to eliminate the cytokines;

[0211] - (vi) culturing the microcompartments in a culture medium devoid of cytokines, for at least one cell division cycle, and

[0212] - (vii) optionally recover the cellular microcompartments obtained.

[0213] Compartmentalization into microcompartments allows the elimination of microcompartments containing more mutated cells than other capsules. Even if the mutated cells grow rapidly, they will reach capsular confluence, which will contain their multiplication. Compartmentalization also allows the entire cell population to be avoided and capsules containing mutant cells to be eliminated at any time, particularly before a reencapsulation step. This sorting can be done either by online analysis or by eliminating capsules that fill more quickly than the others, for example.

[0214] In one embodiment, at least one of the steps (preferably all steps) is carried out at a temperature suitable for cell survival, between 4 and 42°C. The temperature during cell proliferation should preferably be between 32 and 37°C to avoid triggering mutations by lowering the performance of the repair enzymes. Similarly, preferably, the temperature should be low (ideally around T1

[0215] 4°C) to manage cell stress in step (c).

[0216] 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. This verification may be carried out by identifying the expression by at least a portion of the cells contained in the microcompartment, of at least 4 genes specific to the desired phenotype chosen from CD45 RA RO, CCR7, CD62L, CD3, CD4, CD8, CD56, CD16.

[0217] The cellular microcompartments obtained according to the methods of the 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 vehicle (screw-on ampoule or plastic bag) in a warm water bath (preferably 37 degrees) so that the cells thaw fairly quickly. The microcompartments according to the invention, before their use, can be kept at more than 4°C for a limited period before their use, preferably between 4°C and 38°C.

[0218] The implementation of 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 1000, in particular at least 3000 lymphocytes.

[0219] The invention mainly promotes amplification with a high expansion rate, which consequently reduces the culture time and the number of divisions to obtain a very large number of functional lymphocytes.

[0220] According to a second embodiment, the microcompartment according to the invention is obtained from cells capable of differentiating into lymphocytes, and comprises at least the implementation of a method of differentiation into lymphocytes of cells capable of differentiating into lymphocytes (stem cells, in particular induced pluripotent stem cells, hematopoietic stem cells including CD34+ cells, etc.).

[0221] Cells capable of differentiating into lymphocytes are preferentially chosen from:

[0222] * either 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,

[0223] * either progenitor cells capable of differentiating into lymphocytes, at least into T lymphocytes or NK lymphocytes,

[0224] *either differentiated cells capable of undergoing reprogramming so that they become induced pluripotent stem cells capable of differentiating into lymphocytes, at least into T lymphocytes or NK lymphocytes.

[0225] In this variant, the method may comprise at least the implementation of the steps which consist of:

[0226] -produce a cellular microcompartment comprising, inside a hydrogel capsule:

[0227] * preferably at least extracellular matrix elements or an extracellular matrix, natural or synthetic,

[0228] * cells capable of differentiating into lymphocytes, preferably induced pluripotent stem (IPS) cells,

[0229] - induce cellular differentiation into lymphocytes within the cellular microcompartment, so as to obtain at least lymphocytes. : Method according to the invention

[0230] Reagents used:

[0231] -Solution 1: Culture medium used: TexMACS (Miltenyi Biotec) supplemented with 1000 units / ml of recombinant IL-2 (Miltenyi Biotec)

[0232] -Solution 2: CD3 / CD28 Polyantigenic Activation Reagent (Transact™ Miltenyi Biotec)

[0233] -Solution 3: Capsule rinsing medium: DMEM / F-12 + Glutamax

[0234] -Solution 4: Solution for dissolving alginate capsules: non-enzymatic cell detachment buffer (RelesR™ Stem Cell Technology).

[0235] Vials of T lymphocytes sorted from healthy donor peripheral blood were thawed according to the supplier's recommendations (Hu PB Pan-T, Stem Cell Technologies,

[0236] #70024).

[0237] T lymphocytes were counted using the NucleoCounter NC2000 (Standard protocol 1 cassette) and cultured in solution 1 at a density of 1 million cells per ml in a culture flask.

[0238] Solution 2 was added at 1 / 100 to the cell suspension thus prepared.

[0239] The treated cells were left in culture in an incubator at 37°C and 5% CO2 for 3 days.

[0240] Step 3: Change of medium and encapsulation (D3):

[0241] To remove Transcart™ from the medium, cells were centrifuged at 300g for 10 min and the supernatant aspirated.

[0242] Cells were rinsed in solution 1 once.

[0243] A portion of the cell pellet was taken up in solution 1 at a cell density of 5 million cells per ml for encapsulation.

[0244] The other part of the cell pellet was taken up in solution 1 at a cell density of 5 million cells per ml (control).

[0245] Step 4: Encapsulation (D3)

[0246] 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).

[0247] The three necessary solutions are loaded onto three syringe pumps, i) alginate solution (PRONOVA®SLG100 at 2% by mass in distilled water), ii) intermediate solution (sorbitol at 300mM), iii) cell solution (prepared in the previous step); The three solutions are co-injected concentrically using a microfluidic injector that allows the formation of a jet that splits into drops whose outer layer is the alginate solution and the core the cell solution; These drops are collected in a calcium bath (at 100mM) that stiffens the alginate solution to form the shell.

[0248] The invention mainly promotes amplification with a high expansion rate, which consequently reduces the culture time and the number of divisions to obtain a very large number of functional lymphocytes, and therefore limits new mutagenesis.

[0249] A phase contrast microscopy image at 20x magnification of a set after 3 days of culture in capsules is shown in Figure 1a. A phase contrast microscopy image at 10x magnification after 3 days of culture in capsules is shown in Figure 1b.

[0250] The capsules are recovered with a 40 pm cell sieve then after rinsing (1x) with solution 3 they are stored in a 75 cm flask 2 with solution 1 has a cell density of 100,000 cells per ml (condition 1) or 200,000 cells per ml (condition 2).

[0251] The flask is kept in the incubator at 37°C and 5% CO2 for 3 days.

[0252] Unencapsulated cells as prepared during the 3rd step are cultured in parallel under the same atmospheric conditions for 3 days.

[0253] Step 6: Dissolving the capsules and counting (D6)

[0254] -(a): Arm 1: capsule

[0255] -The capsule suspension was collected and allowed to sediment for 5 minutes in a 50ml Falcon tube.

[0256] -The supernatant was carefully removed and discarded.

[0257] -The capsules were then treated with solution 4 for 5 min at 37°C.

[0258] -The cells released from the capsules were centrifuged at 300g for 10 minutes to remove solution 4.

[0259] -The cells were finally counted using the NC2000 (standard protocol 1 cassette) -(b): Arm 2: suspension control

[0260] -The cell suspension was collected and then centrifuged at 300g for 10 min.

[0261] -The supernatant was aspirated.

[0262] -The cells were then counted using the NC2000 (standard 1 cassette protocol).

[0263] The results of encapsulation of T lymphocytes according to the described protocol show a better cellular amplification in capsules compared to a classic culture in suspension. Thus we obtain an amplification factor of times 20 in 3 days of culture in capsule against a factor of times 2 for a classic culture.

[0264] Example 2: Measurement of cytokine secretion from encapsulated or non-encapsulated T lymphocytes

[0265] The detection kit used is the MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi Biotec, 130-125-800)

[0266] The preparation of the various reagents and solutions was done according to the supplier's recommendations in the MACSPlex Filter Plate.

[0267] The culture supernatants of the encapsulated or non-encapsulated cells were collected on day 6 after, undiluted, and incubated in duplicate and incubated in the plate prepared in step 2 of example 1;

[0268] Incubation was carried out in accordance with the supplier's recommendations;

[0269] Data acquisition was performed on a MACSQuant 10 cytometer (Miltenyi Biotec) using a dedicated Express Mode (Miltenyi Biotec).

[0270] Results are expressed as mean fluorescence per million cells (MFI / e6 cells) in Table 1 and Figure 4 (results normalized to GM-CSF and IFNg secretion).

[0271] [Table 1]

[0272] These results confirm in particular that the lymphocytes encapsulated in the microcompartment according to the invention have a lower concentration of extracellular granzyme B, perforin and TNF alpha compared to the lymphocytes in suspension.

[0273] Example 3: Polarization of a microcompartment according to the invention

[0274] The microcompartments according to the invention comprising T lymphocytes were fixed in PFA

[0275] 4% diluted in PBS with calcium and stored at 4°C

[0276] Marking protocol:

[0277] Phalloidin and Lipilight (Membright) labeling were performed at the dilutions recommended by the suppliers for 72 hours under agitation, at room temperature and protected from light. Image acquisition (Figure 5) was performed using confocal microscopy.

[0278] A microcompartment according to the invention can be observed comprising lymphocytes whose nucleus is eccentric. Consequently, the lymphocytes are polarized within the microcompartment and thus exhibit a functional phenotype.

[0279] 4: Method according to the invention

[0280] Reagents used:

[0281] -Solution 1: Basal culture medium for NK expansion (ExCellerate Human NK Cell

[0282] Expansion Media (R&D Systems)) supplemented with 27ng / ml recombinant IL-2, 10ng / ml recombinant IL-12, 10ng / ml recombinant IL-18 and 10ng / ml recombinant IL-21.

[0283] -Solution 2: Solution 1 supplemented with CD2 / NKp46 polyantigenic activation beads

[0284] (eg Cloudz Human NK Cell Expansion Kit (R&D Systems))

[0285] -Solution 3: Capsule rinsing medium: DMEM / F-12 + Glutamax

[0286] -Solution 4: Solution for dissolving alginate capsules: 2.5mM EDTA solution

[0287] 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

[0288] Cell Technologies, #70036).

[0289] NK cells were counted using the NucleoCounter NC2000 (Standard 1 cassette protocol).

[0290] Cells were centrifuged at 300g for 10 min and the supernatant was aspirated.

[0291] A portion of the cell pellet was taken up in solution 2 at a cell density of 15 million cells per ml for encapsulation.

[0292] The other part of the cell pellet was taken up in solution 2 at a cell density between 0.2 and

[0293] 1 million cells per ml (control).

[0294] 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.

[0295] 9, pp. 1593-1604).

[0296] The three necessary solutions are loaded onto three syringe pumps, i) alginate solution

[0297] (PRONOVA®SLG100 at 2% by mass in distilled water), ii) intermediate solution (sorbitol at 300mM), iii) cell solution (prepared in the previous step); The three solutions are co-injected concentrically using a microfluidic injector which allows the formation of a jet which splits into drops whose outer layer is the alginate solution and the core the cell solution; These drops are collected in a calcium bath (at 100mM) which stiffens the alginate solution to form the shell.

[0298] The invention mainly promotes amplification with a high expansion rate, which consequently reduces the culture time and the number of divisions to obtain a very large number of functional lymphocytes, and therefore limits new mutagenesis.

[0299] The capsules are recovered with a 40 pm cell sieve then after rinsing (1x) with solution 3 they are stored in a 75 cm flask2 (Donor A) or in a 30mL minibioreactor (Donor B) with solution 1 at a cell density between

[0300] 200,000 and 1,000,000 cells per ml.

[0301] The 30mL flask or mini-bioreactor is kept in an incubator at 37°C and 5% CO2 for 11 to 14 days. The capsules in the mini-bioreactor are shaken at 100 RPM. The culture medium is renewed according to the suppliers' recommendations.

[0302] Unencapsulated cells as prepared during the 3rd step are cultured in parallel under the same conditions for 11 to 14 days. and corn or J14, depending on the confluence of the ca

[0303] -(a): Arm 1: capsule

[0304] -The capsule suspension was collected and allowed to sediment for 5 minutes in a 50ml Falcon tube.

[0305] -The supernatant was carefully removed and discarded.

[0306] -The capsules were then treated with solution 4 for 5 min at 37°C.

[0307] -The cells released from the capsules were centrifuged at 300g for 10 minutes to remove solution 4.

[0308] -Activation beads are disposed of according to the supplier's recommendations.

[0309] -The cells were finally counted using the NC2000 (standard protocol 1 cassette)

[0310] -(b): Arm 2: suspension control

[0311] -The cell suspension was collected and then centrifuged at 300g for 10 min.

[0312] -The supernatant was aspirated.

[0313] -The activation beads were removed according to the supplier's recommendations. -The cells were then counted using the NC2000 (standard 1-cassette protocol).

[0314] The results of NK encapsulation according to the protocol describe a better cell amplification in capsules compared to a classic culture in suspension, as illustrated in Figure 6. Thus, for donor A, we obtain a 40-fold amplification factor in 14 days of culture in capsules compared to a 3-fold factor for a classic culture. For donor B, we obtain a 110-fold amplification factor in 11 days of culture in capsules compared to a 70-fold factor for a classic culture.

[0315] Figure 10 shows that the viability during the culture of the NK obtained according to the invention is better than that of the NK cultured in 2D. For donor A, the viability at D14 is 74% for the capsule culture against 64% for the suspension culture. For donor B, the viability at D11 is 88% for the capsule culture against 62% in suspension.

[0316] Example 5: Measurement of cytokine secretion from encapsulated or non-encapsulated NK lymphocytes

[0317] The detection kit used is the MACSPlex Cytotoxic T / NK Cell Kit (Miltenyi Biotec, 130-125-800)

[0318] The preparation of the various reagents and solutions was done according to the supplier's recommendations in the MACSPlex Filter Plate.

[0319] The culture supernatants of the encapsulated or non-encapsulated cells were collected 14 days later, diluted 10-fold. e or at 100 e , and incubated in duplicate in the plate prepared in step 2 of example 4;

[0320] Incubation was carried out in accordance with the supplier's recommendations;

[0321] Data acquisition was performed on a MACSQuant 10 cytometer (Miltenyi Biotec) using a dedicated Express Mode (Miltenyi Biotec).

[0322] Results are expressed as mean fluorescence per million cells (MFI / e6 cells) in Tables 2 and 3 and in Figures 7a, 7b (Donor A) and 8a, 8b (Donor B). [Table 2]

[0323] [Table 3]

[0324] These results confirm in particular that the NK lymphocytes encapsulated in the microcompartment according to the invention have a lower concentration of granzyme B, perforin, GM-CSF and IL-6 compared to the NK lymphocytes in suspension.

[0325] Example 6: Measurement of the functionality of encapsulated or non-encapsulated NK lymphocytes

[0326] The measurement was carried out by flow cytometry on K562 according to the following protocol.

[0327] After decapsulation, labeling of NK (NK donors B obtained according to the methods of example 4, capsule arm and control arm in suspension) with the PKH-26 probe, then incubation for 4 hours at 37°C 5% CO2 of NK with K562 at different E:T ratios (“effector to target” effector to target): 12.5:1 / 6.25:1 / 3.125:1 / 1.5:1. Annexin V and 7-AAD labeling. The cytometry data analysis strategy was carried out as follows: separation of NK and K562 populations using the PKH-26 probe (K562 = negative population): live K562 are negative for 7aad- / AnnexinV-

[0328] K562 in early apoptosis are 7AAD- / AnnexinV+

[0329] Late apoptotic K562s are 7AAD+ / AnnexinV+

[0330] Necrotic or dead K562 are 7AAD+ / AnnexinV-,

[0331] The percentage of cytotoxicity was calculated as follows:

[0332] %Cytotoxicity = (early apoptosis + late apoptosis + necrosis) / total K562

[0333] The results obtained are presented in Figure 9. It can be seen that the NKs obtained according to the invention have a cytotoxicity similar to the NKs obtained in 2D.

[0334] 7 Method according to the invention

[0335] Reagents used:

[0336] -Solution 1: Basal culture medium for NK expansion (ExCellerate Human NK Cell Expansion Media (R&D Systems)) supplemented with 27ng / ml recombinant IL-2, 10ng / ml recombinant IL-12, 10ng / ml recombinant IL-18 and 10ng / ml recombinant IL-21.

[0337] -Solution 2: Capsule rinsing medium: DMEM / F-12 + Glutamax

[0338] -Solution 3: Solution for dissolving alginate capsules: 2.5mM EDTA solution

[0339] Step 1: thawing of cells (JO)

[0340] 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).

[0341] NK cells were counted using the NucleoCounter NC2000 (Standard 1 cassette protocol).

[0342] Cells were centrifuged at 300g for 10 min and the supernatant was aspirated.

[0343] A portion of the cell pellet was taken up in solution 1 at a cell density between 5 and 15 million cells per ml for encapsulation.

[0344] The other part of the cell pellet was taken up in solution 1 at a cell density between 0.2 and 1 million cells per ml (control).

[0345] 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).

[0346] The three necessary solutions are loaded onto three syringe pumps, i) alginate solution (PRONOVA®SLG100 at 2% by mass in distilled water), ii) intermediate solution (sorbitol at 300mM), iii) cell solution (prepared in the previous step); The three solutions are co-injected concentrically using a microfluidic injector that allows the formation of a jet that splits into drops whose outer layer is the alginate solution and the core the cell solution; These drops are collected in a calcium bath (at 100mM) that stiffens the alginate solution to form the shell.

[0347] The invention mainly promotes amplification with a high expansion rate, which consequently reduces the culture time and the number of divisions to obtain a very large number of functional lymphocytes, and therefore limits new mutagenesis.

[0348] Step 3: Post-encapsulation treatment (PO)

[0349] The capsules are recovered with a 40pm cell sieve then after rinsing (1x) with solution 2 they are stored in a 30mL mini-bioreactor with solution 1 at a cell density of 200,000 cells per ml.

[0350] The 30mL mini-bioreactors are kept in an incubator at 37°C and 5% CO2 for 11 days. The capsules in the mini-bioreactor are shaken at 100 RPM. The culture medium is renewed according to the suppliers' recommendations.

[0351] Unencapsulated cells as prepared during the 3rd step are cultured in parallel under the same conditions for 11 days.

[0352] Step 6: Dissolving the capsules and counting (D 11)

[0353] -(a): Arm 1: capsule

[0354] -The capsule suspension was collected and allowed to sediment for 5 minutes in a 50ml Falcon tube.

[0355] -The supernatant was carefully removed and discarded.

[0356] -The capsules were then treated with solution 3 for 5 min at 37°C.

[0357] -The cells released from the capsules were centrifuged at 300g for 10 minutes to remove solution 3.

[0358] -The cells were finally counted using the NC200Ô (standard protocol 1 cassette)

[0359] -(b): Arm 2: suspension control

[0360] -The cell suspension was collected and then centrifuged at 300g for 10 min. -The supernatant was aspirated.

[0361] -The cells were then counted using the (standard 1 cassette protocol).

[0362] A phase contrast microscopy image at x4 magnification of an assembly on the day of encapsulation is shown in Figure 11a. A phase contrast microscopy image at x10 magnification after 11 days of capsule cultures is shown in Figure 11b.

[0363] The results of NK encapsulation according to the protocol describe a better cell amplification in capsules compared to a conventional suspension culture, as illustrated in Figure 12a. Thus, for donor B, the amplification factor is 135 times in 11 days of culture in capsules compared to a factor of 72 times for a conventional culture. For donor C, the amplification factor is 41 times in 11 days of culture in capsules compared to a factor of 28 times for a conventional culture.

[0364] Figure 12b shows that the viability during the culture of the NK obtained according to the invention is better than that of the NK cultured in 2D. For donor B, the viability at D11 is 82% for the capsule culture compared to 69% for the suspension culture. For donor C, the viability at D11 is 73% for the capsule culture compared to 51% in suspension.

Claims

Demands

1. A closed, three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical shape, or substantially ovoid, cylindrical, spheroid or spherical, the smallest dimension of which is 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 lymphocytes forming a three-dimensional grouped culture and having a TNF alpha content of less than 100 ng / mL of medium.

2. Microcompartment according to any one of the preceding claims, characterized in that the internal part comprises lymphocytes forming a three-dimensional grouped culture without a lumen.

3. Microcompartment according to any one of the preceding claims, characterized in that it was obtained after encapsulation of lymphocytes without addition of extracellular matrix or without addition of extracellular matrix element.

4. Microcompartment according to any one of the preceding claims, characterized in that it was obtained after the encapsulation of previously activated lymphocytes.

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

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

7. Microcompartment according to any one of the preceding claims, characterized in that the number of lymphocytes forming a grouped culture in three dimensions is between 500 and 5000.

8. Microcompartment according to any one of the preceding claims, characterized in that the number of lymphocytes forming a grouped culture in three dimensions is between 1000 and 3000.

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

10. Microcompartment according to any one of the preceding claims, characterized in that the lymphocytes are lymphocytes expressing a chimeric antigen receptor.

11. A microcompartment according to any one of the preceding claims, characterized in that the lymphocytes are T lymphocytes and in that it has: - a perforin content of less than 0.5 pg / ml of medium, and / or - a granzyme B content of less than 1 pg / mL of medium.

12. Microcompartment according to any one of claims 1 to 10, characterized in that the lymphocytes are NK lymphocytes and in that it has: - a perforin content of less than 0.5 pg / ml of medium, and / or - a granzyme B content of less than 1 pg / mL of medium, and / or - an IL6 content of less than 0.5pg / ml of medium and / or - a GM-CSF content of less than 0.5pg / ml of medium.

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

14. Microcompartment according to any one of the preceding claims, characterized in that the inner part comprises a solution comprising IL2 or the combination of IL7 and IL15 or IL12, IL18, IL21 or the combination of at least two cytokines selected from IL2, IL12, IL18 and IL21.

15. Microcompartment according to any one of the preceding claims, characterized in that it comprises T lymphocytes and in that the proportion of naive T lymphocytes and / or Tscm remains greater than 20%.

16. Set 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. A set of microcompartments according to the preceding claim, characterized in that the microcompartments are arranged in a culture medium in a bioreactor.

18. Microcompartment according to any one of claims 1 to 15, for its use as a medicinal product.

19. Microcompartment according to any one of claims 1 to 15, for its use in the prevention or treatment of autoimmune diseases, immunodeficiency syndromes, cancers, viral diseases or inflammatory diseases.

20. A method for preparing a microcompartment according to any one of claims 1 to 15 or a set of cell microcompartments according to any one of claims 16 or 17, comprising the following steps: - (a) incubate lymphocytes in a culture medium containing cytokines, - (b) encapsulate the lymphocytes in their culture medium in a hydrogel layer, preferably without adding extracellular matrix, to form a closed three-dimensional microcompartment of ovoid, cylindrical, spheroid or spherical shape or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 µm, so that each microcompartment comprises at least 5 cells in its internal part, - (c) cultivate the microcompartments obtained in step (b) in a culture medium comprising at least cytokines, for 2 to 6 days.

21. A method for preparing a microcompartment according to the preceding claim, characterized in that it comprises a step prior to the incubation of lymphocytes, which consists of activating said lymphocytes.

22. A method for preparing a microcompartment according to any one of claims 20 to 21, characterized in that the culture media comprise 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.

23. Method of preparing a microcompartment according to any one of claims 20 to 22, characterized in that in step (a) between 0.5 and 2 million lymphocytes are incubated per ml of culture medium.

24. A method according to any one of claims 20 to 23, characterized in that step b) is carried out by co-injection of two solutions: - a hydrogel solution, - the solution from step a) comprising lymphocytes and culture medium, concentrically via a microfluidic injector which allows the formation of a jet at the injector outlet consisting of the mixture of the two solutions, said jet breaking down into droplets, said drops being collected in a calcium bath which stiffens the hydrogel solution to form the outer layer of each microcompartment, the inner part of each drop being made up of the solution from step (a) comprising lymphocytes and culture medium.

25. Method according to claim 24, characterized in that the microfluidic injector has a final opening diameter between 150 and 300 pm.

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

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

28. A 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 agitation.

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

30. The process according to the preceding claim, characterized in that each re-encapsulation consists of removing the outer hydrogel layer, preferably resuspending the lymphocytes in a partially or totally dissociated form in the microcompartments and repeating the steps of the process.

31. A method for preparing a microcompartment according to any one of claims 20 to 30, characterized in that it comprises a step after step (c) which consists of freezing the microcompartments.

32. Method for preparing a microcompartment according to any one of claims 20 to 31, characterized in that step (c) is carried out in a bioreactor.

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