FR3134116A1 - Cellular microcompartments comprising lymphocytes forming a 3D cluster culture and having low TNF alpha content - Google Patents

Abstract

The invention relates to a three-dimensional cellular microcompartment or a set of three-dimensional cellular microcompartments of ovoid, cylindrical, spheroidal or spherical or substantially ovoid, cylindrical, spheroidal or spherical shape, the smallest dimension of which is between 200 and 400µm, comprising an external hydrogel layer defining an internal part, said internal part comprising in a medium between 500 and 5000 lymphocytes forming a grouped culture in three dimensions and having a TNF alpha content less than 100ng/mL of medium. The invention also relates to a process for preparing such a microcompartment or set of microcompartments.

Description

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

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 process and its uses.

Prior art

The progress made over the last few decades has highlighted the value of cell therapy for treating numerous pathologies.

Cell therapy is defined as the administration of living cells to a patient with the aim of preventing, treating or alleviating a pathology. The objective is to obtain a lasting effect thanks to an injection of therapeutic cells.

Many cell therapy approaches can be considered, such as:
- the so-called autologous approach in which the patient is his own donor.
- the so-called allogeneic approach in which the donor is different from the patient.

Cell therapy covers broad therapeutic areas and applies to numerous cell types ranging from pluripotent cells to specialized adult cells with metabolic function ( eg hepatocytes) or mechanical function ( eg myoblasts, chondrocytes), including cell lines. hematopoietic cells such as blood mononuclear cells ( eg T lymphocytes or NK lymphocytes).

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 advance 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.

There is also an alternative using NK (natural killer) lymphocytes. NK lymphocytes are very potent cytotoxic effector cells that have demonstrated an antitumor effect.

In order to be able to develop the potential of cell 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 techniques.

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 cellular exhaustion, making it impossible to produce lymphocytes on a large scale. In vivo , immune cell proliferation occurs primarily in mechanically stable environments such as solid tissues. Since the environment of a bioreactor is filled with non-physiological shear stresses, the expansion rates obtained are generally still insufficient compared to in vivo expansion capacities.

Currently, there is no satisfactory solution to rapidly produce functional lymphocytes, in particular CAR-T or CAR-NK cells, on a large scale.

Several culture methods are mainly used for the expansion of CAR cells: static flask culture, gas-permeable static 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 ).

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

To remedy this problem, the prior art proposes activating the lymphocytes before their cultivation in order to obtain rapid proliferation of the lymphocytes.

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

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)

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

Furthermore, this method does not guarantee the functional phenotype of the lymphocytes and therefore that the action of the lymphocytes is lasting.

The complexity of the differentiation and function of each lymphocyte subpopulation means that the analysis of the phenotype during proliferation is essential data to successfully carry out any lymphocyte-based therapy, in particular therapies using CAR- cells. T or CAR-NK.

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

On the other hand, we also know that the effector T lymphocyte subpopulation is essential to guarantee a significant effect on the target cell(s).

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

Current production processes involving lymphocytes are very expensive and difficult to scale up and result in lymphocytes of mixed quality and reduced persistence.

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.

The objective of the invention is therefore to meet all of these needs and to overcome the disadvantages and limitations of the prior art.

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

For this purpose, the subject of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical shape, the smallest dimension of which is between 200 and 400 μm, comprising a 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 a content of endogenous tumor necrosis factor (TNF alpha) less than 100 ng/mL of the medium.

Preferably, the internal part of the microcompartmentµ comprises a perforin content of less than 0.5 µg/mL of the medium and/or a granzyme B content of less than 1 µg/mL of the medium and/or an IL6 content of less than 0, 5 µg/mL of the medium and/or a GM-CSF content of less than less than 0.5 µg/mL of the medium.

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.

According to a particularly suitable embodiment, the microcompartment according to the invention comprises lymphocytes forming a grouped culture in three dimensions without lumen.

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

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

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 lymphocytes advantageously makes it possible to stimulate the proliferation of lymphocytes in the internal part of the microcompartment according to the invention. According to a variant, the activation of lymphocytes can be carried out in the microcompartment after encapsulation.

According to another particularly suitable embodiment, the microcompartment according to the invention comprises lymphocytes expressing a chimeric antigen receptor.

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

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

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

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 medicine, preferably in the prevention or treatment of autoimmune diseases, immunodeficiency syndromes, cancers, viral diseases or inflammatory patients.

Other characteristics and advantages will emerge from the detailed description of the invention and the examples which follow.

Brief description of the figures

is a phase contrast microscopy image at x20 magnification of a set of microcompartments according to the invention after 3 days of cultures in capsules

is a phase contrast microscopy image at x10 magnification of a set of microcompartments according to the invention after 3 days of capsule cultures

is a representation of the results of tests on the amplification of lymphocytes in microcompartments according to the invention.

is a representation of T cell expansion factor after 6 days of culture with or without encapsulation.

is a comparative representation of the cytokine expression profile during the culture of T Lymphocytes after 6 days of culture with or without encapsulation. Results are presented with values normalized to GM-CSF and IFN-g secretion.

is an image obtained by confocal microscopy of a microcompartment presenting polarized lymphocytes.

is a representation of NK lymphocyte expansion factor after 14 days of culture with encapsulation (“Capsules”) or without encapsulation (“Control”).

is a comparative representation of the expression of cytokines during the culture of NK Lymphocytes after 14 days of culture with or without encapsulation (for a donor A– supernatant diluted 10th). Results are presented with values normalized to IFN-g secretion.

is a comparative representation of the expression of granzyme B during the culture of NK Lymphocytes after 14 days of culture with or without encapsulation (for a donor A– supernatant diluted 100th). Results are presented with values normalized to IFN-g secretion.

is a comparative representation of the expression of cytokines during the culture of NK Lymphocytes after 11 days of culture with or without encapsulation (for a donor B – supernatant diluted 10th). Results are presented with values normalized to IFN-g secretion.

is a comparative representation of the expression of granzyme B during the culture of NK Lymphocytes after 11 days of culture with or without encapsulation (for a donor B– supernatant diluted 100th). Results are presented with values normalized to IFN-g secretion.

Detailed description of the invention

Definitions

By “alginate” within the meaning of the invention is meant linear polysaccharides formed from β-D-mannuronate and α-L-guluronate, salts and derivatives thereof.

By “hydrogel capsule” or “hydrogel microcompartment” within the meaning of the invention, is meant a three-dimensional structure formed from a matrix of polymer chains, swollen by a liquid and preferably water.

By “human cells” within the meaning of the invention is meant human cells or immunologically humanized non-human mammalian cells. Even when this is not specified, the cells, stem cells, progenitor cells and tissues according to the invention are constituted or are obtained from human cells or from immunologically humanized non-human mammalian cells.

By “mutant cell” within the meaning of the invention, we mean a cell carrying at least one mutation.

By “Feret diameter” of a microcompartment (or 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, so that the entire projection of said microcompartment (or said part) is included 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, that is to say 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 included between these two parallel tangents.

By “variable thickness” of a layer, we mean for the purposes of the invention the fact that the layer for the same microcompartment does not have the same thickness everywhere.

By “microcompartment” or “capsule” within the meaning of the invention is meant a partially or completely closed three-dimensional structure, containing several cells.

By “medium” within the meaning of the invention is meant an aqueous solution encompassing cells, compatible with the survival, development and/or metabolism of cells. It may be a culture medium.

By “convective culture medium” within the meaning of the invention is meant a culture medium animated by internal movements.

By “mutation” within the meaning of the invention, we mean a genetic or epigenetic mutation, preferably a functional mutation. It may in particular be a one-off modification of the genetic sequence, a structural variant, an epigenetic modification, or a modification of mitochondrial DNA.

By “functional mutation” within the meaning of the invention is meant 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 leading to a modification of the phenotype of the mutant cell concerned. Very preferably it is a change in the genome sequence 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.

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” in the sense of the invention, we mean a volume of aqueous solution topologically surrounded by cells. Preferably its content is not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment.

By “smallest radius” of the internal part of a microcompartment according to the invention is meant half the value of the smallest Ferret diameter of the internal part of the microcompartment.

By “average radius” of the internal part of a microcompartment according to the invention is meant 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.

By “expansion rate at X days” according to the invention is meant 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).

By “large-scale culture” according to the invention is meant a cell culture method adapted 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.

By “functional phenotype” according to the invention is meant lymphocytes presenting a classic membrane marker expression profile (CD3+, CD4+ or CD8+ for T lymphocytes and CD3-, CD56+ for NK lymphocytes), known to man. of the art and an ability to secrete interleukins (IL2, IL6, IL10, IL12) or other factors of interest (granzyme B, INF gamma, GMCSF, and TNF alpha) under the induction of antigen presentation in vitro.

By “progenitor cell” within the meaning of the invention is meant a stem cell already engaged in cellular differentiation into lymphocytes but not yet differentiated.

By “embryonic stem cell” within the meaning of the invention is meant a pluripotent stem cell derived from 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 invention are obtained without destruction of the embryo from which they are derived, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Possibly human embryonic stem cells may be excluded.

By “pluripotent stem cell” or “pluripotent cell” within the meaning of the invention, is meant a cell which has the capacity to form all the tissues present in the entire organism of origin, without being able to form an entire organism in as such. Human pluripotent stem cells may be referred to as hPSCs in the present application. These may in particular be induced pluripotent stem cells (iPSC or hiPSC for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for “Multilineage-differentiating Stress Enduring”).

By “induced pluripotent stem cell” within the meaning of the invention is meant a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are notably positive for markers of pluripotency, such as alkaline phosphatase staining and expression of NANOG, SOX2, OCT4 and SSEA3/4 proteins. 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) .

Cellular microcompartment

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

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 secreting little or no TNF alpha.

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 environment external to the microcompartment when the latter is in an environment. Indeed, molecules less than 150KDa are in osmotic equilibrium between the internal part of the microcompartment and the environment in which the microcompartment is included.

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

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

According to a preferred embodiment, the internal part of the microcompartment comprises a granzyme B content less than 1µg/mL of medium, in particular less than 0.5µg/mL of medium, more preferably less than 0.1µg/mL, even more preferably less than 50ng/mL. The microcompartment according to the invention therefore comprises at least lymphocytes secreting little or no granzyme B.

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

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

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 μg/ml of medium, preferably less than 0.1 μg/ml, more preferably less than 50ng /mL, even more preferably less than 10ng/mL.

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

According to one embodiment, the subject of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical 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 grouped culture in three dimensions and having a content of less than 100 ng/mL of medium and:
- a perforin content of less than 0.5 μg/ml of medium, and/or
- a granzyme B content of less than 1µg/mL of medium.

According to one embodiment, the subject of the invention is a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical 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 grouped culture in three dimensions and having a TNF-α content of less than 100 ng/mL of medium and:
- a perforin content of less than 0.5 μg/ml of medium, and/or
- a granzyme B content of less than 1 μg/mL of medium, and/or
- an IL6 content of less than 0.5 μg/ml of medium and/or
- a GM-CSF content of less than 0.5 μg/ml of medium.

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 10µg/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.

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 by other cells (notably 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 the target cells.

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

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

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 10µg/mL, even more preferably less than 3ng/mL is particularly sought after in the context of lymphocyte culture.

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

The same applies for a concentration greater than 0.5 μg/ml IL6 and/or a concentration greater than 0.5 μg/ml GM-CSF for NK lymphocytes. IL6 in particular is known to reduce 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(11) :3037-46. doi: 10.1002/art.39295.). Thus, a reduction in the concentration of IL6 advantageously makes it possible to increase the effectiveness of NK.

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

The microcompartment according to the invention advantageously has an expansion rate of T lymphocytes of at least 20 times after 3 days of culturing, and an expansion rate of NK lymphocytes of at least 40 times after 14 days of culturing. in culture.

The microcompartment according to the invention comprises an external hydrogel layer. Preferably the hydrogel used is biocompatible, that is to say it is not toxic to cells. The outer hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartment and allow their survival. According to one embodiment, the external hydrogel layer comprises at least alginate. It can 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.

The outer hydrogel layer makes it possible in particular to protect the cells from the mechanical stress of bioreactors and to limit the anarchic secretion by lymphocytes in culture of potentially toxic cytokines when accumulated in the medium.

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

The microcompartment according to the invention can be obtained after encapsulation of lymphocytes without addition of natural or synthetic extracellular matrix.

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

According to a variant, the microcompartment according to the invention can be obtained after encapsulation of lymphocytes without adding any extracellular matrix element. The extracellular matrix elements may 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.

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.

Advantageously, the absence of extracellular matrix element improves lymphocyte expansion rate.

Within the microcompartment, the lymphocytes group together in three dimensions preferentially in clusters or clusters (“clusters”).

Preferably, the microcompartment according to the invention comprises lymphocytes forming a grouped culture in three dimensions without lumen.

The absence of lumen advantageously makes it possible to limit the autocrine and paracrine signals of lymphocytes, making their phenotype particularly stable from their encapsulation.

Advantageously, the absence of lumen also makes it possible to better control the size of the groups of lymphocytes present in the form of masses or clusters in the microcompartment.

The microcompartment according to the invention can be obtained after encapsulation of previously activated lymphocytes, thus making it possible to increase their expansion rate by stimulating their proliferation. Activation can be carried out by bringing the lymphocytes into contact with Artificial antigen presenting cells called aAPC (artificial antigen presenting cells) very preferentially via CD3/CD28 antibodies for T lymphocytes, via CD2/NKp46 for lymphocytes NK.

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.

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

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, e.g. activation beads coated with suitable antibody(ies) (e.g. Cloudz Human NK Cell Expansion Kit (R&D Systems ) or NK Cell Activation/Expansion Kit (Miltenyi Biotec)).

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

Advantageously, activation is carried out in the microcompartment by controlling the ratio of the number of lymphocytes / quantity of antigen necessary so as to activate the lymphocytes while limiting cellular exhaustion.

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

Thanks to the mechanical isolation provided by the microcompartment, the activation of lymphocytes cannot be done homogeneously or continuously. In this way, activation approaches the conditions of in vivo activation of lymphocytes, which promotes their polarization.

Preferably, the lymphocytes forming the 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 in the cell. Furthermore, polarized lymphocytes can present anisotropies in the distribution of the Dlg, Scrib and Lgl proteins.

Lymphocyte polarization in the microcompartment may be an indicator of functional phenotype. Indeed, if a lymphocyte contains a sufficient quantity of granules and vesicles comprising interleukins, in particular TNF alpha, then the lymphocytes are polarized.

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

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

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

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

A naive lymphocyte and/or Tscm concentration of less than 20% would indicate that the lifespan of the lymphocytes is too short and does not guarantee a persistent effect.

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.

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 hydrogel layer of at least one lymphocyte.

Preferably, the lymphocytes present in the microcompartment according to the invention were obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division after encapsulation in an external layer of hydrogel, 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 were obtained after at least six cycles of cell division after the encapsulation of the cells in the outer 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.

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

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, a re-encapsulation corresponds to a new passage and each encapsulation cycle corresponds to a passage.

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.

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

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.

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 cell encapsulation. Preferably the microcompartment according to the invention is in a spherical or elongated or substantially spherical or elongated shape. It may have the shape of an ovoid, a cylinder, a spheroid or a sphere or substantially this shape.

It is the outer layer of the microcompartment, that is to say the hydrogel layer, which gives its size and shape to the microcompartment according to the invention. 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.

Its largest dimension is preferably greater than 10 µm, more preferably between 10 µm and 1m, even more preferably between 10 µm and 50 cm.

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

Preferably, the lymphocytes included in the microcompartment according to the invention express a chimeric antigen receptor.

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

The microcompartment according to the invention can optionally be frozen for storage. It should then preferably be thawed before use.

The invention also relates to several microcompartments together.

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

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 culturing lymphocytes can be used, such as for example “TexMACS (Miltenyi Biotec)” or “VIVO 15-20 (Lonza)” or “CTS™ OpTmizer™ (ThermoFisher)” or NKMACS ( Miltenyi Biotec) or ExCellerate Human NK Cell Expansion Media (R&D Systems), as long as the concentration of dissolved salts is compatible with maintaining the crosslinking of the alginate by the divalent cations.

According to a particularly suitable embodiment, the subject of the invention is 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, preferentially, the microcompartments are arranged in a culture medium in a closed bioreactor.

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

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

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 patients.

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

According to a particular embodiment, the invention aims at a closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical 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 forming a three-dimensional grouped culture in a medium comprising:
- a TNF alpha content less than 100ng/mL of medium; And
- a granzyme B content of less than 1µg/mL of medium; and or
- a perforin content of less than 0.5µg/mL of medium.

In this embodiment, the proportion of naïve T lymphocyte and/or Tscm remains greater than 20%.

Preferably, the microcompartment is obtained after encapsulation of lymphocytes with the addition of extracellular matrix or without the addition of extracellular matrix element.

Process for preparing microcompartments according to the invention

The invention also relates to a process for preparing microcompartments according to the invention.

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.).

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

The process for preparing a microcompartment or a set of microcompartments according to the invention may comprise the following steps:
-(a) incubate the lymphocytes in a culture medium comprising cytokines
-(b) encapsulating the lymphocytes washed in a culture medium in a hydrogel layer, preferably without adding extracellular matrix or without adding an extracellular matrix element, to form a closed three-dimensional microcompartment of ovoid, cylindrical shape, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical, the smallest dimension of which is between 200 and 400µm, so that each microcompartment includes at least 5 lymphocytes in its internal part.
-(c) cultivate the microcompartments obtained in step (b) in a culture medium comprising cytokines, for 2 to 6 days.

Incubation step (a) of the process 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.

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

Preferably, the preparation process 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.

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 can be used, such as the “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.

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

The cytokines contained in the culture medium may for example be 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 or other cytokines known to those skilled in the art to stimulate the proliferation of lymphocytes.

Preferably, the culture media used in the preparation process 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.

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.

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

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 groupings of lymphocytes should not be too large in relation to the size of the microcompartment because too large a dimension of these initial cell clusters could lead, during cell divisions, to an 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.

Encapsulation step (b) is implemented according to techniques known to those skilled in the art. Indeed, any method of producing cellular microcompartments containing cells and cells inside a hydrogel capsule can be used for implementing the preparation process 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, p. 1593-1604) , following the steps described below.

Preferably step (b) is implemented without adding extracellular matrix, natural or synthetic. According to a variant, step (b) is implemented without adding any extracellular matrix element.

Preferably step (b) is implemented in a device capable of generating hydrogel capsules using a microfluidic chip. For example, the device can include syringe pumps for several solutions injected concentrically using a microfluidic injector which makes it possible to form a jet which is split into drops then collected in a calcium bath. According to a particularly suitable embodiment, two or three solutions loaded on two or three syringe pumps:
- a hydrogel solution, for example alginate,
- 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,
- the solution resulting from step (a) comprising lymphocytes and culture medium.

The three solutions are co-injected (injected simultaneously) concentrically using a microfluidic injector or microfluidic chip which makes it possible to form a jet which is split into drops whose outer layer is the hydrogel solution and the core is the hydrogel solution. 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.

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

According to the invention, it is necessary to generate capsules whose internal part has an average radius or smaller radius of at least 100µm. 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 co-extrusion chip. By flow rate we mean the flow rate of each solution which arrives at the injector. By final opening of the co-extrusion chip is meant the internal opening of the output channel of the chip.
- for the flow rate: we preferentially 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,
- for the diameter of the final opening of the coextrusion chip (microfluidic injector) which goes from a value preferably between 50 and 120 µm for the standard sizes of capsules known from the prior art, to a diameter value comprised between 150 and 300 µm, preferably between 180 and 240 µm.

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

According to one embodiment, steps (a) and/or (c) are carried out with permanent or sequential stirring. This agitation is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusive gradient. For example, it allows homogeneous control of cellular oxygenation level; thus avoiding the phenomena of necrosis linked to hypoxia, or oxidative stress linked to hyperoxia.

The process according to the invention is preferably implemented in a closed enclosure such as a closed bioreactor.

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

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

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

Each re-encapsulation may include:
- a step which consists of dissociating the microcompartment or the series of microcompartments to obtain a suspension of lymphocytes or a suspension of clusters of lymphocytes; the elimination of the external hydrogel layer can be carried out in particular by hydrolysis, dissolution, drilling and/or rupture by any biocompatible means, that is to say non-toxic for the cells. For example, removal can be accomplished using phosphate buffer saline, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel includes alginate and/or laser microdissection, and
-a step of activating the lymphocytes using an artificial activation system (aAPC) preferably using CD3/CD28 antibodies;
-a step of re-encapsulating all or part of the lymphocytes or clusters of lymphocytes in a hydrogel capsule. Re-encapsulation is a suitable means for increasing cellular amplification obtained from the pluripotent stage, and reducing the risk of mutation.

Re-encapsulation consists of removing the outer layer of hydrogel, preferably resuspending partially or totally dissociated cells which were in the form of cysts in the microcompartments and restarting the steps of the process.

According to one embodiment, re-encapsulation comprises the following steps:
- (i) remove the outer hydrogel layer,
- (ii) resuspend 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
- (iii) encapsulate the lymphocyte solution in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical shape, comprising an external hydrogel layer defining an internal part , the smallest radius or the average radius of said internal part being at least 100 µm;
- (iv) cultivate the microcompartments obtained in a culture medium containing cytokines,
- (vi) preferably rinse the microcompartments, so as to eliminate the cytokines;
- (vi) cultivating the microcompartments in a culture medium devoid of cytokines, for at least one cycle of cell division, and
- (vii) optionally recover the cellular microcompartments obtained.

Compartmentalization in microcompartments makes it possible to eliminate microcompartments containing more mutated cells than other capsules. Even if the mutated cells grow rapidly, they will reach the capsular confluence which will contain their multiplication. Compartmentalization also makes it possible not to contaminate the entire cell population, and also to eliminate capsules containing mutant cells, at any time, in particular before a re-encapsulation step. This sorting can be done either by online analysis, or by eliminating filled capsules more quickly than others, for example.

In one mode of implementation, at least one of the steps (preferably all the steps) is carried out at a temperature adapted to the survival of the cells, between 4 and 42°C. The temperature during cell proliferation should preferably be between 32 and 37°C to avoid triggering mutations by reducing the performance of repair enzymes. Likewise, preferably, the temperature must be low (ideally around 4°C) to manage the stress of the cells in step (c).

At any time, the method according to the invention may include a step consisting of checking the phenotype of the cells contained in the microcompartment. This verification can be carried out by identifying the expression by at least part 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, D56, CD16.

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 (screwable vial or plastic bag) in a lukewarm water bath (preferably 37 degrees) so that the cells thaw fairly quickly. The microcompartments according to the invention before their use can be maintained at more than 4°C for a limited period before their use, preferably between 4°C and 38°C.

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.

The invention especially favors 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.

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 differentiating cells capable of differentiating into lymphocytes into lymphocytes. (stem cells, including induced pluripotent stem cells, hematopoietic stem cells including CD34+ cells, etc.).

The cells capable of differentiating into lymphocytes are preferentially chosen from:

* 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,
* either progenitor cells capable of differentiating into lymphocytes, at least into T lymphocytes or NK lymphocytes,
*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.

In this variant, the method may include at least the implementation of the steps which consist of:

-produce a cellular microcompartment comprising, inside a hydrogel capsule:
* preferably at least extracellular matrix elements or an extracellular matrix, natural or synthetic,
* cells capable of differentiating into lymphocytes, preferably induced pluripotent stem cells (IPS),
- induce cell differentiation into lymphocytes within the cellular microcompartment, so as to obtain at least lymphocytes.

Example 1:P processed according to the invention (T lymphocytes)

Reagents used :
-Solution 1: Culture medium used: TexMACS (Miltenyi Biotec) supplemented with 1000 units/ml of recombinant IL-2 (Miltenyi Biotec)
-Solution 2: CD3/CD28 polyantigenic activation reagent (Transact™ Miltenyi Biotec)
-Solution 3: Capsule rinsing medium: DMEM/F-12 + Glutamax
-Solution 4: Solution to dissolve alginate capsules: non-enzymatic cell detachment buffer (RelesR™ Stem Cell Technology)

Step 1: thawing the cells (J 0 )

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

Step 2: Activation of T lymphocytes (D0)

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

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

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

Step 3: Change of environment and encapsulation (D3):

In order to eliminate the Transcart™ in the medium, the cells were centrifuged at 300 g for 10 min and the supernatant aspirated.

Cells were rinsed in solution 1 once.

Part of the cell pellet was taken up in solution 1 at a cell density of 5 million cells per ml for encapsulation.

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

Step 4: Encapsulation (J3)

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).

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 which makes it possible to form a jet which is split into drops whose outer layer is the alginate solution and the core is the cell solution; These drops are collected in a calcium bath (at 100mM) which stiffens the alginate solution to form the shell.

The invention especially favors 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 further mutagenesis.

Step 5: treatment after encapsulation (D3)

The capsules are recovered with a 40µm cell sieve then after rinsing (1x) with solution 3 they are stored in a 75cm² flask with solution 1 having a cell density of 100,000 cells per ml (condition 1) or 200,000 cells per ml (condition 2).

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

The non-encapsulated cells as prepared during the 3rd step are cultured in parallel under the same atmospheric conditions for 3 days.

Step 6: Dissolving the capsules and counting (D6)

-(a): Arm 1: capsule
-The capsule suspension was taken and left to sediment for 5 minutes in a 50ml Falcon tube.
-The supernatant was carefully collected and discarded.
-The capsules were then treated with solution 4 for 5 min at 37°C.
-The cells released from the capsules were centrifuged at 300 g for 10 minutes to eliminate solution 4.
-The cells were finally counted using the NC2000 (standard 1 cassette protocol)

-(b): Arm 2: suspended control
-The cell suspension was collected then centrifuged at 300g for 10min.
-The supernatant was aspirated.
-The cells were then counted using the NC2000 (standard 1 cassette protocol).

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

Example 2: Measure of there secretion of cytokines of lymphocytes T encapsulated or not

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

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

The culture supernatants of the encapsulated or non-encapsulated cells were taken on day 6 later, undiluted, and incubated in duplicate and incubated in the plate prepared in step 2 of Example 1;

The incubation was carried out in accordance with the supplier's recommendations;

Data acquisition was carried out on a MACSQuant 10 cytometer (Miltenyi Biotec) using a dedicated Express Mode (Miltenyi Biotec).

The results are expressed as average fluorescence per million cells (MFI/e6 cells) in Table 1 and in the (results normalized to GM-CSF and IFNg secretion).

Undiluted supernatant GM-CSF Granzyme B IFNg IL-21 IL-6 MCP-1 Perforin TNF-α T lymphocytes in suspension 499.3 1339.4 28.9 1.1 4.6 18.9 65.4 108.5 Encapsulated T lymphocytes (invention) 22.2 0.9 1.2 0.1 0.6 1.6 1.8 0.2 Suspended T cells normalized to GMCSF and IFNg secretion 1.890571753 5.071563802 0.109428247 0.004165089 0.017417645 0.071563802 0.247633472 0.410829231 Encapsulated T cells normalized to GMCSF and IFNg secretion 1.897435897 0.076923077 0.102564103 0.008547009 0.051282051 0.136752137 0.153846154 0.017094017 Invention/suspension ratio 1.003630724 0.015167526 0.937272647 2.052059052 2.94425864 1.910912133 0.621265585 0.041608571

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

Example 3: Polarization of a microcompartment according to the invention

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

Marking protocol:

Phalloidin and Lipilight (Membright) labeling were carried out at the dilutions recommended by the suppliers for 72 hours with stirring, at room temperature and protected from light.

Image acquisition ( ) was carried out using confocal microscopy.

We can observe a microcompartment according to the invention comprising lymphocytes whose nucleus is eccentric. Consequently, lymphocytes are polarized within the microcompartment and thus exhibit a functional phenotype.

Example 4: Process according to the invention (NK lymphocytes)

Reagents used:

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

-Solution 2: Solution 1 supplemented with CD2/NKp46 polyantigenic activation beads (e.g. Cloudz Human NK Cell Expansion Kit (R&D Systems))

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

-Solution 4: Solution to dissolve alginate capsules: 2.5mM EDTA solution

Step 1: thawing the cells (J 0 )

Ampoules of NK sorted from peripheral blood of healthy donors (donor A and donor B) were thawed according to the supplier's recommendations (Hu PB NK, Stem Cell Technologies, #70036).

NK were counted using the NucleoCounter NC2000 (Standard 1 cassette protocol).

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

Part of the cell pellet was taken up in solution 2 at a cell density of 15 million cells per ml for encapsulation.

The other part of the cell pellet was taken up in solution 2 at a cell density between 0.2 and 1 million cells per ml (control).

Stage 2 : Encapsulation (J 0 )

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

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 which makes it possible to form a jet which is split into drops whose outer layer is the alginate solution and the core is the cell solution; These drops are collected in a calcium bath (at 100mM) which stiffens the alginate solution to form the shell.

The invention especially favors 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 further mutagenesis.

Stage 3 : treatment after encapsulation (J 0 )

The capsules are recovered with a 40µm cell sieve then after rinsing (1x) with solution 3 they are stored in a 75cm² flask (Donor A) or in a 30mL mini-bioreactor (Donor B) with solution 1 at a density cell size between 200,000 and 1,000,000 cells per ml.

The 30mL flask or mini-bioreactor is kept in the incubator at 37°C and 5% CO2 for 11 to 14 days. The capsules in a mini-bioreactor are stirred at 100 RPM. Renewal of the culture medium is carried out according to the suppliers' recommendations.

The non-encapsulated cells as prepared during the 3rd step are cultured in parallel under the same conditions for 11 to 14 days.

Step 6: Dissolving the capsules and counting (J 11 or D14, depending on the confluence of the capsules )

-(a): Arm 1: capsule

-The capsule suspension was taken and left to sediment for 5 minutes in a 50ml Falcon tube.

-The supernatant was carefully collected and discarded.

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

-The cells released from the capsules were centrifuged at 300 g for 10 minutes to eliminate solution 4.

-The activation beads are disposed of according to the supplier's recommendations.

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

-(b): Arm 2: suspended control

-The cell suspension was collected then centrifuged at 300g for 10min.

-The supernatant was aspirated.

-The activation beads are disposed of according to the supplier's recommendations.

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

The results of NK encapsulation according to the protocol describe better cellular amplification in capsules compared to a conventional suspension culture, as illustrated in the . Thus, for donor A, we obtain an amplification factor of 40 times in 14 days of capsule culture compared to a factor of 3 for a conventional culture. For donor B, we obtain an amplification factor of 110 times in 11 days of culture in capsules compared to a factor of 70 times for a conventional culture.

Example 5 : Measurement of lymphocyte cytokine secretion N.K. encapsulated or not

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

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

The culture supernatants of the encapsulated or non-encapsulated cells were taken 14 days later, diluted ^10th or ^100th , and incubated in duplicate in the plate prepared in step 2 of Example 4;

The incubation was carried out in accordance with the supplier's recommendations;

Data acquisition was carried out on a MACSQuant 10 cytometer (Miltenyi Biotec) using a dedicated Express Mode (Miltenyi Biotec).

The results are expressed as average fluorescence per million cells (MFI/e6 cells) in Tables 2 and 3 and in Figures 7a, 7b (Donor A) and 8a, 8b (Donor B).

Supernatant diluted 10th GM-CSF IL-10 IL-17A IL-6 MCP-1 Perforin NK, donor A in suspension 168.3 1.2 8 635 3.8 94.7 NK, donor A in capsule (invention) 14.8 5.5 0.8 25 7.7 27 NK, donor B in suspension 60.9 3.9 2.7 9.6 2.8 110.3 NK, donor B in capsule (invention) 3.3 0.3 0.9 0.5 0.4 3.5 NK, donor A suspension normalized to IFNg secretion 0.014763158 0.000105263 0.000701754 0.055701754 0.000333333 0.008307018 NK, donor A in capsule normalized to IFNg secretion 0.001679337 0.000624078 9.0775E-05 0.002836718 0.000873709 0.003063656 NK, donor B suspension normalized to IFNg secretion 0.022989807 0.001472254 0.001019253 0.003624009 0.001057003 0.041638354 NK, donor B in capsule normalized to IFNg secretion 0.013414634 0.001219512 0.003658537 0.00203252 0.001626016 0.014227642 Invention/donor suspension ratio A 0.113751906 5.928741632 0.129354363 0.050926915 2.621127879 0.368803358 Invention/donor suspension ratio B 0.583503544 0.828330206 3.589430894 0.560848577 1.538327526 0.341695597

Supernatant diluted 100th Granzyme IFNg NK, donor A in suspension 3700 11400 NK, donor A in capsule (invention) 208 8813 NK, donor B in suspension 474 2649 NK, donor B in capsule (invention) 4 246 NK, donor A suspension normalized to IFNg secretion 0.324561404 1 NK, donor A in capsule normalized to IFNg secretion 0.023601498 1 NK, donor B suspension normalized to IFNg secretion 0.178935447 1 NK, donor B in capsule normalized to IFNg secretion 0.016260163 1 Invention/donor suspension ratio A 0.072718128 1 Invention/donor suspension ratio B 0.090871668 1

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.

Claims (31)

Closed three-dimensional cellular microcompartment of ovoid, cylindrical, spheroid or spherical 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 part internal comprising in a medium between 500 and 5000 lymphocytes forming a grouped culture in three dimensions and having a TNF alpha content less than 100ng/mL of medium, the microcompartment not comprising embryonic stem cells of human beings. Microcompartment according to the preceding claim, characterized in that the internal part comprises lymphocytes forming a grouped culture in three dimensions without lumen. Microcompartment according to 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. Microcompartment according to one of the preceding claims, characterized in that it was obtained after the encapsulation of previously activated lymphocytes. Microcompartment according to one of the preceding claims, characterized in that the thickness of the external layer is variable and between 20 and 60µm. Microcompartment according to one of the preceding claims, characterized in that the lymphocytes are polarized. Microcompartment according to one of the preceding claims, characterized in that the number of lymphocytes forming a grouped culture in three dimensions is between 1000 and 3000. Microcompartment according to one of the preceding claims, characterized in that the lymphocytes are T lymphocytes or NK lymphocytes. Microcompartment according to one of the preceding claims, characterized in that the lymphocytes are lymphocytes expressing a chimeric antigen receptor. Microcompartment according to one of the preceding claims, characterized in that the lymphocytes are T lymphocytes and in that it presents:
- a perforin content of less than 0.5 μg/ml of medium, and/or
- a granzyme B content of less than 1 μg/mL of medium. Microcompartment according to one of claims 1 to 9, characterized in that the lymphocytes are NK lymphocytes and in that it presents:
- a perforin content of less than 0.5 μg/ml of medium, and/or
- a granzyme B content of less than 1 μg/mL of medium, and/or
- an IL6 content of less than 0.5 μg/ml of medium and/or
- a GM-CSF content of less than 0.5 μg/ml of medium. Microcompartment according to one of the preceding claims, characterized in that the external layer comprises alginate. Microcompartment according to one of the preceding claims, characterized in that the internal part comprises a solution comprising IL2 or the combination of IL7 and IL15 or IL12, IL18, IL21 or the combination of at least two selected cytokines among IL2, IL12, IL18 and IL21. Microcompartment according to one of the preceding claims, characterized in that it comprises T lymphocytes and in that the proportion of naive T lymphocyte and/or Tscm remains greater than 20%. Set of microcompartments comprising at least two cellular microcompartments in three dimensions, characterized in that at least one microcompartment is a microcompartment according to one of claims 1 to 14. Set of microcompartments according to the preceding claim, characterized in that the microcompartments are arranged in a culture medium in a bioreactor. Microcompartment according to one of claims 1 to 14, for its use as a medicine. Microcompartment according to one of claims 1 to 14, for its use in the prevention or treatment of autoimmune diseases, immunodeficiency syndromes, cancers, viral diseases or inflammatory diseases. Process for preparing a microcompartment according to one of claims 1 to 14 or a set of cellular microcompartments according to one of claims 15 or 16, comprising the following steps:
(a) incubating lymphocytes in a culture medium comprising cytokines,
(b) encapsulating 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 or substantially ovoid, cylindrical, spheroid or spherical, the smallest dimension of which is between 200 and 400µm, so that each microcompartment includes at least 5 cells in its internal part,
(c) cultivating the microcompartments obtained in step (b) in a culture medium comprising at least cytokines, for 2 to 6 days. Process for preparing a microcompartment according to the preceding claim, characterized in that it comprises a step prior to the incubation of the lymphocytes, which consists of activating said lymphocytes. Process for preparing a microcompartment according to one of claims 19 or 20, characterized in that the culture media comprise from 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. Process for preparing a microcompartment according to one of claims 19 to 21, characterized in that in step (a) between 0.5 and 2 million lymphocytes are incubated per ml of culture medium. Method according to one of claims 19 to 22, characterized in that step b) is carried out by co-injection of two solutions:
- a hydrogel solution,
- the solution resulting from step a) comprising lymphocytes and culture medium,
concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of the two solutions, said jet breaking into drops, said drops being collected in a calcium bath which stiffens the hydrogel solution to form the external layer of each microcompartment, the internal part of each drop being constituted by the solution resulting from step (a) comprising lymphocytes and culture medium. Method according to claim 23, characterized in that the microfluidic injector has a final opening diameter of between 150 and 300µm. Method according to one of claims 19 to 24, characterized in that the flow rate of each of the two solutions is between 45 and 150 mL/h. Method according to one of claims 19 to 25, characterized in that all of the cells initially encapsulated in step (b) represent a volume less than 50% of the volume of the microcompartment in which they are encapsulated. Method according to one of claims 19 to 26, characterized in that steps (a) and/or (c) are carried out with permanent or sequential stirring. Method according to one of claims 19 to 27, characterized in that the method comprises at least one re-encapsulation of the cells after step (c). Method according to the preceding claim, characterized in that each re-encapsulation consists of eliminating the external layer of hydrogel, preferably resuspending the lymphocytes in partially or totally dissociated form in the microcompartments and restarting the steps of the process. Process for preparing a microcompartment according to one of claims 19 to 29, characterized in that it comprises a step after step (c) which consists of freezing the microcompartments. Process for preparing a microcompartment according to one of claims 19 to 30, characterized in that step (c) is carried out in a bioreactor.