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  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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  • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

• the invention relates to a method for preparing microcompartmental cells comprising malignant haematopoietic cells.
• the invention also relates to such cellular micro-compartments and to their use, in particular in the pharmaceutical field, for the screening and identification of molecules of interest likely to treat a hematological malignancy.
• Cancers of hematopoietic tissues, or hematological malignancies, are characterized by a disorder of the multiplication and differentiation of cells of a blood line.
• the most common malignant hemopathies include leukemias and lymphomas.
• Leukemia is a cancer of bone marrow cells, which is characterized by abnormal and massive proliferation of precursors of incompletely differentiated white blood cells, to the detriment of red blood cells, normal white blood cells and platelets.
• ALL acute lymphoblastic leukemia
• CLL chronic lymphocytic leukemia
• AML acute myeloblastic leukemia
• CML chronic myeloid leukemia
• Lymphoma is a group of cancers of the lymphatic system that originates in a secondary lymphoid organ and can spread to all parts of the lymphatic system.
• lymphoma There are two main types of lymphoma: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL).
• NHLs are cancers whose incidence has been increasing for 40 years in developed countries and which ranks as the 10th most cancers in terms of frequency.
• lymphomas are formed and evolve within the secondary lymphoid organs comprising several types of cells (microenvironmental cells and lymphoma cells), which interact with each other within an extracellular matrix via soluble and membrane molecules, and are subjected to bio-mechanical forces. All of these factors affect the development of lymphoma, but also the response to treatment. However, 2D models do not reproduce these phenomena and are therefore only weakly representative of the pathophysiological processes of lymphomas.
• animal models have been developed in which human cancer cells are grafted or injected. Such animal models are however expensive, difficult to reproduce, and generally not representative of the physiological phenomena of human hematological malignancies.
• 3D cancer cell cultures have been developed. These 3D cultures are particularly interesting for studying the mechanisms of progression of cancers and better testing anti-cancer treatments. Indeed, cells cultured in 3D within a matrix or in aggregates, have an architecture closer to the tissue and the tumor and show an expression of their genes similar to that of the tumor in vivo (Gravelle et al, 2014 Am. J. Pathol 184: 2082-295, Weiswald et al., 2009, Br. J. Cancer 101: 473-482). In addition, models of 3D co-cultures mimicking cancer cell / stromal cell interactions can at least partially reproduce the tumor niche and study the consequences on tumor progression or drug resistance.
• 3D models of lymphomas are generally obtained using collagen sponges (Kobayashi et al., Trends Immunol 31: 422-428), the so-called “hanging drop” technique (Gravelle et al., 2014. Am., J. Pathol. : 282-295) or a polystyrene architecture (Caicedo-Carvajal et al. J. Tissue Eng 2011: 362326).
• these techniques have many disadvantages, both in terms of costs and reproducibility, making them irrelevant for the industrial scale study of new drugs.
• the developed 3D cultures do not include any element of the tumor microenvironment, thus limiting their relevance.
• the inventors have found that by coextruding a hydrogel solution with a solution of cells comprising lymphoma cells, and optionally lymphoid-type stromal cells, i.e., close to stromal cells that infiltrate lymphomas, and extracellular matrix, said cells aggregated within the hydrogel capsule to organize themselves into a cell mass approximating the cellular organization within a lymphoma. Furthermore, depending on the type of cells coextruded with the hematopoietic malignant cells, the inventors have discovered that it is possible to recreate a tumor niche mimicking a tumor niche in vivo.
• microcompartments in which the nature of the cells and the intercellular interactions are substantially similar to those observed within a lymphomatous tumor niche, or leukemia.
• the cellular microcompartments developed according to the invention are particularly relevant as 3D models of hematological malignancies, in particular for the screening and the identification of new candidate molecules for the treatment of lymphomas and / or leukemias.
• the process according to the invention makes it possible to obtain very large quantities of microcompartment of perfectly controlled dimensions.
• the microcompartments obtained are easy to manipulate, making them particularly suitable for use on a large scale, particularly in the pharmaceutical field.
• the subject of the invention is therefore a method for preparing a cellular microcompartment comprising an aggregate of cells containing malignant hematopoietic cells encapsulated in a hydrogel layer, according to which a hydrogel solution and a solution of cells comprising hematopoietic cells. malignancies are co-extruded concentrically and then crosslinked.
• the cell solution comprises lymphoma cells or leukemic cells.
• the invention also relates to a microcompartment cell capable of being obtained by the method according to the invention, wherein said microcompartment comprises an aggregate of cells comprising at least malignant haematopoietic cells, encapsulated in a hydrogel layer.
• a cellular microcompartment consists of a single cell aggregate encapsulated in a hydrogel layer.
• said microcompartment comprises an aggregate of cells, consisting solely of lymphoma cells, encapsulated in a hydrogel layer. In another embodiment, the microcompartment comprises an aggregate of cells, consisting solely of leukemic cells, encapsulated in a hydrogel layer.
• said microcompartment comprises an aggregate of cells composed in particular of lymphoma cells and lymphoid-type stromal cells, as well as an extracellular matrix layer between the cell aggregate and the hydrogel layer.
• said microcompartment comprises an aggregate of cells composed in particular of leukemic cells and medullary-type stromal cells, as well as an extracellular matrix layer between the cell aggregate and the hydrogel layer.
• the invention also relates to a cellular microcompartment comprising a cell aggregate encapsulated in a hydrogel layer, wherein the cell aggregate comprises malignant hematopoietic cells, such as lymphoma cells or leukemic cells, and stromal cells, said microcompartment further comprising an extracellular matrix layer between the cell aggregate and the hydrogel layer.
• a cellular microcompartment comprising a cell aggregate encapsulated in a hydrogel layer, wherein the cell aggregate comprises malignant hematopoietic cells, such as lymphoma cells or leukemic cells, and stromal cells, said microcompartment further comprising an extracellular matrix layer between the cell aggregate and the hydrogel layer.
• the invention also relates to a method for screening or identifying a compound for the treatment and / or prevention of a lymphoma comprising the steps of: (a) contacting a cellular microcompartment according to the invention, optionally free of a hydrogel layer, with a test compound;
• the invention also relates to a use of a cellular microcompartment according to the invention for the screening or identification of a compound for the treatment of a hematological malignancy, such as lymphoma or leukemia.
• Figure 1 Encapsulation of SUDHL4 and HLY1 lymphoma cells in an alginate capsule.
• SUDHL4 and HLY1 cells express GFP.
• Figure 1A shows images of the phase contrast and fluorescence capsules at different times (J1-J11) after encapsulation, the images being acquired with an Olympus CKX41 microscope (x10 objective).
• Figure 1B shows the growth curves measured for the cell clusters inside the alginate capsules from the photos, using the ImageJ® software.
• FIG. 2 Cell microcompartment formation according to the invention comprising only lymphomatous or leukemic cells (A) or lymphomatous or leukemic cells, and stromal cells (B).
• A lymphomatous or leukemic cells
• B stromal cells
• 1 hydrogel capsule
• 2 capsule light
• 3 lymphoma cells
• 4 growth phase
• S hydrogel capsule containing a cluster of lymphoma cells
• 6 dissolution step of the hydrogel capsule
• 7 cluster of lymphoma cells
• 8 extracellular matrix layer
• 9 stromal cells
• 10 growth phase
• 11 hydrogel capsule containing a cluster of lymphoma and stromal cells
• 12 cluster of lymphoma and stromal cells.
• FIG 3 Microcompartmental cells comprising a cluster of follicular lymphoma cells of the DOHH2 line and of lymphoid-type stromal cells (RESTO, Ame-Thomas Blood 2007; 109: 693) in an alginate capsule covered with an internal layer of Matrigel ® to J9 after encapsulation. The images were obtained in phase contrast using a Leica DMI8 microscope (x10 objective).
• Figure 4 Dissolution of the cell microcellular alginate capsule comprising a cluster of follicular lymphoma cells of line DOHH2 (A) and a cluster of follicular lymphoma cells of line DOHH2 and RESTO cells (B) on day 9 after encapsulation.
• Figure S Flow cytometric analysis of dead cells in clusters of cells comprising SUDHL4 (SA) lymphoma cells or HLY1 (B) lymphoma cells after different encapsulation times.
• Figure 6 Analysis of the residual effect of the stromal tumor niche on the growth of lymphoma cells. Images of cellular micro-compartments in an alginate capsule coated with an internal Matrigel® layer comprising only RESTO (A) cells, only DOHH2 (B) lymphoma cells, RESTO cells and DOHH2 (C) lymphoma cells. The images were obtained in phase contrast using a Leica DMI8 microscope (x10 objective). The scale bar is the same for all three images.
• FIG. 7 Comparative effects of Etoposide (A) and Cisplatin (B) on cell death after contacting, for 48 hours, a suspension of HLY1 cells (suspension) or a HLY1 cell cluster derived from a cellular microcompartment according to the invention, comprising only lymphomatous cells, with increasing doses of etoposide ( ⁇ g / ml) or cisplatin ( ⁇ M).
• FIG. 8 Microcompartment cell showing differentiation of stromal cells into pro-tumoral lymphoid stroma.
• Cellular microcomponents comprising a cluster of follicular lymphoma cells of the DOHH2 line and lymphoid-type stromal cells RESTO in an alginate capsule coated with an internal layer of Matrigel® at D8 after encapsulation.
• CD20 (revealing B-cell), GFP (revealing RESTO cells) and TG2 markings were made on a fixed spheroid and embedded in paraffin.
• the image was taken under a confocal microscope LSM510 objective x20.
• the image on the right is the superposition of the first three images.
• Arrows indicate RESTO cells (GFP +) expressing TG2.
• Figure 9 Study of the diffusion of doxorubicin in SUDHL4 cells cultured in suspension or in spheroids.
• SUDHL4 cells cultured in suspension or after 7 days in 3D in the presence or absence of extracellular matrix (ECM) (Mg: matrigel) and RESTO cells are treated for 24 hours with doxorubicin ( ⁇ ). After dissociation of the cells cultured in 3D, the fluorescence intensity of the cells was analyzed by flow cytometry.
• ECM extracellular matrix
• Figure 10 Representative images showing the penetration of antibodies in the cellular microcompartment.
• SC-GFP + Microcompartment containing DLBCL SUDHL4 cells cultured in 3D in the presence of ECM.
• B Labeling with Rituximab (RTX) - 633.
• the capsules are incubated for 12h with AC, then they are imaged with a microscope confocal Zeiss LSMS10 to the x25 lens.
• the nuclei are marked with DAPI in blue.
• the outline of the capsules is visible (marked at the periphery on the images).
• Figure 11 Comparison of the cytotoxic effects of doxorubicin and etoposide on cells of DLBCL SUDHL4 grown in 2D (suspension) or in 3D ⁇ ECM ⁇ Resto. Cells or spheroids at J7 post-encapsulation are treated for 48 hours with drugs. At the end of the treatment, the cell viability is measured using the CellTiter-Glo® 3D Cell Viability Assay® kit (Promega).
• FIG. 12 Cellular Cell Micromembrane Formation According to the Invention as a Function of Time from T Cells in Primary Culture from a Patient With Sezary Lymphoma
• the cells are labeled with calcein-AM to visualize live cells and with propidium iodide (PI) to visualize dead cells.
• PI propidium iodide
• the nuclei are stained with DAPI.
• D10 days after encapsulation
• the invention relates to a cellular microcompartment, in 3D, comprising an aggregate, or cluster, of malignant hematopoietic cells encapsulated in a hydrogel envelope.
• the terms “hydrogel layer”, “hydrogel capsule” or “hydrogel envelope” denote a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, and preferentially some water.
• the polymer or polymers of the hydrogel layer are crosslinkable polymers when subjected to a stimulus, such as a temperature, a pH, ions, etc.
• the hydrogel used is biocompatible, in that it is not toxic to the cells.
• the hydrogel layer must allow the diffusion of oxygen and nutrients to feed cells in the cell microcompartment and allow their survival.
• the hydrogel layer also passes test molecules, such as pharmaceutical molecules.
• the polymers of the hydrogel layer may be of natural or synthetic origin.
• the outer layer of hydrogel contains one or more polymers among sulfonate-based polymers, such as sodium polystyrene sulfonate, acrylate-based polymers, such as sodium polyacrylate, polyethylene glycol diacrylate, the gelatin methacrylate compound, polysaccharides, and especially polysaccharides of bacterial origin, such as gellan gum, or of plant origin, such as as pectin or alginate.
• the outer hydrogel layer comprises at least one of alginate.
• the outer layer of hydrogel comprises only alginate.
• alginate is understood to mean linear polysaccharides formed from ⁇ -D-mannuronate (M) and ⁇ -L-guluronate (G), salts and derivatives thereof.
• the alginate is a sodium alginate, composed of more than 80% of G and less than 20% of M, with an average molecular mass of 100 to 400 kDa (for example: PRONOVA® SLG100) and at a total concentration of between 0.5% and 5% by weight (weight / volume).
• the hydrogel layer comprises polymers capable of limiting cell adhesion ("cell-repellent"), such as natural polysaccharides (for example sodium alginate), or polymers comprising polyethylene glycol units, so that to facilitate, where appropriate, the separation of said hydrogel layer from the cluster of cells that it covers or its degradation without affecting the structure of the cell aggregate.
• cell-repellent such as natural polysaccharides (for example sodium alginate), or polymers comprising polyethylene glycol units
• the cell compartment according to the invention is characterized by the presence, in the internal volume of the hydrogel envelope, of an aggregate of cells organized in a cohesive cluster in which the cells interact.
• the cell aggregate comprises malignant hematopoietic cells.
• malignant hematopoietic cells is meant cancer cells derived from the differentiation of lymphoid (i.e., lymphocytes) or myeloid (i.e., erythrocyte, leukocyte, platelet) progenitors.
• lymphoid i.e., lymphocytes
• myeloid i.e., erythrocyte, leukocyte, platelet
• the hematopoietic malignant cells are chosen from lymphomatous cells and leukemic cells.
• the cell aggregate contained in the hydrogel outer envelope comprises lymphoma cells.
• lymphoma cells refer to lymphoid malignant cells.
• the lymphoma cells are chosen from follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, peripheral T lymphoma, lymphoblastic lymphoma, anaplastic lymphoma, lymphoma of the zone marginal, lymphoma of MALT ("Mucosa- associated lymphoid tissue"), lymphoma of the lymphoplasmocyte, and / or lymphoma of the spleen, cutaneous T-cell lymphoma, cutaneous B-cell lymphoma.
• the cell aggregate contained in the hydrogel outer envelope comprises leukemic cells.
• leukemic cells denote malignant blood cells.
• the leukemic cells may be selected from acute myeloblastic leukemia cells, chronic myeloid leukemias, chronic lymphoid leukemias, acute leukemias.
• malignant hematopoietic cells can come from cellular models, but also be obtained from patients.
• the use of lymphoma or leukemia cells from a particular patient may be particularly interesting in the context of personalized medicine, for the identification of molecule (s) particularly adapted (s) to the treatment of lymphoma or leukemia of said patient .
• the tumor cells of the patient are advantageously purified before use.
• the purification is done by negative selection, in order to avoid the introduction of antibodies into the cell culture.
• the cell aggregate of the cellular microcompartment comprises only lymphoma cells.
• the organization of lymphoma cells into a cohesive cell cluster within the hydrogel capsule makes it possible to confer on these cells resistance to the penetration of external molecules that is close to the resistance observed in the cells of a lymphoma.
• the cell aggregate of the cellular microcompartment comprises only leukemic cells.
• the cell aggregate comprises, in addition to malignant hematopoietic cells, stromal cells.
• the nature of the stromal cells chosen depends advantageously on the nature of the associated hematopoietic malignant cells.
• the microcompartment further comprises an extracellular matrix layer. Indeed, the inventors have shown that the presence of an extracellular matrix is necessary for the adhesion and survival of stromal cells and for the formation of the aggregate of mixed cells within the hydrogel capsule.
• the extracellular matrix layer advantageously tapes the inner face of the hydrogel envelope.
• the extracellular matrix layer comprises a mixture of proteins and extracellular compounds promoting cell culture, and more particularly that of stromal cells.
• the extracellular matrix comprises structural proteins, such as laminins containing the ⁇ 1, ⁇ 4 or ⁇ 5 subunits, the ⁇ or ⁇ 2 subunits, and the ⁇ or ⁇ 3 subunits of entactin and vitronectin. , fibronectin, laminin, collagen, as well as growth factors, such as TGF-beta and / or EGF.
• the extracellular matrix layer consists of, or contains Matrigel® and / or Geltrex®.
• the malignant hematopoietic cells are lymphoma cells and the stromal cells are lymphoid stromal cells, such as adipose tissue stem cells (ADSCs), or more particularly, cells of the type RESTO.
• Stromal cells of the RESTO type are lymphoid stromal cells derived from tonsils ("tonsil-derived stromal cells"). Isolation and characterization of RESTO cells can be done according to the protocol described in the publication Amé-Thomas et al. Blood 2007. The tonsils are cut into pieces and then incubated in a solution containing DNAse I and collagenase IV. The cell suspension is then deposited on a Percoll® gradient.
• RESTO stomal cells
• CD4S hematopoietic cell markers
• CD10S mesenchymal cell markers
• RESTO cells are known to have characteristics close to those of fibroblastic reticular cells of secondary lymphoid organs: secretion of chemokines, fibronectin and a transglutaminase network in response to TNF ⁇ (Tumor Necrosis Factor) and LTaip2 (LymphoToxin) .
• TNF ⁇ Tumor Necrosis Factor
• LTaip2 LTaip2
• the cell aggregate may comprise, in addition to lymphoma cells, cells normally present in the microenvironment of a lymphoma.
• the term "cells of the microenvironment" refers to the cells present in the aggregate of cells that are not lymphomatous cells.
• the cell aggregate can also comprise, in addition to stromal cells, cells of the immune system, such as macrophages.
• malignant hematopoietic cells are leukemic cells and stromal cells are medullary stromal cells, such as HS-S line cells, or bone marrow Mesenchymal Stromal Cells (MSCs).
• stromal cells are medullary stromal cells, such as HS-S line cells, or bone marrow Mesenchymal Stromal Cells (MSCs).
• the ratio of lymphoma cells / stromal cells in the cell aggregate is between 1/1 and 1000/1.
• the ratio may vary over time, the amount of lymphoma cells tending to increase exponentially compared to stromal cells.
• the ratio of lymphoma cells / stromal cells is between 1/1 and 10000/1.
• the cell density in the cellular microcompartment at D8 is between one hundred and several thousand cells.
• a microcompartment with a diameter of 200 ⁇ preferably comprises 100 to 10,000 cells.
• the microcompartment cell is closed. It is the outer layer of hydrogel that gives its size and shape to the cellular microcompartment.
• the microcompartment can have any shape compatible with encapsulation of cells, including a spheroidal, ovoid or tabular shape.
• the cell aggregate is constrained in the internal volume of said hydrogel layer, and once the cells are confluent, the aggregate can no longer increase in volume.
• the microcompartment cell has a diameter or a smaller dimension between 50 ⁇ and 600 ⁇ . "Smallest dimension" means twice the minimum distance between a point on the outer surface of the hydrogel layer and the center of the microcompartment.
• the thickness of the hydrogel outer layer represents 5% to 30% of the radius of the microcompartment.
• the "thickness" of a layer is the dimension of said layer extending radially with respect to the center of the microcompartment.
• the cellular microcompartment has a diameter or a smaller dimension of between 50 ⁇ and 300 ⁇ . Such dimensions ensure that all of the cells in the cell aggregate, including those in the center of said cell aggregate, have sufficient access to oxygen and nutrients that diffuse through the hydrogel layer. Thus, no phenomenon of hypoxia and / or necrosis is observed within such a microcompartment, all cells having sufficient access to small molecules that diffuse through the hydrogel envelope.
• the cellular microcompartment has a diameter or a smaller dimension between 500 ⁇ and 600 ⁇ .
• cells in the center of the cell aggregate have little or no access to oxygen and nutrients that diffuse through the hydrogel wrap.
• Such microcompartments are particularly interesting for the study of the phenomena of hypoxia and / or necrosis which can sometimes occur in a lymphoma.
• the invention also relates to methods of preparing microcompartmental cells for obtaining cell microcompartments comprising an aggregate of cells containing malignant hematopoietic cells encapsulated in an outer hydrogel envelope. After encapsulation of the cells, they will reorganize within the hydrogel envelope, so as to form a cohesive cluster.
• the encapsulation is done by means of a concentric coextrusion process, in which the hydrogel solution is coextruded with the cell solution, before being crosslinked by means of a crosslinking solution capable of crosslinking the hydrogel.
• Concentric coextrusion it is meant that the solutions are coextruded so that one solution surrounds the other solution.
• the concentric coextrusion is such that the hydrogel solution surrounds the cell solution.
• drops of coextruded solutions then fall into the crosslinking solution, comprising a crosslinking agent capable of crosslinking the hydrogel and thus forming a hydrogel capsule around the cells.
• the solutions are coextruded directly in the crosslinking solution, so as to form an outer hydrogel tube in which the cells will be organized.
• drops of coextruded solutions pass through a crosslinking aerosol (made from a crosslinking solution) so as to allow at least partial crosslinking of the hydrogel layer around the drop of solution of cells.
• the partially crosslinked microcompartments then fall into a crosslinking solution where the crosslinking is terminated.
• any extrusion process for concentrically coextruding hydrogel and cells can be used.
• the process according to the invention is implemented by means of a concentric double-wall extrusion device as described in patent FR2986165.
• crosslinking solution means a solution comprising at least one crosslinking agent adapted to crosslink a hydrogel comprising at least one hydrophilic polymer, such as alginate, when it is applied. contact with it.
• the crosslinking solution may be, for example, a solution comprising at least one cation divalent.
• the crosslinking solution may also be a solution comprising another known crosslinking agent of the alginate or of the hydrophilic polymer to be crosslinked, or a solvent, for example water or an alcohol, adapted to allow crosslinking by irradiation or by any other means. other technique known in the art.
• the crosslinking solution is a solution comprising at least one divalent cation.
• the divalent cation is a cation that makes it possible to crosslink alginate in solution.
• H may be for example a divalent cation selected from the group consisting of Ca 2+ , Mg 2+ , Ba 2+ and Sr 2 "1" , or a mixture of at least two of these divalent cations.
• the divalent cation, such as Ca 24 ⁇ may be associated with a counterion to form, for example, CaCl or CaCCh type solutions, well known to those skilled in the art.
• the crosslinking solution may also be a solution comprising CaC0 3 coupled to Glucono delta-lactone (GDL) forming a solution of CaCOs-GDL.
• the crosslinking solution may also be a mixture of CaCO 3 -CaSO 4 -GDL.
• the crosslinking solution is a solution comprising calcium, in particular in the Ca 2+ form.
• the crosslinking solution may also be a solution comprising polylysine.
• the divalent cation concentration in the crosslinking solution is between 10 and 1000 mM.
• the crosslinking solution may comprise other constituents, which are well known to those skilled in the art, than those described above, in order to improve the crosslinking of the hydrogel sheath under the conditions, in particular time and / or temperature, special.
• the hydrogel solution is coextruded with a solution of cells.
• the cell density in the cell solution is between 1 ⁇ 10 6 and 100 ⁇ l cells / ml.
• the cell solution used for coextrusion contains only lymphomatous cells suspended in culture medium.
• the cell solution used for coextrusion contains only leukemic cells suspended in culture medium.
• the cell solution used for coextrusion comprises lymphoma cells and lymphoid stromal cells, suspended in an extracellular matrix.
• the number ratio of lymphoma cells / stromal cells in the cell solution is between 1/1 and 1/2.
• such a solution may also comprise immune cells, preferentially chosen from macrophages.
• the number ratio of lymphoma cells / microenvironment cells in the cell solution is advantageously between 1/1 and 1/2.
• the cell solution used for coextrusion comprises leukemic cells and medullary stromal cells, suspended in an extracellular matrix.
• the ratio in number of leukemic cells / stromal cells in the cell solution is between 1/1 and 1/2.
• the cell suspension advantageously represents between 50 and 95% of the volume of the solution, while the extracellular matrix represents between 5 and 50% of said volume.
• coextrusion also involves an intermediate solution, including sorbitol.
• the coextrusion is carried out so that the intermediate solution is extruded between the hydrogel solution and the cell solution.
• the extrusion rate of the hydrogel solution is between 5 and 100 mlVh, preferably between 15 and 60 mIJh.
• the extrusion rate of the cell solution is between 5 and 100 mJh, preferably between 10 and 50mIVh.
• the extrusion rate of the intermediate solution is between 5 and 100 mJh, preferably between 10 and 50 mlVh.
• the coextrusion rate of the different solutions can be easily modulated by those skilled in the art, so as to adapt the diameter or the smallest dimension of the cellular microcompartment and / or the thickness of the hydrogel layer.
• the extrusion rates of the cell solution and the intermediate solution are identical.
• the extrusion rate of the hydrogel solution is substantially equal to the extrusion rate of the cell solution and optionally of the intermediate solution.
• the hydrogel solution, the intermediate solution and the cell solution are loaded into three concentric compartments of a coextrusion device, so that the solution of Hydrogel, forming the first stream, surrounds the intermediate solution that forms the second stream, which itself surrounds the cell solution that forms the third stream.
• the tip of the device extrusion, through which the three flows out, opens above the crosslinking solution.
• the tip of the extrusion device is located about 50 cm, +/- 10 cm, of the crosslinking solution.
• An electric field is generated at the output of the coextrusion device, to allow the formation of microdroplets.
• a copper ring is disposed about 1 cm at the exit of the coextrusion device.
• Microdroplets thus fall sequentially into the crosslinking bath where the hydrogel layer is crosslinked, forming an outer shell around the cells.
• the tip of the extrusion device opens into a crosslinking aerosol, formed by microdroplets of crosslinking solution, so that the hydrogel layer of the microdroplets begins to crosslink in contact with the microdroplets of the aerosol .
• the crosslinking may, if appropriate, continue in a crosslinking solution in which the microdroplets are received.
• the method according to the invention makes it possible very rapidly to obtain several thousand microcompartments that are substantially identical in terms of size and composition.
• the method according to the invention makes it possible to encapsulate malignant hematopoietic cells, such as lymphomatous cells, in an external hydrogel envelope. After only a few hours, the cells contained in the hydrogel envelope reorganize, so as to aggregate and form a cluster of cells that becomes cohesive after a few days.
• the microcompartment cell obtained by coextrusion is maintained in a suitable culture medium for two to twelve days before being used, preferably between four and ten days. This latency advantageously allows the cells to aggregate and form a cell cluster mimicking the cluster of cells in a lymphoma.
• the cell microcompartment obtained by coextrusion as such, that is to say with the external hydrogel shell. It is otherwise possible to proceed before any use to hydrolysis of the hydrogel shell, in order to recover the aggregate of cells. It is also possible to freeze the cell microcompartment obtained by coextrusion (with the hydrogel casing) for later use.
• microcompartment cell according to the invention can be used for many applications, particularly for pharmacological purposes.
• microcompartmental cells according to the invention can in particular be used for identification and / or validation tests of candidate molecules having an action on hematological malignancies.
• candidate molecules having an action on hematological malignancies.
• the permeability of certain hydrogels is sufficient to pass molecules having a molecular weight less than or equal to 200 kDa. It is therefore possible to study these molecules directly on the microcompartment cell. In the case of molecules of higher molecular weight, it is possible to hydrolyze the outer hydrogel shell before performing the tests. Thus, ⁇ study is done directly on ⁇ clusters of cells.
• the hydrolysis of the hydrogel casing is performed 6 or 10 days after the coextrusion, so as to ensure that the cluster of cells is well formed and that the cells are cohesive.
• the cellular microcompartments according to the invention can also be used in personalized medicine, using cells of a subject presenting a lymphoma or leukemia, in order to specifically test the reaction of said subject to different treatments, before selecting the most appropriate treatment for said subject.
• Hydrogel Solution 2.5% Alginate w / v (LF200FTS) in 0.5mM SDS
• Extracellular matrix Matrigel® classic (without phenol red and with growth factors)
• Exudation device - 3 x 12ml hamilton syringes containing respectively 2.5% sterile alginate and the other two 300mM sterile sorbitol syringes
• the alginate capsules are obtained according to the method described in Alessandri et al (PNAS 2013, DOI: 10.1073 / pnas.1309482110 and LOC 2016 DOI: 10.1039 / c61c00133e) and in the application WO2013113855.
• the microfluidic coextrusion device for the production of capsules is placed about 50 cm above a petri dish containing the crosslinking solution.
• the alginate solution, the sorbitol solution and the cell solution are then co-injected into the microfluidic coextrusion device, to form composite droplets which are crosslinked by falling into the calcium bath.
• the coextrusion device is operated for 10 seconds, and produces about 5000 alginate capsules per second, totaling about 50,000 capsules.
• the alginate capsules are then recovered by filtering the calcium bath with a 40 ⁇ mesh cell strainer which retains the capsules. These are rinsed with the base of medium then re-suspended in the final medium.
• a potential of + 2kV was applied via an electrode in the alginate.
• a copper ring with a mass of 3 cm in diameter is positioned approximately 1 cm from the tip of the microfluidic coextrusion device to generate the electric field necessary for the electro-formation of the droplets.
• Microcompartmental cells comprising only SUDHL4 or HLY1 lymphoma cells
• the lymphoma cells Prior to encapsulation, the lymphoma cells are cultured in DMEM supplemented with 10% fetal calf serum (FCS) in a humid atmosphere at 37 ° C in the presence of 5% CO 2.
• FCS fetal calf serum
• the cells are centrifuged and resuspended in sorbitol (300 mM) at a rate of 6 to 100.10 6 10.10 ceUules / mL.
• the number of cells per capsule varies between 30 and 100.
• the capsules are cultured in DMEM medium supplemented with 10% FCS in a CO2 oven at 37 ° C.
• the medium is changed every 2 to 3 days.
• the capsules are divided into 96-well plates, one capsule per well.
• the clusters of cells are then periodically imaged (every other day) in phase contrast and in fluorescence if the cell line expresses a fluorescent protein.
• the area of cell clusters is measured using ImageJ® software and growth curves are established ( Figure 1B).
• lymphocytic cells In order to get closer to the microenvironment of a lymph node, lymphocytic cells (DOHH2) are co-cultured with Matrigel® extracellular matrix and "RESTO" type stromal cells.
• DOHH2 lymphocytic cells
• the RESTO cells were previously cultured in DMEM medium supplemented with 10% fetal calf serum in a humid atmosphere at 37 ° C with 5% CCh. At the time of encapsulation, the cells are detached from the support by action of trypsin, and then they are resuspended in the extracellular matrix in the presence of lymphoma cells at a ratio of 1/1.
• the cell density of RESTO cells and lymphoma cells may vary from 10-50.10 6 cells / ml.
• the coextrusion encapsulation method makes it possible to obtain a coating of the inner wall of the alginate capsules by the extracellular matrix.
• This coating allows the adhesion of stromal cells and promotes the formation of the tumor niche. In a few days, the cells organize themselves freely and form a cluster of cohesive cells after 4-10 days of culture (FIG. 3).
• Micro-compartments were made according to the invention from lymphocyte cells from patients with Sezary syndrome (cutaneous T-cell leukemic form). After encapsulation, the cells grow inside the alginate capsules and form a cluster of cells after about ten days ( Figure 12). This confirms that the method according to the invention makes it possible to obtain cellular microcompartments from primary cells of patients, which makes it possible to envisage the use of this method in personalized medicine.
• Example 2 Analysis of cellular microcompartments
• Capsules containing clusters of SUDHL4 or HLY1 cells are placed in agarose wells coated with DMEM medium without phenol red (on D1-D6). The images are acquired using a ZEISS lsm 510 confocal microscope. Two types of analysis are performed: a one-time analysis after marking the microcompartment with a fluorophore, and an analysis using intermittent imaging, after labeling or non-labeling of cells with a fluorophore. The cells are labeled with calcein-AM and propidium iodide to visualize the dead cells; the cell nuclei are marked in blue with Hoechst 33342.
• the paraffin-embedded immunofluorescence technique was adapted to the analysis of the capsules: capsules containing the cell clusters are removed and included in a gelled solution of agarose with a low melting point of 2%. Once gelled, the agarose block containing the capsules is immersed in a 4% paraformaldehyde fixative for 30 minutes. After fixation, the samples are treated according to the protocols conventionally described for immunohistochemistry.
• Ki67 protein which is a marker of cell activation
• cleaved caspase 3 which makes it possible to evaluate cell death
• the extracellular matrix consists mainly of fibronectin, collagen I and laminin. Immunofluorescence analysis showed the presence of these three types of matrix in the cell clusters of microcompartments, whereas the same cells grown in suspension do not express these matrices. D] Dissolution of alginate capsules
• FIG. 4 shows an example of dissolution of a capsule containing a cluster of cells comprising only SUDHL4 lymphoma cells (FIG. 4A) and a capsule containing a cluster of cells comprising DOHH2 lymphoma cells and stromal cells (FIG. 4B). .
• the alginate capsules are dissolved and the clumps of cells dissociated before being incubated with a marker of the apoptosis (TMRM) and then analyzed by flow cytometer in the presence of counting beads (Figure S). Over time, it is observed that the number of cells increases, while the proportions of dead cells within the cell clusters decrease between J3 and J10, which is correlated with the increase in the volume of the spheroid described in Figure 1A .
• TMRM marker of the apoptosis
• FIG. 6 the reconstitution of a tumor niche similar to that found in the lymph node (C: RESTO + DOHH2 cells) promotes the growth of tumor B cells.
• C RESTO + DOHH2 cells
• stromal cells differentiate into pro-tumoral lymphoid stroma (Thomazy et al, 2003, Ohe et al., 2016) expressing, inter alia, transglutaminase 2 (TG2) which has a role in the stabilization of extracellular matrix and cell adhesion.
• TG2 transglutaminase 2
• the Resto-type stromal cell (FRC) culture in the presence of tumor B cells and extracellular matrix leads to the expression of TG2 revealed by immunolabeling.
• FRC Resto-type stromal cell
• TMRM the alginate capsules are previously dissolved and the clumps of dissociated cells
• Preclinical cancer drug research has the worst success rate of any therapeutic trial, with less than 5% of candidate compounds passing phase m clinical trials.
• One of the explanations for these failures is the lack of a relevant model capable of reproducing the spread of drugs within a tumor. Indeed, one of the hypotheses to explain the decrease in the effectiveness of molecules during the transition from pre-clinical tests to clinical trials is that cell density within tumors would slow the penetration and spread of drugs, decreasing their effectiveness.
• the 3D model according to the invention is relevant for studying the diffusion of drugs within a lymphomatous tumor niche.
• doxorubicin which is otherwise used in the standard treatment of B-cell lymphoma
• ECM extracellular matrix
• stromal cells of RESTO type were incubated 24h in the presence or absence of Doxorubicin ( ⁇ ).
• the fluorescence intensity of the cells was then measured by flow cytometry.
• Standard treatment for B-cell lymphoma is conventional multidrug therapy combined with CD20-directed immunotherapy expressed on the surface of mature B cells.
• CD20-directed immunotherapy expressed on the surface of mature B cells.
• the spheroids were incubated on post-encapsulation day-7 in the presence of anti-CD19 AC directly coupled to phycoerythrin (PE) (very strongly expressed in B cells), or
• the diffusion of drugs is impaired in the 3D structures according to the invention.
• the purpose of the present experiment is to verify if this alteration is correlated with a chemoresistance.
• the efficacy of two conventional chemotherapies doxorubicin and etoposide was tested in parallel on cells cultured in suspension (2D) and on clumps of cells from microcompartmental cells according to the invention, containing lymphomatous cells alone. or in the presence of ECM, with or without RESTO stromal cells, treated with increasing concentrations of these chemotherapy molecules.

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