Method for treating crosslinked microcompartments comprising biological elements

Abstract

The present invention relates to the treatment of three-dimensional microcompartments comprising biological elements encapsulated in a crosslinked hydrogel. In particular, the invention relates to a method for treating a three-dimensional microcompartment comprising at least one biological element encapsulated in a crosslinked hydrogel, so as to reinforce the mechanical properties of said microcompartment, characterized in that it comprises a step of rinsing the microcompartment in a rinsing solution comprising at least one cation.

Description

Process for treating cross-linked microcompartments comprising biological elements technical field

[0001] The present invention relates to the processing of three-dimensional microcompartments comprising biological elements encapsulated in a cross-linked hydrogel. In particular, the invention relates to a process for processing said microcompartments, a process for preparing said microcompartments, a microcompartment obtained by said preparation process, and a kit for implementing said process. Previous art

[0002] The present invention relates to the field of microcompartments containing biological elements and their use in various industrial applications. Microcompartments, three-dimensional structures that are partially or totally enclosed and include one or more biological elements, play a crucial role in many biological and biotechnological processes.

[0003] These three-dimensional structures allow the encapsulation and culture of various biological elements in a controlled environment, thus offering new perspectives for cell therapy, tissue engineering and the production of molecules of interest.

[0004] However, the large-scale exploitation of microcompartments faces a major challenge: their fragility under mechanical stress, particularly due to changes in their properties caused by their storage environment (culture media, for example). Indeed, these structures are susceptible to damage, which can lead to their rupture, during routine manipulations such as pipetting, centrifugation, or filtration. This sensitivity to mechanical stress significantly limits their potential use in industrial settings, where robustness and reproducibility are essential.

[0005] Therefore, there is a need to develop solutions to improve the mechanical resistance of microcompartments while preserving their functional properties and in particular the viability of the encapsulated biological elements.

[0006] Therefore, there is a need for an innovative solution to improve the mechanical strength of three-dimensional microcompartments containing biological elements encapsulated in a cross-linked hydrogel, without compromising their essential functional properties, such as the viability of the encapsulated biological elements. This solution should be scalable, economically viable, and compatible with existing industrial processes, in order to unlock the full potential of microcompartments in various biotechnological and industrial applications. Summary of the invention

[0007] To meet this need, the invention proposes a method for treating a three-dimensional microcompartment comprising at least one biological element encapsulated in a cross-linked hydrogel, so as to strengthen the mechanical properties of said microcompartment.

[0008] To achieve this, the process according to the invention includes a step of rinsing said microcompartment in a rinsing solution comprising at least one cation.

[0009] Advantageously, this rinsing step with a solution containing at least one cation strengthens the structure of the cross-linked hydrogel, thereby improving the mechanical strength of the microcompartment. This allows for more efficient handling and processing of the microcompartments without damaging them.

[0010] This approach solves the problem of the fragility of microcompartments under mechanical stress by strengthening the ionic bonds within the cross-linked hydrogel, thereby increasing its structural stability and resistance to mechanical stress.

[0011] The rinsing solution can be a solution in which at least one cation has a concentration between 0.5 and 50mM.

[0012] Advantageously, this concentration range allows for effective reinforcement of the cross-linked hydrogel structure without compromising the viability of the encapsulated biological components. Indeed, the cation concentration in the rinsing solution is high enough to improve the mechanical strength of the microcompartment, but low enough not to disrupt the environment of the encapsulated biological components.

[0013] At least one cation of the rinsing solution may be chosen from among the ions calcium, iron, barium, strontium, copper, lead, aluminium, magnesium, manganese, zinc, cobalt, nickel and their combinations.

[0014] Advantageously, these cations are particularly effective at enhancing the mechanical properties of microcompartments via interaction with the crosslinked hydrogel commonly used in the manufacture of microcompartments, such as Iginate.

[0015] By using cations that interact strongly with the functional groups of cross-linked hydrogen I, optimal structural reinforcement is achieved without requiring high concentrations that could be harmful to biological elements. This reinforcement can be obtained by combining various improvements in mechanical properties, such as self-healing, malleability, elasticity, plasticity, deformability, resistance to point loads, shear strength, and compressive strength, depending on the cations used. Regardless of the specific properties improved, the end result is a reduction, or even prevention, of damage to the microcompartments during mechanical stress, thanks to the rinsing step according to the invention.

[0016] At least one cation in the rinsing solution may be a divalent or trivalent cation.

[0017] Advantageously, divalent and trivalent cations are particularly effective at creating strong ionic bonds within the cross-linked hydrogel, thus significantly strengthening its structure.

[0018] According to one embodiment, the rinsing solution comprises calcium at a concentration between 0.5 and 10 mM, preferably between 2.5 and 5 mM.

[0019] Advantageously, calcium is a particularly effective divalent cation for strengthening the structure of cross-linked hydrogels, especially in the case of alginate-based hydrogels. Calcium forms ionic bonds with the alginate chains, thereby increasing the rigidity and mechanical strength of the microcompartment.

[0020] According to one embodiment, the rinsing solution comprises zinc at a concentration between 0.1 and 5 mM, preferably between 0.3 and 1 mM, even more preferably between 0.4 and 0.6 mM.

[0021] Advantageously, zinc is a divalent cation particularly effective for strengthening the mechanical properties of microcompartments while preserving the viability and proliferation capacity of encapsulated biological elements, including pluripotent stem cells.

[0022] According to a particular embodiment, the rinsing solution comprises zinc sulfate (ZnSO4).

[0023] According to another embodiment, the rinsing solution comprises copper at a concentration between 0.1 and 5 mM, preferably between 0.3 and 2 mM, even more preferably between 0.5 and 1 mM.

[0024] Advantageously, copper helps to strengthen the structure of the cross-linked hydrogel while maintaining conditions compatible with cell growth and the maintenance of the pluripotency of the encapsulated cells.

[0025] According to a particular embodiment, the rinsing solution comprises copper chloride (CuCl2).

[0026] According to one embodiment, the rinsing solution comprises calcium and zinc, preferably calcium at a concentration between 0.5 and 10 mM and zinc at a concentration between 0.1 and 1 mM.

[0027] According to another embodiment, the rinsing solution comprises calcium and copper, preferably calcium at a concentration between 0.5 and 10 mM and copper at a concentration between 0.1 and 2 mM.

[0028] Advantageously, these combinations of cations allow for a synergistic effect on strengthening the mechanical properties of the microcompartment.

[0029] According to one variant, the microcompartment can be hollow and include an outer hydrogel layer defining an inner part comprising at least one biological element.

[0030] Advantageously, this hollow structure offers a space suitable for the biological elements present within the internal part.

[0031] The microcompartment may be hollow, and consist of an outer hydrogel layer encapsulating a hollow inner part comprising at least one or more biological element(s), and possibly culture medium and / or extracellular matrix elements.

[0032] Advantageously, this configuration allows for the creation of a controlled microenvironment for biological elements.

[0033] This variant of the invention thus makes it possible to create an environment favorable to biological elements while ensuring mechanical protection. Indeed, the hollow inner part can be filled with culture medium and extracellular matrix elements adapted to the specific needs of the biological elements, while the outer layer reinforced by cations ensures the protection and integrity of the microcompartment.

[0034] According to another variant, the microcompartment can be a solid hydrogel ball encapsulating at least one biological element.

[0035] Advantageously, this configuration offers maximum protection to the encapsulated biological elements and allows for uniform distribution of cations throughout the volume of the microcompartment.

[0036] This variant of the invention thus makes it possible to protect biological elements in mechanically demanding environments. Indeed, the solid structure provides a complete physical barrier around the biological elements, while the treatment with cations improves the mechanical resistance of the entire microcompartment.

[0037] The hydrogel constituting the microcompartment can be chosen in particular from alginates, carrageenans, pectins, gelatins, collagen, hyaluronic acid, chitosan, and their combinations.

[0038] Advantageously, these hydrogels are biocompatible and exhibit good versatility, both in terms of their mechanical properties and their encapsulation capacity.

[0039] According to one embodiment, the hydrogel of the microcompartment has a shear modulus between 30 and 80 kPa, preferably between 40 and 70 kPa.

[0040] Advantageously, this shear module gives the microcompartment a mechanical resistance suitable for industrial handling while maintaining sufficient flexibility to preserve the integrity of the encapsulated biological elements.

[0041] The Young's modulus E of the hydrogel can be expressed as a function of the shear modulus G according to the relation E = 2G(l+v), where v is Poisson's ratio. For an incompressible hydrogel where v = 0.35, this relation simplifies to E = 3G.

[0042] According to one embodiment, the hydrogel has: - a shear modulus between 30 and 80 kPa, preferably between 40 and 70 kPa; and / or - a Young's modulus between 100 and 250 kPa, preferably between 100 and 200 kPa.

[0043] Thus, depending on the specific needs of the encapsulated biological elements, each type of hydrogel offers unique characteristics in terms of porosity, rigidity and interactions with biological elements, allowing the choice of the most suitable material for each application.

[0044] At least one biological element encapsulated in the microcompartment may be chosen in particular from among the following biological elements: a cell, a cell organelle, a cell fragment, a cell aggregate, an organoid, DNA, RNA, a protein, a microorganism, a vector, a vesicle, an exosome, a micro tissue, a spheroid, an embryoid body and their combinations.

[0045] Advantageously, this wide range of encapsulable biological elements offers numerous application possibilities in the fields of regenerative medicine and tissue engineering.

[0046] The microcompartment can have various three-dimensional shapes, including ovoid, cylindrical, spheroidal, spherical, teardrop, or fiber-like. The microcompartment can be closed or partially closed.

[0047] Advantageously, these different shapes allow the microcompartments to be adapted to various experimental or therapeutic applications and constraints.

[0048] The smallest dimension of the microcompartment can be between 1 pm and 1.5 mm, preferably between 100 pm and 800 pm.

[0049] The largest dimension of the microcompartment can be between 1pm and 1.5mm, preferably between 120pm and 800pm, preferably between 200pm and 600pm, even more preferably between 200pm and 500pm, especially between 200pm and 250pm.

[0050] In the context of the invention, the largest dimension of the microcompartment is always greater than the smallest dimension of the microcompartment.

[0051] Advantageously, this range of sizes makes it possible to create microcompartments small enough to be easily handled and / or administered, yet large enough to contain a significant number of biological elements.

[0052] The process may also include a step of mechanical stressing said rinsed microcompartment, in which the mechanical stressing may be carried out in particular by filtration, agitation in liquid medium, centrifugation, aspiration or a combination thereof.

[0053] The mechanical stressing step can be carried out without degrading the microcompartments, thanks to the strengthening of the mechanical properties obtained during the rinsing step with the solution containing cations.

[0054] The invention allows the rinsed microcompartments to be subjected to various controlled mechanical stresses while preserving their structural integrity. The mechanical stress can serve different functions depending on the context, such as a change of culture medium, filtration, sampling, flow through a restricted conduit, agitation in a bioreactor, or centrifugation to group biological elements.

[0055] The mechanical stress step may include the application of a centrifugal acceleration of at least 10g on at least one rinsed cell microcompartment according to the invention.

[0056] Advantageously, this centrifugal acceleration allows the encapsulated biological elements to be grouped together, significantly reducing the number of isolated biological elements.

[0057] The rinsing step and / or the mechanical stressing step can be carried out using at least one device comprising a body (2) including at least a first port (5) and a second port (6) and a filter membrane (3) extending along a plane P in the body (2) thus defining two compartments and capable of preventing the passage of microcompartments of biological elements from one compartment to the other, the first port (5) being intended for the injection and withdrawal of cellular microcompartments bathed in a rinsing solution and / or a solution A and the second port (6) being intended for the withdrawal and injection of solution A and / or the rinsing solution, characterized in that the injection into the first port (5) is carried out along a direction D not substantially orthogonal to the plane P of the filter membrane (3).

[0058] Advantageously, this device allows the rinsing and / or mechanical stressing steps to be carried out efficiently, preferably simultaneously, while minimizing the risk of damage to the microcompartments.

[0059] This approach solves the problem of delicate handling of microcompartments during rinsing and mechanical stressing steps by performing an injection not orthogonal to the plane of the filter membrane, creating a tangential flow that reduces the risks of clogging and damage to the microcompartments, while ensuring effective rinsing and controlled mechanical stressing.

[0060] The invention also proposes a method for preparing a three-dimensional microcompartment comprising the implementation of the following steps: a) Encapsulation of at least one biological element in a cross-linked hydrogel to form a three-dimensional microcompartment; e b) Treatment of the microcompartment obtained in step a) by any one of the embodiments of the treatment process according to the invention.

[0061] Advantageously, this preparation process allows for the optimization of the production of microcompartments containing biological elements in a controlled and reproducible manner.

[0062] In this way, the preparation process according to the invention makes it possible to increase the production capacity of the microcompartments by increasing the mechanical stresses / forces on the microcompartments.

[0063] The encapsulation step a) may include a crosslinking step of the hydrogel using a crosslinking solution comprising at least one crosslinking agent.

[0064] Preferably, at least one crosslinking agent is calcium.

[0065] Advantageously, this crosslinking step allows the initial structure of the hydrogel to be formed, which will then be reinforced by treatment with the rinsing solution containing cations during step b) of microcompartment treatment.

[0066] Preferably, at least one crosslinking agent is calcium and has a concentration in the crosslinking solution between 0.5 and 300 mM, preferably a concentration between 25 and 150 mM.

[0067] Advantageously, this range of concentrations allows for fine control of the degree of crosslinking of the hydrogel and therefore its initial mechanical properties.

[0068] At least one crosslinking agent from step a) can be distinct from at least one cation from step b).

[0069] Advantageously, this distinction between the initial crosslinking agent and the cations used for strengthening allows finer control of the microcompartment properties at each stage of its preparation.

[0070] At least one crosslinking agent from step a) may be identical to at least one cation from step b).

[0071] Advantageously, using the same agent for initial crosslinking and subsequent strengthening can simplify the process and ensure optimal compatibility between the two steps.

[0072] According to one embodiment, step a) may include the following steps: 1) Formation of a hollow hydrogel microcompartment encapsulating biological elements, using an encapsulation device, preferably comprising a microfluidic and / or millifluidic device and solutions, said solutions preferably being the following: - a hydrogel solution - possibly an intermediate solution, preferably isotonic; and - a solution comprising biological elements, culture medium and possibly extracellular matrix and / or extracellular matrix substitute; 2) Crosslinking of the hydrogel, preferably using a crosslinking solution, such as a crosslinking bath.

[0073] Advantageously, this process can enable the formation of hollow microcompartments with a well-defined structure, comprising an outer layer of cross-linked hydrogel and a hollow inner part containing the biological elements.

[0074] This approach solves the problem of creating microcompartments with a complex and controlled architecture using a microfluidic device that allows multilayer structures to be formed in a single step, while collection in a crosslinking solution ensures rapid gelation of the outer layer, thus preserving the structure of the microcompartment.

[0075] The invention also relates to a three-dimensional cellular microcompartment, obtained by any one of the embodiments of the preparation process according to the invention.

[0076] Advantageously, this three-dimensional cellular microcompartment benefits from all the characteristics and advantages described above, particularly in terms of mechanical resistance, biocompatibility and application versatility.

[0077] The invention also proposes the use of the cellular microcompartment obtained by any embodiment of the preparation process according to the invention for cell culture, drug testing, regenerative medicine, or tissue engineering.

[0078] Finally, the invention proposes a kit comprising at least one rinsing solution comprising at least one cation in which at least one cation has a concentration between 0.5 and 50mM, for the implementation of a process according to any one of the preceding claims.

[0079] Advantageously, this kit facilitates the implementation of the microcompartment treatment process by providing a ready-to-use rinsing solution with an optimal concentration of cations. Brief description of the Figures

[0080] [Figure 1] is a perspective view of an example of an embodiment of a device capable of performing a rinsing and / or mechanical stressing step in the context of the invention.

[0081] [Figure 2] is a schematic view of an example embodiment of a device capable of performing a rinsing and / or mechanical stressing step in the context of the invention,

[0082] [Figure 3] is a perspective view of an example of an embodiment of a device capable of performing a rinsing and / or mechanical stressing step in the context of the invention,

[0083] [Figure 4] is a half-sectional view of figure 3 along section plane AA,

[0084] [Figure 5] is a half-sectional view of another example of a device capable of performing a rinsing and / or mechanical stressing step in the context of the invention, and

[0085] [Figure 6 A] Figure 6 A is a microscopic observation of the microcompartments washed with a solution not supplemented with calcium, accompanied by a photograph of the cell sieve after rinsing.

[0086] [Figure 6 B] Figure 6 B is a microscopic observation of the microcompartments washed with a calcium-supplemented solution (2mM), accompanied by a photograph of the cell sieve after rinsing.

[0087] [Figure 7] Figure 7 is a set of photographs taken under a microscope of cell microcompartments harvested after rinsing with a rinsing solution comprising different concentrations of calcium, accompanied by a photograph of the filter after rinsing.

[0088] [Figure 8] Figure 8 is a microscopic observation of a microcompartment obtained from rinsing with a rinsing solution comprising different concentrations of calcium at different flow rates.

[0089] [Figure 9] Figure 9 is a series of photos from video-microscopy of microcompartments containing cells from cell line no. 1.

[0090] [Figure 10] Figure 10 is a series of photos from video-microscopy of microcompartments containing cells from cell line #2.

[0091] [Figure 11] Figure 11 is a series of photos from video-microscopy of microcompartments containing cells from cell line no. 3.

[0092] [Figure 12] Figure 12 is a graphical representation of the measurement of cell viability of cells from different cell lines from microcompartments obtained after a rinsing step using a rinsing solution containing iron.

[0093] [Figure 13] Figure 13 is a graphical representation of the measurement of the amplification factor at 5 days of cells from different cell lines from microcompartments obtained after a rinsing step using a rinsing solution containing iron.

[0094] [Figure 14] Figure 14 is a graphical representation of the pluripotency measurement of cells belonging to cell line No. 3 from microcompartments obtained after a rinsing step using a rinsing solution containing iron.

[0095] [Figure 15] Figure 15 is a graphical representation of the pluripotency measurement of cells belonging to cell line No. 2 from microcompartments obtained after a rinsing step using a rinsing solution containing iron.

[0096] [Figure 16] Figure 16 is a diagram illustrating the experimental protocol for washing cell capsules with different solutions containing zinc (ZnSO4) or copper (CuCl2) at different concentrations.

[0097] [Figure 17] Figure 17 is a series of microscopic observations of the cellular microcompartments during culture (day 1 to day 5) after rinsing with different solutions: CaCl23 mM (control), ZnSO40.5 mM, CuCl20.5 mM and CuCl21 mM.

[0098] [Figure 18] Figure 18 is a graphical representation of lactate production expressed in mmol / mL of capsules over time for the different rinsing conditions.

[0099] [Figure 19] Figure 19 is a graphical representation of the measurement of cell pluripotency, showing the expression of the OCT4, SOX2 and OCT4 / SOX2 markers for the different rinsing conditions.

[0100] [Figure 20] Figure 20 is a graphical representation of rheological measurements showing the storage modulus and normal strength as a function of sample thickness, as well as a bar chart of the shear modulus for different washing conditions. Description of the invention

[0101] Definitions:

[0102] For the purposes of this invention, "centrifugal acceleration" refers to the acceleration that occurs when an object or body is in circular motion around a central point or along a curved path. This acceleration is directed outwards, away from the center of rotation, and results from the inertia of the moving object. The centrifugal force results from the mass of the object or body multiplied by this centrifugal acceleration. Thus, centrifugal acceleration is related to the acceleration acquired by a body or object under the effect of a centrifugal force.

[0103] For the purposes of this invention, "alginate" means linear polysaccharides formed from α3-D-mannuronate and αL-guluronate, salts and derivatives thereof.

[0104] By "differentiated" cells within the meaning of the invention, we mean cells that exhibit a particular phenotype, as opposed to pluripotent stem cells that are not differentiated or progenitor cells that are in the process of differentiation.

[0105] For the purposes of this invention, "micro-fluidic device" means any device or combination of devices having one or more inlets and one or more outlets connected together by a plurality of channels with a cross-section on the order of a hundred micrometers and capable of directing a flow of one or more fluids from the inlet(s) to the outlet(s).

[0106] For the purposes of this invention, "milli-fluidic device" means any device or combination of devices having one or more inlets and one or more outlets connected together by a plurality of channels with a cross-section on the order of millimeters and capable of directing a flow of one or more fluids from the inlet(s) to the outlet(s).

[0107] For the purposes of this invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even where not explicitly stated, the cells, stem cells, progenitor cells, and tissues according to this invention are constituted or obtained from human cells or from immunologically humanized non-human mammalian cells.

[0108] For the purposes of this invention, "progenitor cell" means a stem cell already engaged in cell differentiation but not yet differentiated.

[0109] For the purposes of this invention, a "pluripotent stem cell" or "pluripotent cell" is defined as a cell that has the capacity to form all the tissues present in the entire organism of origin, without, however, being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the context of this invention. These may include, in particular, induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells, or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0110] For the purposes of this invention, "induced pluripotent stem cell" refers to a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are notably positive for pluripotency markers, such as alkaline phosphatase staining and the expression of the proteins NANOG, SOX2, OCT4, and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 2007, 131(5): 861-872), and Nakagawa et al. (Nat Biotechnol, 2008, 26(1): 101-106).

[0111] For the purposes of this invention, a "cell layer" or "cell layer" refers to several cells forming a layer or layer that can be structured around a lumen. This could be, for example, a tissue or micro-cellular tissue, or a three-dimensionally grouped culture. The thickness of the cell layer can be variable. This layer is organized three-dimensionally within the microcompartment.

[0112] By "Feret diameter" of a microcompartment, we mean the distance "d" between two tangents to said microcompartment, these two tangents being parallel, such that the whole of the projection of said microcompartment is contained between these two parallel tangents.

[0113] For the purposes of this invention, a "droplet" also refers to a three-dimensional structure formed from at least one liquid solution comprising the constituents of a non-crosslinked hydrogel (polymerization precursors, non- or partially crosslinked polymer chains, etc.), and hydrogel precursor elements. Furthermore, the droplet represents a transitional state between the co-injection of the various constituents and the microcompartment according to the invention.

[0114] The term "microcompartment" or "capsule" in the context of this invention also refers to a partially or totally enclosed three-dimensional structure. This structure is formed from a matrix of polymer chains, a hydrogel, for example, alginate, containing one or more biological elements. These biological elements can be diverse, including cells. The encapsulated cells can take various forms, including single cells, cell aggregates, cellular microtissues, multicellular aggregates, cellular tissues, or any other configuration that allows for the encapsulation, confinement, or culture of cells. The microcompartment can be designed to be filled with one or more biocompatible materials, such as alginate, thus forming a solid microcompartment, such as a solid sphere or ball.Alternatively, the microcompartment can be hollow, thus forming an internal cavity in which biological elements can be contained or cultured. The structure therefore consists of a rigidified outer hydrogel layer and an inner part comprising at least one cell and a hydrogel layer or mesh suitable for culturing biological elements, particularly cell culture.

[0115] By "largest dimension" of a microcompartment within the meaning of the invention, we mean the value of the largest diameter of Feret of said microcompartment.

[0116] By "smallest dimension" of a microcompartment or cell layer within the meaning of the invention, we mean the value of the smallest diameter of Feret of said microcompartment.

[0117] For the purposes of this invention, "tissue" or "biological tissue" refers to the common biological meaning of tissue, that is, the intermediate level of organization between the cell and the organ. A tissue is a group of similar cells of the same origin (most often derived from a common cell lineage, although they may originate from the association of distinct cell lineages), grouped into clusters, networks, or bundles (fibers). A tissue forms a functional unit, meaning that its cells work together to perform the same function. Biological tissues regenerate regularly and are assembled to form organs.

[0118] “Strengthening mechanical properties” in the context of the invention means improving the ability of a material to withstand mechanical stresses, such as strengthening the elastic modulus and / or strengthening the fracture parameters.

[0119] For the purposes of this invention, "rinsing solution" refers to a solution used to treat microcompartments in order to enhance their mechanical properties. The rinsing solution differs from a culture medium in its function: it is not intended to maintain the growth of biological elements but to strengthen the structure of the cross-linked hydrogel. The rinsing solution may consist of a culture medium supplemented with cations, or an aqueous solution containing at least one cation at a concentration suitable for mechanical reinforcement. When the rinsing solution is a supplemented culture medium, the concentration of at least one cation is higher than the concentration naturally present in said culture medium.As an example, when the culture medium is DMEM / F-12, which naturally contains about 0.9 mM of calcium, the rinsing solution comprises a calcium concentration greater than 0.9 mM, preferably greater than 1.5 mM, more preferably of at least 2 mM.

[0120] For the purposes of this invention, "biological element" means any entity of biological origin or involved in biological processes, including, but not limited to, cells (e.g., stem cells, differentiated cells, immune cells), cellular components (organelles, membranes), vectors such as transduction agents (a viral particle, a viral pseudoparticle), biomolecules (proteins, peptides, enzymes, nucleic acids, lipids, carbohydrates), or derived entities (exosomes, extracellular vesicles, lysates).

[0121] Process for treating a microcompartment

[0122] The present invention relates to a method for treating a three-dimensional microcompartment comprising at least one biological element encapsulated in a cross-linked hydrogel so as to enhance the mechanical properties of said microcompartment.

[0123] To achieve this, the treatment process according to the invention includes a step of rinsing said microcompartment in a rinsing solution comprising at least one cation.

[0124] Advantageously, the rinsing step strengthens the structure of the cross-linked hydrogel by creating additional ionic bonds, improving the mechanical properties of the microcompartment.

[0125] According to one variant, the treatment process according to the invention includes a step of rinsing said microcompartment in a rinsing solution comprising at least one cation.

[0126] According to another variant, the treatment process according to the invention comprises several steps of rinsing said microcompartment in a rinsing solution comprising at least one cation.

[0127] Preferably, the treatment process according to the invention comprises at least one, at least two, at least three, at least four, or at least five rinsing steps of said microcompartment in a rinsing solution comprising at least one cation.

[0128] Unlike simple culture where microcompartments are maintained in a culture medium to allow the growth of biological elements, the rinsing step according to the invention has as its main objective the strengthening of the microcompartment's mechanical properties. This strengthening allows the microcompartments to withstand subsequent mechanical stresses, such as high-flow filtration, centrifugation, or aspiration, without being damaged. The inventors discovered that microcompartments cultured in a standard culture medium, without an additional rinsing step with a cation-enriched solution, exhibit increased fragility during mechanical handling.

[0129] In the context of the invention, the rinsing step is preferably carried out by completely immersing a microcompartment in the rinsing solution comprising at least one cation. This total immersion ensures uniform contact between the rinsing solution and the entire surface of the microcompartment.

[0130] In the context of the invention, the rinsing step differs from simply culturing the microcompartments in a culture medium. The rinsing step according to the invention is a specific treatment step, carried out to enhance the mechanical properties of the microcompartment before subsequent handling, such as mechanical stress. This rinsing step can be performed with a rinsing solution separate from the culture medium, or with a culture medium supplemented with cations at a concentration higher than that naturally present in said culture medium.

[0131] According to one embodiment, the rinsing step is carried out for a period of between 10 seconds and 10 minutes, preferably between 15 seconds and 5 minutes, even more preferably between 15 seconds and 2 minutes.

[0132] According to one embodiment, the rinsing step is carried out at a flow rate between 30 and 300 mL / min, preferably between 50 and 250 mL / min.

[0133] According to one embodiment, the volume of rinsing solution used is between 10 and 100 times the volume of microcompartments to be treated.

[0134] According to one embodiment, the rinsing solution comprises at least one cation selected from calcium, iron, barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations.

[0135] Preferably, at least one cation in the rinsing solution is a divalent or trivalent cation.

[0136] According to a particular embodiment, the rinsing solution comprises at least one divalent cation and at least one trivalent cation.

[0137] According to a particular embodiment, the rinsing solution comprises at least 2, 3, 4, 5 distinct cations.

[0138] The rinsing solution can have different variations in terms of composition and cation concentration.

[0139] According to one embodiment, the rinsing solution is a solution in which at least one cation has a concentration between 0.5 and 50mM, in particular between 0.5 and 25mM.

[0140] A cation concentration below 0.5 mM is insufficient to provide satisfactory reinforcement of the microcompartment's mechanical properties. Conversely, a concentration above 50 mM may negatively affect the viability of the encapsulated biological components.

[0141] Advantageously, within the range of values ​​between 0.5 and 50 mM, different concentrations can be used according to specific needs depending on the cations used.

[0142] The cation concentration in the rinsing solution can be adjusted within the described concentration range according to the specific needs of the microcompartment and the biological elements it contains. A higher cation concentration may lead to greater enhancement of mechanical properties, while a lower concentration may be preferable to maintain the viability of certain sensitive biological elements.

[0143] Preferably, the rinsing solution comprises at least calcium. In a particular embodiment, the rinsing solution comprises only one type of cation: calcium.

[0144] Calcium is a particularly advantageous cation because it strengthens the structure of cross-linked hydrogels, especially alginate-based hydrogels. Calcium forms ionic bonds with the alginate chains, increasing the rigidity and mechanical strength of the microcompartment.

[0145] Preferably the rinsing solution includes at least one divalent calcium (Ca 2+ ).

[0146] In a particularly suitable manner, the rinsing solution includes calcium at a concentration between 0.5 and 10mM, preferably between 2.5 and 5mM.

[0147] In particular, the rinsing solution may include calcium at a concentration between 1 and 8 mM, preferably between 1 and 6 mM, more preferably between 2 and 6 mM, and even more preferably between 2.5 and 5 mM.

[0148] According to one embodiment, the rinsing solution comprises calcium at a concentration between 0.5 and 2mM, in particular between 0.5 and ImM.

[0149] Advantageously, calcium supplementation of the rinsing solution allows the microcompartments to withstand increased mechanical stress. In particular, a calcium concentration of at least 2 mM enables the microcompartments to withstand filtration flow rates of at least 180 mL / min without damage. In comparison, microcompartments treated with a solution containing less calcium, or with no supplementation, may be damaged under mechanical stress.

[0150] In another embodiment, the rinsing solution contains at least iron. In a particular embodiment, the rinsing solution contains only one type of cation: iron.

[0151] In a particularly suitable manner, the rinsing solution includes iron at a concentration between 0.5 and 10mM, preferably between 0.5 and 5mM.

[0152] According to one embodiment, the rinsing solution comprises a ferrous cation (Fe 2+ ) and / or a ferric cation (Fe 3+ ).

[0153] In another embodiment, the rinsing solution comprises calcium and at least one cation selected from the iron ions (preferably Fe). 2+ and / or Fe 3+ ), barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations.

[0154] According to one embodiment, the rinsing solution comprises between 0.5 and 10 mM of calcium and at least one cation selected from among the iron ions (preferably Fe 2+ and / or Fe 3+), barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations at a concentration between 0.5 and 50mM, in particular between 0.5 and 25mM, preferably between 0.5 and 10mM, more preferably between 0.5 and 5mM.

[0155] In one embodiment, the rinsing solution comprises calcium ions (preferably Ca²⁺). 2+ ) and iron ions (preferably Fe2+ and / or Fe3+).

[0156] Preferably, in this embodiment, the rinsing solution comprises between 0.5 and 10 mM of calcium, preferably Ca 2+ ) and between 0.5 and 9mM of iron (preferably Fe2+ and / or Fe3+).

[0157] In another embodiment, the rinsing solution comprises at least zinc. In a particular embodiment, the rinsing solution comprises only one type of cation: zinc.

[0158] In a particularly suitable manner, the rinsing solution includes zinc at a concentration between 0.1 and 5 mM, preferably between 0.3 and 1 mM, even more preferably between 0.4 and 0.6 mM.

[0159] According to one embodiment, the rinsing solution comprises zinc sulfate (ZnSO4).

[0160] Zinc is a divalent cation (Zn 2+ ) which interacts with the carboxylate groups of alginate to form ionic bonds, strengthening the structure of the cross-linked hydrogel. Furthermore, zinc is a trace element naturally present in cell culture media, making it particularly compatible with maintaining cell viability.

[0161] According to another embodiment, the rinsing solution comprises at least copper. According to a particular embodiment, the rinsing solution comprises only one type of cation: copper.

[0162] In a particularly suitable manner, the rinsing solution includes copper at a concentration between 0.1 and 5 mM, preferably between 0.3 and 2 mM, even more preferably between 0.5 and 1 mM.

[0163] According to one embodiment, the rinsing solution comprises copper chloride (CuCl2).

[0164] Copper is a divalent cation (Cu 2+ ) which forms ionic bonds with the alginate chains, thus contributing to the mechanical reinforcement of the microcompartment. At appropriate concentrations, copper helps maintain cell viability and the proliferation capacity of encapsulated cells.

[0165] According to one embodiment, the rinsing solution comprises calcium and zinc, preferably calcium at a concentration between 0.5 and 10 mM and zinc at a concentration between 0.1 and 1 mM.

[0166] According to another embodiment, the rinsing solution comprises calcium and copper, preferably calcium at a concentration between 0.5 and 10 mM and copper at a concentration between 0.1 and 2 mM.

[0167] Advantageously, the combined use of several divalent cations can make it possible to obtain optimized mechanical properties according to the specific needs of the intended application.

[0168] According to one embodiment, the microcompartment obtained at the end of the rinsing step has a Young's modulus between 3kPa and 1.2MPa, preferably 3 and 800kPa, in particular between 3 and 600kPa, between 3 and 500kPa, preferably between 3 and 350kPa, even more preferably between 100 and 200kPa.

[0169] According to one embodiment, the microcompartment resulting from the rinsing step has a breaking stress between 10 and 500kPa, in particular between 200 and 500kPa.

[0170] In the context of the invention, the breaking strength of the microcompartment can be measured using a compression test, a torsion test or a tensile test.

[0171] According to one embodiment, the microcompartment from the rinsing step has: * a Young's modulus between 3kPa and 1.2MPa, preferably between 3 and 800kPa, in particular between 3 and 600kPa, between 3 and 500kPa, preferably between 3 and 350kPa; and / or * a tensile strength between 10 and 500kPa, in particular between 200 and 500kPa.

[0172] In one embodiment, the microcompartment is hollow and comprises an outer hydrogel layer defining an inner portion, said inner portion comprising at least one biological element. This hollow structure allows for the encapsulation of a significant volume of biological elements while maintaining an outer hydrogel layer. The cross-linked hydrogel outer layer acts as a semi-permeable barrier, allowing the exchange of nutrients and metabolites with the external environment while protecting the encapsulated biological elements.

[0173] According to one embodiment, the microcompartment obtained at the end of the rinsing step is hollow and comprises an outer hydrogel layer having a Young's modulus between 3kPa and 1.2MPa, preferably between 3 and 800kPa, in particular between 3 and 600kPa, between 3 and 500kPa, preferably between 3 and 400kPa, more preferably between 3 and 300kPa and defining an inner part, said inner part comprising at least one biological element.

[0174] According to one variant, the microcompartment obtained after the rinsing step comprises: - an outer hydrogel layer, and - an internal part comprising at least: * at least one biological element, and *a layer or mesh of hydrogel arranged between the outer layer and at least one biological element.

[0175] According to one embodiment, the microcompartment obtained after the rinsing step comprises: - an outer layer of hydrogel, and - an internal part comprising at least: * at least one biological element, and *a layer or mesh of hydrogel arranged between the outer layer and at least one biological element, said layer or mesh of hydrogel having a Young's modulus between 0.01 and 200kPa, in particular between 0.1 and 60kPa, even more preferably between 0.1 and 5kPa.

[0176] According to one embodiment, the microcompartment obtained after the rinsing step comprises: - an outer layer of hydrogel, said hydrogel having a Young's modulus between 3kPa and 1.2MPa, preferably between 3 and 800kPa, in particular between 3 and 600kPa, 3 and 500kPa, preferably between 3 and 400kPa, more preferably between 3 and 300kPa, and - an internal part comprising at least:* at least one biological element, and *a layer or mesh of hydrogel arranged between the outer layer and at least one biological element, said layer or mesh of hydrogel having a Young's modulus between 0.01 and 200kPa, in particular between 0.1 and 60kPa, even more preferably between 0.1 and 5kPa.

[0177] Preferably, the three-dimensional microcompartment is hollow and consists of an outer hydrogel layer encapsulating a hollow inner part comprising at least: - one or more biological element(s), and - possibly culture medium and / or extracellular matrix elements.

[0178] When the internal part of the microcompartment includes a culture medium, it is suitable for the encapsulated biological elements.

[0179] Advantageously, the culture medium provides the nutrients and factors necessary for the survival, growth, and function of the biological elements. Therefore, the composition of the culture medium can be adjusted according to the specific needs of the encapsulated biological elements.

[0180] When the inner part of the microcompartment contains extracellular matrix elements, these can be selected from proteins such as collagen, fibronectin, and laminin, or polysaccharides such as hyaluronic acid and their combinations. The presence of extracellular matrix elements can promote the adhesion, proliferation, and differentiation of encapsulated cells.

[0181] The composition of the microcompartment's internal components can be tailored to create specific microenvironments mimicking different tissue or organ types. This flexibility allows microcompartments to be used for a variety of applications, such as cell culture, drug testing, regenerative medicine, and tissue engineering.

[0182] According to another embodiment, the microcompartment is solid, i.e. not hollow, for example a solid hydrogel ball, encapsulating at least one biological element.

[0183] This solid structure offers increased mechanical resistance compared to a hollow structure. The solid hydrogel sphere also allows for a homogeneous distribution of biological elements throughout the entire volume of the microcompartment.

[0184] First, the solid hydrogel structure provides enhanced protection for the encapsulated biological elements. The hydrogel forms a continuous three-dimensional matrix that completely surrounds the biological elements, isolating them from the external environment. This physical barrier can help protect the biological elements from mechanical stress or other potentially damaging factors.

[0185] Secondly, the solid sphere configuration allows for a more uniform distribution of cations throughout the entire volume of the microcompartment. During the rinsing step with a solution containing at least one cation, the cation can diffuse homogeneously throughout the hydrogel structure. This uniform diffusion of cations results in a more balanced mechanical reinforcement of the entire microcompartment.

[0186] The microcompartment can be formed from different types of hydrogels, each with specific properties suited to various applications.

[0187] According to one embodiment, the hydrogel is selected from alginates, carrageenans, pectins, gelatins, collagen, hyaluronic acid, chitosan, and combinations thereof.

[0188] According to a preferred embodiment, the hydrogel comprises or is made exclusively of alginate.

[0189] The alginate may, in particular, be sodium alginate, composed of 80% α-L-guluronate and 20% α-3-D-mannuronate, having a Young's modulus greater than 100 kPa, preferably greater than 60 kPa, and more preferably greater than 100 kPa. In one embodiment, the outer hydrogel layer comprises alginate, said alginate having an average molecular weight of 100 to 400 kDa, more preferably a molecular weight between 150 and 250 kDa.

[0190] When the hydrogel of the outer layer of the microcompartment is alginate, the concentration of the alginate solution intended to form said outer layer of the microcompartment is preferably between 0.5 and 5% by mass, more preferably the concentration is equal to 2% (plus or minus 0.5%) by mass.

[0191] Advantageously, the use of a rinsing solution containing at least one cation, in particular divalent cations such as calcium, helps to strengthen the structure of the already cross-linked alginate hydrogel forming the microcompartment.

[0192] The cations in the rinsing solution bind to the alginate chains, increasing the crosslinking density and improving the mechanical properties of the microcompartment.

[0193] According to one embodiment, at least one biological element encapsulated in the microcompartment is chosen: a cell, a cell organelle, a cell fragment, a cell aggregate, an organoid, DNA, RNA, a protein, a microorganism, a vector, a vesicle, an exosome, a microtissue, a spheroid, an embryoid body and their combinations.

[0194] The choice of biological elements depends on the intended application and the desired properties of the microcompartment.

[0195] When the microcompartment contains at least one cell, it can be any type of cell, but in particular, at least one cell must be a eukaryotic cell. More preferably, at least one cell must be a human, plant, or animal cell.

[0196] In one particular embodiment, the microcompartment comprises pluripotent stem cells. A pluripotent stem cell is defined as a cell that has the capacity to form all the tissues present in the entire organism of origin, without, however, being able to form an entire organism as such. Pluripotent stem cells may, in particular, be induced pluripotent stem cells (iPSCs), MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem cells (ESCs). According to one embodiment, the microcompartment of the invention does not comprise embryonic stem cells (ESCs).

[0197] According to a particularly adapted variant of the invention, the microcompartment according to the invention comprises human or animal induced pluripotent stem cells.

[0198] In another particular embodiment, the microcompartment according to the invention comprises human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells and / or cells undergoing differentiation. The multipotent cells and / or progenitor cells were preferably obtained from pluripotent stem cells, in particular human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile was artificially modified to match that of particular multipotent cells and / or progenitors, typically by forced expression of transcription factors specific to the target cell phenotype.Preferably, multipotent and / or progenitor cells were obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells.

[0199] According to another embodiment, the microcompartment according to the invention comprises differentiated human or animal cells. The differentiated cells were preferably obtained from pluripotent stem cells or progenitor cells, in particular human pluripotent stem cells or human progenitor cells, or possibly from non-pluripotent human cells whose transcriptional profile has been artificially modified to match that of particular differentiated cells, typically by forced expression of transcription factors specific to the target cell phenotype. Preferably, the differentiated cells were obtained from pluripotent or multipotent stem cells or progenitor cells after contact with a solution capable of initiating the differentiation of said stem cells.According to one variant, the cellular content of the microcompartment includes homogeneous or mixed cellular identities.

[0200] Differentiated cells can, in particular, be present as at least one layer of cells, or as a three-dimensional tissue, cell aggregate, or microtissue, or as several tissues or microtissues within the microcompartment. This tissue or microtissue may be compacted or uncompacted, with or without light.

[0201] The microcompartment can therefore include several types of cells. In particular, the microcompartment can include, for example, induced pluripotent stem cells and / or multipotent cells and / or progenitor cells and / or cells undergoing differentiation and / or differentiated cells.

[0202] Thus, when the microcompartment includes at least one cell, it is chosen from among stem cells, progenitor cells, differentiated cells, cancer cells, and their combinations.

[0203] According to one variant, the microcompartment includes: - an outer layer of hydrogel, and - an internal part comprising at least: * at least one layer of cells, and * a layer or mesh of hydrogel arranged between the outer layer and the cell layer.

[0204] According to this variant, the layer or mesh of the inner hydrogel portion, preferably alginate, may include other constituents. Thus, said layer or mesh of the inner hydrogel portion preferably includes at least one peptide sequence, and more preferably a peptide sequence of interest capable of interacting with the cells constituting, in particular, the cell layer present in the microcompartment. For example, the peptide sequence may be a peptide or a protein. According to a particularly preferred design, the peptide sequence is a YIGSR motif and / or an RGD motif. The YIGSR motif is a peptide derived from the pi chain of laminin with the sequence Tyrosine-Isoleucine-Glycine-Serine-Arginine, facilitating cell adhesion to the layer or mesh of the inner hydrogel portion.The RGD motif is an Arginine-Glycine-Asparagine sequence peptide which also facilitates cell adhesion to the mesh of the inner hydrogel part.

[0205] According to the same variant, the layer or mesh of the inner part in hydrogel may comprise at least a second hydrogel distinct from the first hydrogel of the layer or mesh of the inner part, more preferably this is chosen from fibrin, laminin, fibronectin, entactin, hyaluronic acid, and collagen.

[0206] In this embodiment, the process according to the invention relates to the treatment of a three-dimensional microcompartment, delimited by the outer hydrogel layer and within said outer layer, an inner part comprises at least one cell and the hydrogel layer or mesh.

[0207] According to one embodiment, the microcompartment encapsulates at least one human biological element.

[0208] In one embodiment, the microcompartment is closed. A closed microcompartment offers several advantages for the encapsulation of biological elements.

[0209] The closed configuration creates a protective barrier that isolates the encapsulated biological elements from the external environment. This isolation protects the biological elements from potentially harmful external factors, such as contaminants or undesirable chemical or biological agents.

[0210] The sealed nature of the microcompartment also prevents leakage or escape of the encapsulated biological elements. This retention is beneficial for maintaining the desired concentration and localization of biological elements such as cells within the microcompartment.

[0211] Furthermore, a closed microcompartment configuration facilitates the handling and processing of encapsulated biological elements as a single unit. The closed structure allows the microcompartment to be transferred, washed, or subjected to various treatments while keeping the internal contents intact and protected.

[0212] According to one embodiment, the microcompartment has the shape of an ovoid, a cylinder, a spheroid, a sphere, a fiber or a teardrop.

[0213] Each of these geometric shapes has specific characteristics in terms of mechanical strength, heat exchange surface area, and hydrodynamic behavior. The choice of microcompartment shape can be adapted to the specific requirements of the intended application.

[0214] According to one embodiment, the smallest dimension of the microcompartment is between 1pm and 1.5mm, preferably between 100 and 800pm, even more preferably between 200 and 700pm.

[0215] According to one embodiment, the process according to the invention also includes a step of mechanical stressing said rinsed microcompartment, in which the mechanical stressing is carried out by filtration, agitation in liquid medium, centrifugation, aspiration or a combination thereof.

[0216] Advantageously, the rinsing step strengthens the microcompartments, enabling them to better withstand the mechanical stresses applied to them. Indeed, the properties of the cross-linked microcompartments can be altered by their environment, for example, the composition of the culture medium in which they are placed. The invention makes it possible to strengthen the mechanical properties of the microcompartments, thereby increasing the possibilities for industrial-scale production by reducing the limitations related to their fragility. Thus, the microcompartments can be subjected to steps such as filtration, aspiration, or agitation while minimizing losses, or even eliminating them completely.

[0217] According to one embodiment, the mechanical stressing step is carried out simultaneously with the microcompartment rinsing step.

[0218] According to one embodiment, the mechanical stressing step applies a centrifugal acceleration of at least 10g on at least one rinsed microcompartment.

[0219] Advantageously, when the mechanical stressing step uses the centrifugal effect, by any means, it allows for the optimization of growing conditions in a three-dimensional system.

[0220] Indeed, the centrifugal effect promotes the formation of clusters or aggregates of biological elements, preferentially of cells within microcompartments, in order to improve cell survival and growth, as well as the appearance of better cell structuring for the initiation of 3D amplification.

[0221] Preferably, when the microcompartments include cells and are derived from the mechanical stress step applying a centrifugal acceleration of at least 10g, they comprise less than 5% of single cells by number of all encapsulated cells, preferably less than 1%.

[0222] Indeed, in the context of the invention, it is preferable for the encapsulated cells to be in the form of clusters in order to optimize amplification and improve cell survival.

[0223] Advantageously, a centrifugal acceleration of at least 10g is suitable to promote the appearance of cell clusters without affecting cell viability.

[0224] In the context of the invention, the centrifugal acceleration exerted on at least one biological element and / or at least one microcompartment can be calculated using the following formula: [Math 1] G = R * fl 2 where R is the radius of the trajectory expressed in meters, Q is the angular velocity expressed in radians per second, and G is the centrifugal acceleration expressed in m / s²

[0225] Preferably, the centrifugal acceleration applied to at least one biological element and / or at least one microcompartment is between 10 and 3000g, even more preferably between 50 and 1000, in particular between 200 and 400g.

[0226] According to one embodiment, the centrifugal acceleration applied to at least one biological element and / or at least one microcompartment is between 10 and 500g, preferably between 10 and 400g.

[0227] According to one embodiment, the application of centrifugal acceleration is carried out by centrifugation, in particular using a centrifuge.

[0228] According to another embodiment, the application of centrifugal acceleration is carried out using a filtration device.

[0229] According to one embodiment, the rinsing step and / or the mechanical stressing step are carried out using at least one device comprising a body (2) including at least a first port (5) and a second port (6) and a filter membrane (3) extending along a plane P in the body (2) thus defining two compartments and capable of preventing the passage of microcompartments from one compartment to the other, the first port (5) being intended for the injection and withdrawal of microcompartments c bathed in a solution A and / or a rinsing solution and the second port (6) being intended for the withdrawal and injection of solution A and / or the rinsing solution, characterized in that the injection into the first port (5) is carried out along a direction D not substantially orthogonal to the plane P of the filter membrane (3).

[0230] For illustrative purposes, the said device is designated by reference 1 in its entirety and illustrated in figure 1 relates to a filtration device comprising a body 2 having a filter membrane 3 which extends in the body 2 along a plane P.

[0231] According to one embodiment, the rinsing and mechanical stressing steps are carried out simultaneously using at least one device 1.

[0232] According to one embodiment, the body 2 of the device 1 comprises an inner envelope 4 having rounded edges.

[0233] According to one embodiment of the invention illustrated in particular in Figures 2 and 5, the inner envelope 4 has the shape of a burger, that is to say a cylindrical shape with a rounded upper edge surface and a rounded lower edge surface.

[0234] According to the embodiment illustrated in figures 3 and 4, the inner envelope 3 of the body 2 has the shape of a donut, that is to say a toric shape.

[0235] According to the embodiment illustrated in Figure 2, the device 1 is symmetrical with respect to the filter membrane 3. Other non-symmetrical embodiments illustrated in Figures 3, 4, and 5 are also compatible with the invention. For example, the portion intended to receive microcompartments may have larger volumetric dimensions than the portion on the other side of the filter membrane 3, or vice versa.

[0236] The filter membrane 3 extends along a plane P within the body 2 of the device 1. The filter membrane 3 extends to the edges of the inner casing 4 of the body 2 of the device 1. In the illustrated embodiments, the effective cross-section of the filter membrane 3 is circular. In other, unillustrated embodiments, the effective cross-section of the filter membrane 3 is elliptical.

[0237] In order to retain the microcompartments, the filter membrane 3 includes a fibrous membrane capable of preventing the passage of the microcompartments.

[0238] The filter membrane 3 is therefore adapted according to the type of microcompartments implemented.

[0239] According to one embodiment of the invention, the filter membrane 3 comprises a microfabric covered with a fiber membrane. The fiber membrane contains a wide-mesh textile material, nylon, or PET. Other filter materials for the microcompartments may be used within the scope of the invention.

[0240] According to one embodiment of the invention, for microcompartments having a larger dimension between 120 and 700 micrometers, the filter membrane comprises porosities of less than 100 micrometers.

[0241] According to one embodiment of the invention, for microcompartments, having a largest dimension less than 600 micrometers, the filter membrane comprises porosities less than 100 micrometers.

[0242] According to one embodiment of the invention, the filter membrane 3 is capable of filtering at least one type of microcompartment and is also capable of allowing at least one other type of microcompartment to pass through. The mesh can thus be adapted according to the dimensions of the microcompartments to be filtered.

[0243] The body 2 of the device 1 includes a first port 5 intended for tangential injection of the microcompartments, bathed in a soaking solution.

[0244] The first port 5 comprises for its purposes a cylindrical shape which extends substantially in a direction D belonging to a plane P' parallel to the plane P of the filter membrane 3 as shown in Figure 1.

[0245] The second port 6 is located in the second part of the body 2, on the other side of the filter membrane 3. According to the embodiment illustrated in Figures 1 and 2, the second port 6 does not allow tangential injection into the body 2 of the device 1. According to the embodiment illustrated in particular in Figures 3, 4 and 5, the second port 6 is also suitable for tangential injection into the body 2 of the device 1.

[0246] According to the embodiment illustrated in Figure 1, the first port 5 includes an inlet 7 and an injection channel 8.

[0247] The injection channel 8 allows tangential injection into the inner envelope 4 of the body 2 of the device 1.

[0248] According to other unillustrated embodiments, the body 2 of the device 1 includes more than two inlet and outlet ports, thus allowing the injection of different types of microcompartment solutions on each side of the filter membrane 3.

[0249] Other forms of architecture can be shaped according to the use of the invention.

[0250] In the context of the invention, solution A can be any type of solution capable of comprising microcompartments.

[0251] According to one embodiment, solution A is a culture medium suitable for keeping the microcompartments in suspension or a crosslinking solution.

[0252] According to one embodiment, the rinsing and mechanical stressing steps are carried out continuously using at least two devices 1 according to any of the preceding embodiments. Preferably, when the rinsing and mechanical stressing steps are carried out continuously using at least two devices 1 according to any of the preceding embodiments, said devices are arranged in series.

[0253] Method for preparing a microcompartment:

[0254] According to another aspect, the invention relates to a method for preparing a three-dimensional microcompartment comprising carrying out the following steps: a) Encapsulation of at least one biological element in a cross-linked hydrogel to form a three-dimensional microcompartment; and b) Treatment of the microcompartment obtained in step a) according to the treatment process of any of the preceding embodiments.

[0255] Advantageously, the preparation process according to the invention makes it possible to form a microcompartment exhibiting improved resistance to mechanical stresses undergone in particular during filtration, centrifugation, aspiration or their combinations.

[0256] According to a preferred embodiment, the preparation process comprises the following steps in this order: a) Encapsulation of at least one biological element in a cross-linked hydrogel to form a three-dimensional microcompartment; b) Optionally, culture of the microcompartment in a culture medium; c) Rinsing the microcompartment in a rinsing solution comprising at least one cation at a concentration higher than that of the culture medium in step b); d) Mechanical stress on the rinsed microcompartment.

[0257] This sequence helps to strengthen the mechanical properties of the microcompartment just before mechanical handling, thus maximizing the protective effect of rinsing.

[0258] Preferably, at least one three-dimensional microcompartment obtained at the end of step a) is hollow in that it has an outer layer and a hollow inner portion comprising at least one biological element. Preferably, at least one microcompartment obtained at the end of step a) has an outer hydrogel layer and an inner portion comprising at least one cell.

[0259] According to one embodiment, the microcompartment obtained at the end of step a) has an outer hydrogel layer and an inner part comprising an internal matrix and at least one biological element, preferably a cell. Preferably, the internal matrix is ​​an extracellular matrix or an extracellular matrix substitute.

[0260] The microcompartment resulting from step a) comprises at least one biological element, preferably at least one cell, encapsulated in hydrogel, said hydrogel being biocompatible, i.e., non-toxic to biological elements, preferably to cells. The hydrogel must allow the diffusion of oxygen and nutrients to supply the biological elements when necessary, particularly to the cells contained within the microcompartment, and enable their survival. In a particularly preferred embodiment, the outer hydrogel layer of the microcompartment resulting from step a) comprises at least α1-ginate. In a particular embodiment, the outer hydrogel layer of the microcompartment resulting from step a) may consist exclusively of alginate.

[0261] The alginate may in particular be a sodium alginate, composed of 80% a-L-guluronate and 20% pD-mannuronate, having a Young's modulus greater than 100 kPa, preferably greater than 60 kPa, more preferably greater than 100 kPa.

[0262] According to one embodiment, the outer hydrogel layer of the microcompartment from step a) comprises alginate, said alginate advantageously having an average molecular weight of 100 to 400 kDa, more preferably the outer layer having a molecular weight between 150 and 250 kDa. When the hydrogel of the outer layer of the microcompartment from step a) is alginate, the concentration of the alginate solution intended to form said outer layer of the microcompartment is preferably between 0.5 and 5% by mass, more preferably the concentration is equal to 2% (plus or minus 0.5%) by mass.

[0263] When the concentration of the alginate solution intended to form the outer layer of the microcompartment in step a) is equal to 2%, the viscosity of the alginate is preferentially between 500 and 1600 mPa / s, more preferably between 1000 and 1400 mPa / s.

[0264] Advantageously, the outer hydrogel layer of the microcompartment from step a) is devoid of biological elements, preferably devoid of cells.

[0265] The outer hydrogel layer of the microcompartment produced in step a) thus protects the biological components, primarily cells, from the external environment. When the microcompartment produced in step a) contains cells, the outer hydrogel layer also limits cell proliferation through this physical barrier.

[0266] According to one embodiment, the microcompartment resulting from step a) comprises at least one biological element selected from: a cell, a cell organelle, a cell fragment, a cell aggregate, an organoid, DNA, RNA, a protein, a microorganism, a vector, a vesicle, an exosome, a microtissue, a spheroid, an embryoid body and combinations thereof.

[0267] When the microcompartment resulting from step a) contains cells, the cells can be any type of cell, but in particular, they are eukaryotic cells. More preferably, the cells are human, plant, or animal cells.

[0268] In one particular embodiment, the microcompartment resulting from step a) comprises pluripotent stem cells. A pluripotent stem cell is understood to be a cell that has the capacity to form all the tissues present in the organism of origin, without, however, being able to form an entire organism as such. Pluripotent stem cells may, in particular, be induced pluripotent stem cells (iPSCs), MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem cells (ESCs). According to one embodiment, the microcompartment according to the invention does not comprise embryonic stem cells (ESCs).

[0269] According to a particularly suitable variant of the invention, the microcompartment from step a) comprises human or animal induced pluripotent stem cells.

[0270] In another particular embodiment, the microcompartment resulting from step a) comprises human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells and / or cells undergoing differentiation. The multipotent cells and / or progenitor cells were preferably obtained from pluripotent stem cells, in particular human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile was artificially modified to match that of particular multipotent cells and / or progenitors, typically by forced expression of transcription factors specific to the target cell phenotype.Preferably, multipotent and / or progenitor cells were obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells.

[0271] According to another variant, the microcompartment resulting from step a) comprises differentiated human or animal cells. The differentiated cells were preferentially obtained from pluripotent stem cells or progenitor cells, in particular human pluripotent stem cells or human progenitor cells, or possibly from non-pluripotent human cells whose transcriptional profile was artificially modified to match that of particular differentiated cells, typically by forced expression of transcription factors specific to the target cell phenotype. Preferably, the differentiated cells were obtained from pluripotent or multipotent stem cells or progenitor cells after contact with a solution capable of initiating the differentiation of said stem cells.According to one variant, the cellular content of the microcompartment includes homogeneous or mixed cellular identities.

[0272] Differentiated cells can, in particular, be present as at least one layer of cells, or as a three-dimensional tissue, cell aggregate, or microtissue, or as several tissues or microtissues within the microcompartment. This tissue or microtissue may be compacted or uncompacted, with or without light.

[0273] The microcompartment resulting from step a) can therefore include several types of cells. In particular, the microcompartment resulting from step a) can include, for example, induced pluripotent stem cells and / or multipotent cells and / or progenitor cells and / or cells undergoing differentiation and / or differentiated cells.

[0274] According to one variant, the microcompartment resulting from step a) comprises: - an outer layer of hydrogel, and - an internal part comprising at least: * at least one layer of cells, and *a layer or mesh of hydrogel arranged between the outer layer and the cell layer.

[0275] According to this variant, the layer or mesh of the inner hydrogel portion, preferably alginate, may include other constituents. Thus, said layer or mesh of the inner hydrogel portion preferably includes at least one peptide sequence, and more preferably a peptide sequence of interest capable of interacting with the cells constituting, in particular, the cell layer present in the microcompartment resulting from step a). For example, the peptide sequence may be a peptide or a protein. According to a particularly preferred design, the peptide sequence is a YIGSR motif and / or an RGD motif. The YIGSR motif is a peptide derived from the pi chain of laminin with the sequence Tyrosine-Isoleucine-Glycine-Serine-Arginine, facilitating cell adhesion to the layer or mesh of the inner hydrogel portion.The RGD motif is an Arginine-Glycine-Asparagine sequence peptide that also facilitates cell adhesion to the internal hydrogel mesh.

[0276] According to the same variant, the layer or mesh of the inner part in hydrogel may comprise at least a second hydrogel distinct from the first hydrogel of the layer or mesh of the inner part, more preferably this is chosen from fibrin, laminin, fibronectin, entactin, hyaluronic acid, and collagen.

[0277] The microcompartment obtained at the end of step a) is then a three-dimensional microcompartment, delimited by the outer hydrogel layer and inside said outer layer, an inner part includes at least one cell and the hydrogel layer or mesh.

[0278] The microcompartment obtained at the end of step a) is preferably in various spherical or substantially spherical shapes. Advantageously, the three-dimensional microcompartment is hollow, more preferably, the hollow microcompartment is in the form of an ovoid, a cylinder, a spheroid, a sphere or a teardrop or in a substantially ovoid, substantially cylindrical, substantially spheroid, substantially spherical or substantially teardrop shape.

[0279] On the one hand, the outer hydrogel layer protects the cells from the external environment, limits uncontrolled cell proliferation, and restricts their differentiation if differentiation occurs; on the other hand, the possible presence of a layer or mesh in the inner part of the hydrogel and / or the extracellular matrix and / or extracellular matrix substitute provides a suitable environment for cell growth and multiplication.

[0280] According to one embodiment, the microcompartment from step a) has a larger dimension between 1pm and 1.5mm, preferably between 120pm and 800pm, preferably between 200pm and 600pm, even more preferably between 200pm and 500pm, in particular between 200pm and 250pm.

[0281] According to one embodiment, the microcompartment from step a) has a larger dimension of between 400 and 500 µm.

[0282] According to one embodiment, the microcompartment from step a) has a smaller dimension between 1 pm and 1.5 mm, preferably between 100 pm and 800 pm.

[0283] In the context of the invention, the largest dimension of the microcompartment is always greater than the smallest dimension of the microcompartment.

[0284] Thus, according to one embodiment, the microcompartment resulting from step a) has: *a larger dimension between 1 µm and 1.5 mm, preferably between 120 µm and 800 µm, preferably between 200 µm and 600 µm, even more preferably between 200 µm and 500 µm, in particular between 200 µm and 250 µm; and / or *a smaller dimension between 1 pm and 1.5 mm, preferably between 100 pm and 800 pm.

[0285] According to one embodiment, the encapsulation step a) includes a hydrogel crosslinking step carried out using a crosslinking solution comprising at least one crosslinking agent.

[0286] According to one embodiment, step a) may include the following steps: 1) Formation of a microcompartment of hydrogel, preferably hollow, encapsulating biological elements, using an encapsulation device, preferably comprising a microfluidic and / or millifluidic device and solutions, said solutions being preferably the following: - a hydrogel solution; - possibly an intermediate solution, preferably isotonic; and - a solution comprising biological elements, culture medium and possibly extracellular matrix and / or extracellular matrix substitute; 2) Crosslinking of the hydrogel, preferably using a crosslinking solution, such as a crosslinking bath.

[0287] Preferably, step a) of encapsulating at least one biological element in a cross-linked hydrogel includes the implementation of the following steps: 1) bring into contact at least one biological element, preferably at least one cell, and a hydrogel solution intended to form an outer layer to form at least one droplet, and 2) Crosslinking of the hydrogel using a crosslinking solution capable of crosslinking said hydrogel solution to form the outer layer of each microcompartment.

[0288] According to one embodiment, encapsulation step a) comprises the following steps: 1) co-injection of two or three solutions to form a hollow hydrogel microcompartment encapsulating biological elements, said solutions being chosen from: - a hydrogel solution; - possibly an intermediate solution, preferably isotonic; - a solution comprising biological elements, culture medium and possibly extracellular matrix and / or extracellular matrix substitute, concentrically via a microfluidic injector which allows the formation of a jet at the injector outlet consisting of the mixture of solutions, said jet breaking into drops; 2) Crosslinking of the hydrogel.

[0289] Preferably, step a) of encapsulating at least one biological element in a cross-linked hydrogel includes the implementation of the following steps: 1) bring into contact at least one biological element, preferably at least one cell, and a hydrogel solution intended to form an outer layer to form at least one droplet, and 2) collect the drop obtained in a crosslinking solution suitable for crosslinking said hydrogel solution to form the outer layer of each microcompartment.

[0290] Once the outer hydrogel layer is crosslinked by the crosslinking solution, the microcompartment is formed.

[0291] According to one embodiment, step a) of encapsulation is carried out by co-injection of two or three solutions: -a hydrogel solution; -possibly an intermediate solution, preferably isotonic; - a solution comprising biological elements, preferably cells, culture medium and possibly extracellular matrix and / or an extracellular matrix substitute, concentrically via a microfluidic injector which allows the formation of a jet at the injector outlet consisting of the mixture of solutions, said jet breaking into drops.

[0292] In this embodiment, in step 2) the drops formed in step 1) are collected in a crosslinking solution which crosslinks the hydrogel solution to form the outer layer of each microcompartment, the inner part of each drop being made up of the solution comprising the biological elements, preferably the cells, of the culture medium and the extracellular matrix.

[0293] Thus, according to a preferred embodiment, the process according to the invention comprises an encapsulation step a) comprising the implementation of the following steps: 1) Encapsulation of at least one biological element, preferably a cell, in hydrogel so as to form a suspension comprising at least one microcompartment in a crosslinking solution, said encapsulation is carried out by co-injection of two or three solutions: - a hydrogel solution; - possibly an intermediate solution, preferably isotonic; - a solution comprising biological elements, preferably cells, culture medium and possibly extracellular matrix and / or an extracellular matrix substitute, concentrically via a microfluidic injector which allows the formation of a jet at the injector outlet consisting of the mixture of solutions, said jet breaking down into droplets, 2) collection of said drops in a crosslinking solution which crosslinks the hydrogel solution to form the outer layer of each microcompartment, the inner part of each drop being made up of the solution comprising the biological elements, preferably cells, culture medium and possibly extracellular matrix.

[0294] In this embodiment, the intermediate solution, preferably isotonic in encapsulation step 1), is preferably a sorbitol solution.

[0295] In the same embodiment, the final opening diameter of the microfluidic injector of encapsulation step 1) is preferably between 50 and 800 pm, preferably between 80 and 240 pm, and the flow rate of each of the solutions is between 0.1 and 2000 mL / h, preferably between 10 and 2000 mL / h, more preferably between 11 and 100 mL / h.

[0296] According to one embodiment, the crosslinking solution comprises calcium.

[0297] Preferably, the crosslinking solution comprises calcium at a concentration between 0.5 and 300mM, preferably between 25 and 150mM, even more preferably between 50 and 120mM.

[0298] According to one embodiment, the crosslinking solution comprises at least one cation, preferably divalent, having a concentration of at least 25mM, preferably at least 50mM.

[0299] According to one embodiment, at least one crosslinking agent of step a) is distinct from at least one cation of step b).

[0300] According to another embodiment, at least one crosslinking agent of step a) is identical to at least one cation of step b).

[0301] According to a preferred embodiment, the crosslinking agent of step a) and the cation of step b) is calcium.

[0302] According to one embodiment, step a) can be implemented using an encapsulation system comprising preferably a microfluidic or millifluidic device.

[0303] In one embodiment, step a) is carried out using an encapsulation system comprising at least one component capable of electrically charging at least one of the solutions with an electrical potential and the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is a hydrogel solution and an internal flow is a solution comprising biological elements, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device being arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow such that this jet is fractionated into microcompartments.

[0304] In this embodiment, the encapsulation device may be a micro-fluidic or milli-fluidic type device capable of generating a concentric jet comprising at its center a solution including biological elements, optionally surrounded by the intermediate solution, itself optionally surrounded by a hydrogel solution.

[0305] The increase in hydrodynamic instabilities in the jet forces the jet to fragment into droplets, this effect being known as Plateau-Rayleigh instability.

[0306] These droplets, once the hydrogel solution has crosslinked, form the microcompartments. Electrically charging at least one of the solutions passing through the encapsulation device improves the breakup of the jet into droplets. This technique is known as "electro-jetting." It should be noted that the relative sizes of the outer and inner layers of the microcompartments can be adjusted by modifying the flow ratios of the two solutions at the dispensers.

[0307] In the case of electro-jetting, an electric field generating device, such as a metallic ring positioned downstream of the encapsulation device's outlet, can be added so that the jet or microcompartments pass through this ring. If necessary, the electric field generating device can be connected to an electrical potential, for example, to ground. This electric field helps to promote the dispersion of the microcompartments.

[0308] In another embodiment, step a) is carried out using an encapsulation system comprising at least one component capable of electrically charging at least one of the solutions with an electrical potential. The encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the hydrogel solution and an internal flow is the solution containing biological elements. A nozzle connected to the body receives this concentric flow and forms the outlet of the encapsulation device. The encapsulation device is arranged to form droplets directly from the concentric flow at the nozzle outlet. Once the hydrogel solution has cross-linked, these droplets form the microcompartments. The encapsulation device is thus of the "electrodripping" type and forms the droplets one after another directly from the nozzle, without a jet.

[0309] In yet another embodiment, step a) is carried out using an encapsulation device comprising a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the hydrogel solution and an internal flow is the solution comprising biological elements, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, and the encapsulation system also comprises an acoustic wave generator coupled to the nozzle and / or the body so that the encapsulation device is arranged to form, directly at the outlet of the nozzle, drops from the concentric flow.In this embodiment, the encapsulation device is of the "acousto-dripping" type, and forms the drops one after another directly from the nozzle under the effect of the acoustic waves emitted by the generator, the dimensions of the drops being determined according to the choice of the frequency and amplitude of the acoustic waves.

[0310] In yet another embodiment, step a) is carried out using an encapsulation device comprising a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the hydrogel solution and an internal flow is the solution comprising biological elements, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, and the encapsulation system also comprises a vibrating element coupled to the nozzle and / or the body so that the encapsulation device is arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow, said jet being broken down into droplets.In this embodiment, the encapsulation device is of the vibrating-jetting type and forms the droplets by breaking the concentric jet due to a Plateau-Rayleigh instability induced by the vibrations generated by the vibrating element. The dimensions of the droplets are determined by the choice of the frequency and amplitude of these vibrations. This vibrating element could, for example, be a piezoelectric actuator.

[0311] In yet another embodiment, step a) is carried out using an encapsulation device comprising a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the hydrogel solution and an internal flow is the solution containing biological elements, and a nozzle connected to the body for receiving said concentric flow and forming the outlet of the encapsulation device. The encapsulation system also includes a cutting element disposed downstream of the nozzle outlet, such that the encapsulation device is arranged to form, at the nozzle outlet, a concentric jet from the concentric flow, said jet being fractionated by the cutting element. In this embodiment, the encapsulation device is thus of the "jet cutting" type and forms the drops by cutting the concentric jet with the cutting element.The cutting element could, for example, be a rotating blade, with the dimensions of the drops being determined according to the rotation speed and the dimensions of the rotating blade.

[0312] In another embodiment, step a) is carried out using an encapsulation device comprising a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the hydrogel solution and an internal flow is the solution comprising biological elements, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, and the encapsulation system comprises an electromechanical element coupled to the nozzle, the encapsulation device being arranged to form, directly at the outlet of each nozzle, drops from the concentric flow under the effect of a vibration applied by the electromechanical element.

[0313] In this embodiment, the encapsulation device is of the "inkjet printing" type, forming the droplets one after another directly from each nozzle. This electromechanical element could, for example, be a piezoelectric actuator, the droplet dimensions being determined by the frequency and amplitude of the vibrations applied by this element.

[0314] Regardless of the embodiment envisaged, step a) can be carried out by an encapsulation system comprising a collection tank containing a crosslinking solution, preferably containing calcium, and arranged to collect the microcompartments formed by the encapsulation device. If necessary, the collection tank may be arranged downstream of the encapsulation device to collect the microcompartments formed by the encapsulation device, the stiffening solution being arranged to cause stiffening of the outer layer of each microcompartment upon immersion in this solution.

[0315] For example, the outlet of the encapsulation device may be positioned above the collection tank, so that the microcompartments fall by gravity into this collection tank. Advantageously, the collection tank and the encapsulation device are arranged at a distance from each other such that the microcompartments formed by the encapsulation device pass through a gaseous volume, in particular air, defined by a closed and sterile enclosure before being collected by the collection tank.

[0316] In yet another embodiment, step a) is carried out using an encapsulation device comprising a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the hydrogel solution and an internal flow is the solution comprising biological elements, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device and the collection tank being arranged so that the nozzle of the encapsulation device is in contact with a collection fluid, such as an oil, contained in a collection tank and / or so that the nozzle of the encapsulation device is immersed in a collection fluid contained in a collection tank, the encapsulation device being arranged to form, directly in the collection fluid, drops or a jet, which then break into drops, from the concentric flow.The gelling solution can then be cross-linked, for example via a stiffening solution, to form the microcompartments.

[0317] Regardless of the embodiment considered, the body and / or nozzle may be made of glass. Alternatively, the body and / or nozzle may be made of polymer or metal. The body and nozzle may be a single piece, or they may be manufactured separately and then assembled to form the encapsulation device.

[0318] Advantageously, the body comprises a first inlet connected to the first dispenser to receive the solution comprising biological elements and at least a second inlet connected to the second dispenser to receive the hydrogel solution, as well as a single outlet connected to the nozzle, the body comprising a main channel comprising a substantially straight portion defining a central axis of the encapsulation device, the main channel connecting the first inlet to the single outlet and at least one secondary channel connecting the second inlet to the single outlet, said secondary channel subdividing into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet in a single circular portion concentric with the first channel, said single circular portion and the first channel joining to form the single outlet of the body.

[0319] In another embodiment of the invention, step a) is carried out using an encapsulation device comprising a first body equipped with a nozzle, the first body being arranged to form at the outlet of the nozzle a first jet from the solution comprising biological elements supplied by the first dispenser, this first jet being fractionated into drops, and a second body equipped with a nozzle, the second body being arranged to form at the outlet of the nozzle a second jet from the hydrogel solution supplied by the second dispenser, the first and second bodies being arranged so that the drops from the first jet interact with the second jet to form the microcompartments.

[0320] In this embodiment, the first jet fragments into droplets due to a Plateau-Rayleigh instability. These droplets encounter the second continuous jet, which then encapsulates them, via the Marangoni effect, to form the microcompartments. Crosslinking of the outer layer of the microcompartments, formed by the hydrogel solution, can then be achieved in a gaseous environment, such as air, for example using ultraviolet radiation.

[0321] It may also be envisaged any combination of the embodiments described above, or even other embodiments of the encapsulation device allowing the generation of microcompartments without departing from the scope of the present invention, and in particular encapsulation devices allowing the formation of drops one after the other at the outlet of the encapsulation device and equipped with a means of controlling the ejection of the drops and of controlling the dimensions of the drops during their ejection, encapsulation devices allowing the formation of a concentric jet at the outlet of the encapsulation device and equipped with a means of separating the jet, after its exit from the encapsulation device, into drops, or even encapsulation devices allowing the coating of drops or a jet from a first device with drops or a jet from another device.

[0322] According to one embodiment, the preparation process according to the invention also includes a step of mechanical stressing said rinsed microcompartment, in which the mechanical stressing is carried out by filtration, agitation in liquid medium, centrifugation, aspiration or a combination thereof.

[0323] According to one embodiment, the preparation process according to the invention also includes a step of culturing the biological elements, preferably cells, encapsulated in the microcompartment. This culture step is preferably carried out within a bioreactor.

[0324] According to one embodiment, the preparation process also includes a cryopreservation step of the microcompartments obtained at the end of step b). Cryopreservation allows the long-term storage of the microcompartments while preserving the viability of the encapsulated cells.

[0325] According to one embodiment, the cryopreservation step of the microcompartments obtained at the end of step b) includes the implementation of the following steps: - Suspension of the microcompartments obtained at the end of step b) in a cryopreservant; - Optionally, homogeneous distribution of the microcompartments in the cryopreservant solution; - Gradual decrease in the temperature of the cryopreservant solution, including the microcompartments, down to 4°C, adjusted according to the biological elements contained in the microcompartments. - Rapid decrease in the temperature of the cryopreservant solution, including the microcompartments, from -4°C down to -180°C, adjusted according to the biological elements contained in the microcompartments. This could be, for example, at a rate of 1°C per minute down to -80°C, passively or in a controlled manner.

[0326] The invention also relates to the use of at least one divalent or trivalent cation to enhance the mechanical properties of a three-dimensional microcompartment comprising at least one biological element encapsulated in a cross-linked hydrogel. All embodiments described for the treatment process according to the invention, particularly with regard to the nature of the cations, their concentrations, the composition of the rinsing solution, and the characteristics of the microcompartment and the hydrogel, apply mutatis mutandis to this use.

[0327] Microcompartment according to the invention

[0328] The invention also relates to a three-dimensional microcompartment, obtained by any one of the embodiments of the preparation process according to the invention or any one of the embodiments of the treatment process according to the invention.

[0329] The microcompartment according to the invention thus presents unique characteristics combining mechanical resistance, biocompatibility and versatility of application, making it particularly suitable for various uses in the biomedical and biotechnological field.

[0330] According to one embodiment, the microcompartment according to the invention has a Young's modulus between 3kPa and 1.2MPa, preferably 3 and 800kPa, in particular between 3 and 600kPa, between 3 and 500kPa, preferably between 3 and 350kPa.

[0331] According to one embodiment, the microcompartment according to the invention has a breaking stress of between 10 and 500kPa, in particular between 200 and 500kPa.

[0332] In the context of the invention, the breaking strength of the microcompartment can be measured using a compression test, a torsion test or a tensile test.

[0333] According to one embodiment, the microcompartment according to the invention has: * a Young's modulus between 3 kPa and 1.2 MPa, preferably between 3 and 800 kPa, particularly between 3 and 600 kPa, between 3 and 500 kPa, preferably between 3 and 350 kPa; and / or * a breaking stress between 10 and 500kPa, in particular between 200 and 500kPa.

[0334] According to one embodiment, the microcompartment according to the invention is hollow and comprises an outer hydrogel layer having a Young's modulus between 3kPa and 1.2MPa, preferably between 3 and 800kPa, in particular between 3 and 600kPa, between 3 and 500kPa, preferably between 3 and 400kPa, more preferably between 3 and 300kPa and defining an inner part, said inner part comprising at least one biological element.

[0335] According to one embodiment, the microcompartment according to the invention comprises: - an outer layer of hydrogel, and - an internal part comprising at least: * at least one biological element, and *a layer or mesh of hydrogel arranged between the outer layer and at least one biological element, said layer or mesh of hydrogel having a Young's modulus between 0.01 and 200kPa, in particular between 0.1 and 60kPa, even more preferably between 0.1 and 5kPa.

[0336] According to one embodiment, the microcompartment according to the invention comprises: - an outer layer of hydrogel, said hydrogel having a Young's modulus between 3kPa and 1.2MPa, preferably between 3 and 800kPa, in particular between 3 and 600kPa, 3 and 500kPa, preferably between 3 and 400kPa, more preferably between 3 and 300kPa, and - an internal part comprising at least: * at least one biological element, and *a layer or mesh of hydrogel arranged between the outer layer and at least one biological element, said layer or mesh of hydrogel having a Young's modulus between 0.01 and 200kPa, in particular between 0.1 and 60kPa, even more preferably between 0.1 and 5kPa.

[0337] According to one embodiment, the microcompartment according to the invention is obtained by the preparation process according to the invention comprising the implementation of the following steps: a) Encapsulation of at least one biological element in a cross-linked hydrogel to form a three-dimensional microcompartment comprising the following steps: 1) Encapsulation of at least one biological element, preferably a cell, in hydrogel so as to form a suspension comprising at least one microcompartment in a crosslinking solution, said encapsulation is carried out by co-injection of two or three solutions: - a hydrogel solution; - possibly an intermediate solution, preferably isotonic; - a solution comprising biological elements, preferably cells, culture medium and possibly extracellular matrix and / or an extracellular matrix substitute, concentrically via a microfluidic injector which allows the formation of a jet at the injector outlet consisting of the mixture of solutions, said jet breaking down into droplets, 2) Crosslinking of the hydrogel; etb) Treatment of the microcompartment obtained in step a) by a process comprising a step of rinsing the microcompartment in a rinsing solution comprising at least one cation, preferably comprising calcium, and more preferably comprising calcium at a concentration between 0.5 and 10mM.

[0338] Use of the microcompartment according to the invention

[0339] The invention also relates to the use of a microcompartment according to the invention for cell culture, drug testing, regenerative medicine or tissue engineering.

[0340] Kit

[0341] The invention also relates to a kit for implementing the preparation or treatment process according to the invention comprising at least one rinsing solution containing at least one cation.

[0342] Advantageously, the kit according to the invention allows the user to have a suitable solution for strengthening the mechanical properties of three-dimensional microcompartments comprising cross-linked hydrogel.

[0343] According to one embodiment, the concentration of at least one cation in the rinsing solution is between 0.5 and 50 mM.

[0344] Preferably, at least one cation of the rinsing solution may be chosen from calcium, iron, barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations.

[0345] According to a particular embodiment, the kit according to the invention comprises a rinsing solution comprising at least 2, 3, 4, 5 distinct cations.

[0346] Preferably, at least one cation in the rinsing solution is a divalent or trivalent cation.

[0347] According to a particular embodiment, the rinsing solution of the kit comprises at least one divalent cation and at least one trivalent cation.

[0348] The kit's rinsing solution may have different variations in terms of composition and cation concentration.

[0349] According to one embodiment, the kit rinsing solution is a solution in which at least one cation has a concentration between 0.5 and 50mM, in particular between 0.5 and 25mM.

[0350] Advantageously, within the range of values ​​between 0.5 and 50 mM, different concentrations can be used according to specific needs depending on the cations used.

[0351] The cation concentration in the kit's rinsing solution can be adjusted within the described concentration range according to the specific needs of the microcompartment and the biological elements it contains. A higher cation concentration may lead to greater enhancement of mechanical properties, while a lower concentration may be preferable to maintain the viability of certain sensitive biological elements.

[0352] Preferably, the kit's rinsing solution contains at least calcium. In a particular embodiment, the rinsing solution contains only one type of cation: calcium.

[0353] Preferably, the kit's rinsing solution includes at least one divalent calcium (Ca 2+ ).

[0354] In a particularly suitable manner, the rinsing solution includes calcium at a concentration between 0.5 and 10mM, preferably between 2.5 and 5mM.

[0355] In particular, the rinsing solution may include calcium at a concentration between 1 and 8 mM, preferably between 1 and 6 mM, more preferably between 2 and 6 mM, and even more preferably between 2.5 and 5 mM.

[0356] According to one embodiment, the rinsing solution comprises calcium at a concentration between 0.5 and 2mM, in particular between 0.5 and ImM.

[0357] In another embodiment, the rinsing solution contains at least iron. In a particular embodiment, the rinsing solution contains only one type of cation: iron.

[0358] In a particularly suitable manner, the rinsing solution includes iron at a concentration between 0.5 and 10mM, preferably between 0.5 and 5mM.

[0359] According to one embodiment, the rinsing solution comprises a ferrous cation (Fe 2+ ) and / or a ferric cation (Fe 3+ ).

[0360] In another embodiment, the rinsing solution comprises calcium and at least one cation selected from the iron ions (preferably Fe). 2+ et / ou Fe 3+ ), barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations.

[0361] According to one embodiment, the rinsing solution comprises between 0.5 and 10 mM of calcium and at least one cation selected from among the iron ions (preferably Fe 2+ and / or Fe 3+ ), barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations at a concentration between 0.5 and 50mM, in particular between 0.5 and 25mM, preferably between 0.5 and 10mM, more preferably between 0.5 and 5mM.

[0362] In one embodiment, the rinsing solution comprises calcium ions (preferably Ca²⁺). 2+ ) and iron ions (preferably Fe2+ and / or Fe3+).

[0363] Preferably, in this embodiment, the rinsing solution comprises between 0.5 and 10 mM of calcium, preferably Ca 2+ ) and between 0.5 and 9mM of iron (preferably Fe2+ and / or Fe3+).

[0364] According to one embodiment, the kit according to the invention comprises a rinsing solution comprising calcium at a concentration between 0.5 and 10mM.

[0365] According to one embodiment, the kit may include at least two rinsing solutions with different cation concentrations, allowing the process to be adjusted according to specific needs.

[0366] Thus, according to this embodiment, the kit according to the invention comprises a rinsing solution comprising calcium having a concentration of 0.5 and 10mM and another rinsing solution comprising at least one cation selected from iron, barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel and their combinations having a concentration between 0.5 and 50mM.

[0367] According to one embodiment, the kit according to the invention comprises a rinsing solution comprising calcium having a concentration of 0.5 and 10mM and another rinsing solution comprising iron having a concentration between 0.5 and 5mM.

[0368] Examples

[0369] Example 1: Demonstration of the effect of the treatment process according to the invention (Calcium) on the mechanical resistance of the microcompartments, in the context of a filtration carried out using a cellular sieve.

[0370] A volume of 2.6 mL of microcompartments was deposited in a reversible cell sieve with a porosity of 100 pm.

[0371] A rinsing solution was used, comprising a culture medium, DMEM / F-12 initially containing around 0.9mM of Calcium, supplemented or not with calcium at a concentration of 2mM CaCl. Thus, the calcium concentration of the rinsing solution comprising the DMEM / F-12 culture medium supplemented with calcium is 2.9mM.

[0372] The microcompartments were rinsed at a flow rate of 50 mL / min for 1 minute.

[0373] Results

[0374] The results of this example are illustrated in Figures 6A and 6B:

[0375] Figure 6A: In the absence of calcium supplementation, the cellular microcompartments exhibit increased fragility, as well as a greater tendency to clump together on the filter.

[0376] Figure 6B: Conversely, the microcompartments washed with the calcium-supplemented solution show better mechanical resistance and a reduction in agglomeration on the filter.

[0377] This example highlights the beneficial impact of the treatment process according to the invention via a rinsing step using a rinsing solution comprising calcium on the resistance and stability of the cellular microcompartments during their handling.

[0378] Example 2: Demonstration of the effect of the treatment process according to the invention (Calcium) on the mechanical resistance of the microcompartments, in the context of a rinsing and filtration carried out using device 1.

[0379] A 4 mL volume of microcompartments was introduced into device 1. The microcompartments were then rinsed with DM EM / F-12 supplemented with different concentrations of calcium, between 2 mM and 4 mM, for 15 seconds at a flow rate of 250 mL / min.

[0380] After the rinsing step, a photograph of the microcompartments recovered in DMEM / F-12 was taken by post-rinsing microscopy (Figure 7), as well as a photograph of the remaining microcompartments trapped in the filter used at the end of the rinsing. These photographs were obtained using a wide-field microscope at 2X magnification.

[0381] Observations show that the integrity of the microcompartments is preserved. A small proportion of cellular microcompartments were damaged by impact on the filter, indicating increased mechanical resistance.

[0382] Furthermore, no significant aggregation of the microcompartments was observed on the filter, thanks to the calcium supplementation in the rinsing solution. These results demonstrate that calcium supplementation (Ca 2+ ) of the rinsing solution significantly improves the mechanical resistance of the cross-linked cellular microcompartments during rinsing, as illustrated with device 1.

[0383] Example 3: Evaluation of the mechanical resistance of microcompartments during filtration at different rinsing flow rates according to the treatment process of the invention (Calcium).

[0384] A 3 mL volume of microcompartments was loaded onto a reversible cell sieve with a porosity of 100 µm. The microcompartments were then rinsed with different solutions, either DMEM alone or DMEM supplemented with increasing concentrations of calcium, as listed in Table 1. Rinsing was performed for 1 minute at different flow rates.

[0385] After this step, the microcompartments were examined under a phase-contrast microscope at 4X magnification to assess any damage, including ruptures (Figure 8). It was observed that microcompartments rinsed with DMEM alone were damaged at a rinsing flow rate of 60 mL / min. In contrast, the addition of calcium to the DMEM allowed for an increased rinsing flow rate without damaging the microcompartments.

[0386] Specifically, a 1 mM CaCl2 concentration preserved the integrity of the microcompartments up to a flow rate of 120 mL / min, but damage was observed beyond this rate (Figure 8). With 2 mM CaCl2 or higher, the microcompartments remained intact even at higher flow rates, up to 180 mL / min.

[0387] Table 1 summarizes the microscopic observations, showing that microcompartments washed with calcium-supplemented DMEM exhibit improved mechanical resistance. The optimal concentration observed is 2 mM CaCl2 or higher, ensuring protection against damage at high flow rates. [Table 1]

[0388] The symbol - means that microscopic observation did not reveal any damage to the microcompartments.

[0389] The symbol + means that microscopic observation has identified damage to the microcompartments.

[0390] Example 4: Demonstration of the effect of the treatment process according to the invention (Iron) on the mechanical resistance of the microcompartments, in the context of a filtration carried out using a cellular sieve.

[0391] This example illustrates an embodiment of the invention in which the rinsing solution is supplemented with iron (FeCl3). Three distinct cell lines were tested (Cell Line No. 1, 2 and 3) while being mixed with a cell culture medium.

[0392] The co-injection of the solutions was performed using a microfluidic injector with three lines upstream of the nozzle: a first line containing the suspended cells, a second line containing a 2% alginate solution, and a third line containing an intermediate sorbitol solution. After encapsulation, the resulting droplets were collected in a calcium (CaCl2) bath, allowing cross-linking of the alginate and the formation of cell microcompartments.

[0393] After encapsulation, the microcompartments were rinsed with serum-free culture medium. A 1 mL volume of microcompartments was loaded onto a reversible cell sieve (100 µm), and then a DMEM solution supplemented with FeCl3 concentrations of 0.79 mM or 8.9 mM (75 mL) was injected to perform the rinsing step. The rinsed microcompartments were then examined by microscopy.

[0394] The results are illustrated in figures 9 (cell line no. 1), 10 (cell line no. 2) and 11 (cell line no. 3).

[0395] In general, cellular microcompartments exhibit a more rigid and harder outer shell after rinsing with a solution containing iron.

[0396] After 5 days of culture in a flask, the microcompartments were dissolved and the cells were dissociated to assess their viability (Figure 12), their amplification factor (Figure 13) and their pluripotency (Figures 14 and 15).

[0397] Cell lines No. 1 and No. 2 show greater amplification than the controls.

[0398] These results demonstrate improved mechanical resistance of cellular microcompartments subjected to rinsing with a rinsing solution containing iron, without negative impact on their amplification, viability or ability to maintain their pluripotency.

[0399] Example 5: Demonstration of the effect of the treatment process according to the invention (Zinc and Copper) on the mechanical resistance of the microcompartments and cell viability

[0400] This example illustrates an embodiment of the invention in which the rinsing solution is supplemented with zinc (ZnSO4) or copper (CuCl2) at different concentrations.

[0401] Cellular microcompartments containing pluripotent stem cells were prepared by co-injection of solutions using a microfluidic injector, then cross-linked in a calcium bath (CaCl2).

[0402] The microcompartments were then rinsed with different DMEM solutions supplemented as follows: - CaCl23 mM (control) - ZnSO40.5 mm - ZnS041 mM - ZnS0410 mM - CuCl20.5 mM - CuCl21 mM - CuCl210 mM

[0403] The microcompartments were cultured for 5 days with a daily renewal of the culture medium (1 mL).

[0404] The results are illustrated in Figures 16 to 19.

[0405] The cultures that showed satisfactory cell growth were those rinsed with: - CaCl23 mM (control) - ZnSO40.5 mm - CuCl20.5 mM - CuCl21 mM

[0406] In contrast, cultures rinsed with ZnSO41 mM, ZnSO410 mM and CuCl210 mM did not show cell growth, indicating that these concentrations are too high to maintain cell viability.

[0407] Cells cultured in microcompartments rinsed with ZnSO40.5 mM, CuCl20.5 mM and CuCl21 mM show amplification and viability similar to those of the control (CaCl23 mM) after 5 days of culture.

[0408] Lactate production, an indicator of cellular metabolic activity, follows a similar trend for the ZnSO40.5 mM, CuCI20.5 mM and CuCI21 mM conditions compared to the CaCI23 mM control ([Figure 18]).

[0409] Pluripotency measurements show that the cells maintain high expression of the OCT4, SOX2 and OCT4 / SOX2 markers (close to 100%) for all conditions that allowed cell growth (Figure 19).

[0410] These results demonstrate that zinc at a concentration of 0.5 mM and copper at concentrations of 0.5 mM and 1 mM can be used as alternatives or complements to calcium in the rinsing solution, allowing to strengthen the mechanical properties of the microcompartments while preserving the viability, the capacity for proliferation and the pluripotency of the encapsulated cells.

[0411] Example 6: Characterization of the mechanical properties of alginate hydrogels by rheological measurements

[0412] This example illustrates the characterization of the mechanical properties of bulk alginate hydrogels (bulk gels) after different washing treatments.

[0413] Mass production protocol for hydrogels:

[0414] Bulk alginate gels were manufactured using a mold composed of two plastic parts separated by a dialysis membrane (MWCO10 kDa).

[0415] 2 mL of liquid alginate were poured into the mold. The mold was immersed in 30 mL of a 100 mM calcium bath for 1.5 hours, then the bath was renewed and left for 3.5 hours.

[0416] The bath was then replaced with the wash medium for 30 minutes, renewed and left to stand overnight, then renewed and left for 1 hour.

[0417] The washing media tested were as follows: - 100 mM Ca (crosslinking control) - DMEM + 0 mM Ca - DMEM + 0.5 mM Ca - DMEM + 2 mM Ca - DMEM + 10 mM Ca

[0418] Rheological measurements: The gel was removed from the mold and placed in the rheometer geometry (25 mm diameter sandblasted plane-to-plane geometry). The geometry was lowered in 0.2 mm increments, the sample was left in equilibrium for each increment, and then an oscillatory measurement was performed at 1 Hz and 0.1% strain.

[0419] The results are presented in the table below and illustrated in Figure 21: [Table 2]

[0420] The Young's modulus E was calculated from the shear modulus G according to the relation E = 3G, assuming a Poisson's ratio v = 0.35.

[0421] These results show that the calcium concentration in the washing medium influences the mechanical properties of the hydrogel. Washing with a medium containing 10 mM calcium maintains a higher shear modulus (48 kPa) compared to washing without additional calcium (44 kPa) or with intermediate concentrations (40-41 kPa).

[0422] The highest shear modulus (67 kPa) is obtained for gels maintained in the crosslinking bath at 100 mM calcium, which corresponds to the maximum crosslinking of al-ginate.

[0423] These rheological measurements confirm that rinsing treatment with a solution containing cations, particularly calcium, allows for the modulation and strengthening of the mechanical properties of alginate hydrogels.

Claims

Demands

1. A method for treating a three-dimensional microcompartment comprising at least one biological element encapsulated in a cross-linked hydrogel, so as to enhance the mechanical properties of said microcompartment, characterized in that it comprises a step of rinsing the microcompartment in a rinsing solution comprising at least one cation.

2. A method according to the preceding claim, characterized in that the rinsing solution is a solution in which at least one cation has a concentration between 0.5 and 50mM.

3. A method according to any one of the preceding claims, characterized in that at least one cation of the rinsing solution is selected from the following ions: calcium, iron, barium, strontium, copper, lead, aluminum, magnesium, manganese, zinc, cobalt, nickel, and combinations thereof.

4. A method according to any one of the preceding claims, characterized in that the rinsing solution comprises calcium at a concentration of between 0.5 and 10 mM.

5. A method according to any one of the preceding claims, characterized in that the rinsing solution comprises zinc at a concentration of between 0.1 and 5 mM, preferably between 0.3 and 1 mM, and even more preferably between 0.4 and 0.6 mM.

6. A method according to the preceding claim, characterized in that the rinsing solution comprises zinc sulfate (ZnSO4).

7. A process according to any one of the preceding claims, characterized in that the rinsing solution comprises copper at a concentration between 0.1 and 5 mM, preferably between 0.3 and 2 mM, even more preferably between 0.5 and 1 mM.

8. A method according to the preceding claim, characterized in that the rinsing solution comprises copper chloride (CuCl2).

9. A method according to any one of the preceding claims, characterized in that at least one cation of the rinsing solution is a divalent or trivalent cation.

10. A process according to any one of the preceding claims, characterized in that the rinsing solution comprises calcium and zinc, preferably calcium at a concentration of between 0.5 and 10 mM and zinc at a concentration of between 0.1 and 1 mM.

11. A process according to any one of the preceding claims, characterized in that the rinsing solution comprises calcium and copper, preferably calcium at a concentration of between 0.5 and 10 mM and copper at a concentration of between 0.1 and 2 mM.

12. A method according to any one of the preceding claims, characterized in that the microcompartment is hollow and in that it comprises an outer hydrogel layer defining an inner part comprising at least one biological element.

13. A method according to the preceding claim, characterized in that the three-dimensional microcompartment is hollow, and consists of an outer hydrogel layer encapsulating a hollow inner part comprising at least: - one or more biological element(s), and - possibly culture medium and / or extracellular matrix elements.

14. A method according to any one of claims 1 to 11, characterized in that the microcompartment is a solid hydrogel ball encapsulating at least one biological element.

15. A method according to any one of the preceding claims, characterized in that the hydrogel is selected from alginates, carrageenans, pectins, gelatins, collagen, hyaluronic acid, chitosan, and combinations thereof.

16. A method according to any one of the preceding claims, characterized in that the hydrogel comprises alginate.

17. A method according to any one of the preceding claims, characterized in that at least one biological element encapsulated in the microcompartment is selected from: a cell, a cell organelle, a cell fragment, a cell aggregate, an organoid, DNA, RNA, a protein, a microorganism, a vector, a vesicle, an exosome, a microtissue, a spheroid, an embryoid body and combinations thereof.

18. A method according to the preceding claim, characterized in that at least one cell is selected from stem cells, progenitor cells, differentiated cells, cancer cells, and combinations thereof.

19. A method according to any one of the preceding claims, characterized in that the microcompartment encapsulates at least one human biological element.

20. A method according to any one of the preceding claims, characterized in that the microcompartment is closed.

21. A method according to any one of the preceding claims, characterized in that the microcompartment has the shape of an ovoid, a cylinder, a spheroid, a sphere, a fiber or a teardrop.

22. A method according to any one of the preceding claims, characterized in that the smallest dimension of the microcompartment is between 1 pm and 1.5 mm, preferably between 100 pm and 800 pm.

23. A method according to any one of the preceding claims, characterized in that it comprises a step of mechanical stressing said rinsed microcompartment, in which the mechanical stressing is carried out by filtration, agitation in liquid medium, centrifugation, aspiration or a combination thereof.

24. A method according to the preceding claim, characterized in that the mechanical stressing step applies a centrifugal acceleration of at least 10g on at least one rinsed microcompartment.

25. A method according to any one of the preceding claims, characterized in that the rinsing step and / or the mechanical stressing step are carried out using at least one device comprising a body (2) including at least a first port (5) and a second port (6) and a filter membrane (3) extending along a plane P in the body (2) thus defining two compartments and capable of preventing the passage of the cell microcompartments from one compartment to the other, the first port (5) being intended for the injection and withdrawal of the cell microcompartments bathed in a soaking solution and / or a rinsing solution and the second port (6) being intended for the withdrawal and injection of the soaking solution and / or the rinsing solution, characterized in that the injection into the first port (5) is carried out along a direction D not substantially orthogonal to the plane P of the filter membrane (3).

26. Method for preparing a three-dimensional microcompartment comprising carrying out the following steps:. a) Encapsulation of at least one biological element in a cross-linked hydrogel to form a three-dimensional microcompartment; b) Treatment of the microcompartment obtained in step a) according to the process of any one of claims 1 to 25.

27. ​​A method according to the preceding claim, characterized in that the encapsulation step a) comprises a crosslinking step of the hydrogel using a crosslinking solution comprising at least one crosslinking agent.

28. A method according to the preceding claim, characterized in that at least one crosslinking agent is calcium.

29. A process according to the preceding claim, characterized in that at least one crosslinking agent is calcium having a concentration between 0.5 and 300mM, preferably a concentration between 25 and 150mM.

30. A method according to any one of claims 26 to 29, characterized in that at least one crosslinking agent of step a) is distinct from at least one cation of step b).

31. A method according to any one of claims 26 to 30, characterized in that at least one crosslinking agent of step a) is identical to at least one cation of step b).

32. A method according to any one of claims 26 to 31, characterized in that step a) comprises the following steps: 1) Formation of a hydrogel microcompartment encapsulating biological elements, using an encapsulation device, preferably comprising a microfluidic and / or millifluidic device, and solutions, said solutions being preferably the following: - a hydrogel solution; - possibly an intermediate solution, preferably isotonic; and - a solution comprising biological elements, culture medium and possibly extracellular matrix and / or extracellular matrix substitute; 2) Crosslinking of the hydrogel, preferably using a crosslinking solution.

33. A method according to claim 32, characterized in that step a) comprises the following steps 1) Co-injection of two or three solutions to form a hollow microcompartment of hydrogel encapsulating biological elements, said solutions being chosen from: - a hydrogel solution; - possibly an intermediate solution, preferably isotonic; and - a solution comprising biological elements, culture medium and possibly extracellular matrix and / or extracellular matrix substitute, concentrically via a microfluidic injector which allows the formation of a jet at the injector outlet consisting of the mixture of solutions, said jet breaking into drops; 2) crosslinking of the hydrogel.

34. A method according to any one of claims 26 to 33, characterized in that it comprises an additional step of culturing the cells encapsulated in the microcompartment, preferably in a bioreactor.

35. A method according to any one of claims 26 to 34, characterized in that it comprises an additional step of cryopreservation of the microcompartments obtained.

36. Three-dimensional microcompartment, obtained by a process according to one of the preceding claims.

37. Microcompartment according to the preceding claim, characterized in that it presents * a Young's modulus between 3 kPa and 1.2 MPa, preferably between 3 and 800 kPa, particularly between 3 and 600 kPa, between 3 and 500 kPa, preferably between 3 and 350 kPa; and / or * a tensile strength between 10 and 500 kPa, in particular between 200 and 500 kPa.

38. Microcompartment according to the preceding claim, characterized in that the hydrogel exhibits: - a shear modulus between 30 and 80 kPa, preferably between 40 and 70 kPa; and / or - a Young's modulus between 100 and 250 kPa, preferably between 100 and 200 kPa.

39. Use of the microcompartment according to any one of claims 36 to 38, for cell culture, drug testing, regenerative medicine, or tissue engineering.

40. Use of at least one divalent or trivalent cation to enhance the mechanical properties of a three-dimensional microcompartment comprising at least one biological element encapsulated in a cross-linked hydrogel.

41. Use according to the preceding claim, characterized in that the cation is selected from calcium, iron, zinc, copper, barium, strontium, magnesium, manganese, cobalt, nickel and their combinations.

42. Kit comprising at least one rinsing solution comprising at least one cation in which at least one cation has a concentration between 0.5 and 50mM, for carrying out a process according to any one of claims 1 to 35.

43. Kit according to the preceding claim, characterized in that at least one cation is selected from the ions calcium, iron, barium, strontium, copper, lead, aluminium, magnesium, manganese, zinc, cobalt, nickel and their combinations.

44. Kit according to any one of claims 42 or 43, characterized in that it comprises a rinsing solution comprising zinc at a concentration of between 0.1 and 1 mM, preferably zinc sulfate (ZnSO4) at a concentration of about 0.5 mM.

45. Kit according to any one of claims 42 to 44, characterized in that it comprises a rinsing solution comprising copper at a concentration of between 0.1 and 2 mM, preferably copper chloride (CuCl2) at a concentration of between 0.5 and 1 mM.

46. Kit according to any one of claims 42 to 45, characterized in that it comprises a rinsing solution having a calcium concentration of between 0.1 and 10mM and a rinsing solution comprising at least one cation selected from the ions calcium, iron, barium, strontium, copper, lead, aluminium, magnesium, manganese, zinc, cobalt, nickel and their combinations having a concentration of between 0.5 and 50mM.

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