Optimization of three-dimensional cell culture

Abstract

The invention relates to the technical field of three-dimensional cellular microcompartment culture. In particular, the invention relates to a method for preparing a three-dimensional cellular microcompartment comprising the implementation of an encapsulation of at least one cell in hydrogel so as to form a suspension comprising cellular microcompartments in a soaking solution and of at least one step applying a gravity of at least 10 g to at least one cell and / or at least one cellular microcompartment. The invention also relates to a device suitable for implementing at least one part of said method.

Description

[0001] OPTIMIZATION OF CELL CULTURE IN THREE DIMENSIONAL

[0002] Technical field

[0003] The invention relates to the technical field of the culture of three-dimensional cellular microcompartments and devices for filtering suspended mesoscopic objects. In particular, the invention relates to a method for preparing a three-dimensional cellular microcompartment and a device suitable for implementing at least part of said method.

[0004] State of the art

[0005] Three-dimensional (3D) cell culture is a laboratory technique that offers broad application prospects in multiple fields. It offers a valuable alternative to conventional two-dimensional (2D) cell culture methods, allowing cells to be grown in an environment closer to their natural physiological context. This technology has the potential to revolutionize various fields, from basic research to pharmacology and cell therapy.

[0006] Traditional 2D cell culture methods have long been the mainstay of biomedical research, but they have some significant limitations. Cells cultured in 2D are arranged on flat surfaces, which do not accurately reflect the complex three-dimensional environment of living tissues. This discrepancy between 2D cell cultures and in vivo conditions can lead to inadequate or inaccurate results, limiting its relevance.

[0007] 3D cell culture aims to overcome these challenges by allowing cells to grow in an environment that more closely mimics the three-dimensional structure of biological tissues. To achieve this, several methods have been developed, each with advantages and disadvantages depending on the intended use.

[0008] Despite the existence of a great diversity of techniques, 3D cell culture remains in constant evolution with the aim of achieving standardization of processes and overcoming certain technical limits.

[0009] To date, a major drawback of 3D cell culture is that in some cases, cell survival may be insufficient and the amplification factor may be considered low in view of the behavior of cells in vivo. In other words, the implementation of 3D cell culture processes can present many challenges related to cell viability and the production of sufficient quantity of cells depending on the intended use.

[0010] These drawbacks are, in part, closely related to the fact that, in some cases, cells are not properly organized within the 3D cellular structure. When cells are not sufficiently well integrated into the three-dimensional structure, they tend to become isolated or scattered, which can lead to increased cell death.

[0011] When cells are isolated within the 3D cellular structure, they may encounter several difficulties:

[0012] *Insufficient structural support: Isolated cells have poor structural cohesion compared to those properly organized in 3D. As a result, they are more vulnerable to mechanical forces and stresses from the culture medium, which can lead to increased cell mortality.

[0013] *Difficulties with nutrient and oxygen supply: Isolated cells may have difficulty accessing nutrients and oxygen because they do not benefit from the same distribution as those properly organized in the 3D structure. This limitation can compromise their survival and growth.

[0014] *Limited cellular interactions: Isolated cells are less likely to interact appropriately with their neighboring cells, which can negatively affect cell signaling, intercellular communication, and the regulation of essential cellular processes.

[0015] *Limited amplification: The dispersion of cells in 3D culture can make it difficult for them to multiply and amplify, as they are less likely to form colonies, cysts or proliferative aggregates.

[0016] Therefore, the lack of adequate structural organization of cells in 3D cultures may lead to greater cell mortality and limitations in cell production.

[0017] Thus, it appears crucial to develop methods to promote the organization of cells into clusters or aggregates, thus minimizing the aforementioned drawbacks, such as insufficient cell viability and amplification.

[0018] There is therefore a need for a new method for preparing three-dimensional cellular microcompartments that can promote the appearance of cellular clusters or aggregates while improving cell survival and amplification.

[0019] In addition, there is also a need for a device for filtering suspended mesoscopic objects which can be used in particular in the implementation of such a method for preparing three-dimensional cellular microcompartments.

[0020] Mesoscopic cellular objects in suspension, hereinafter referred to as OCMS, comprise a set of objects whose dimensions vary between 5 pm and 5 mm, which are in suspension and which comprise at least one eukaryotic cell, encapsulated or not. For example, OCSM comprise human cells in suspension, microtissues, spheroids, cell aggregates, embryoid bodies, encapsulated or not.

[0021] In the field of the present invention, OCMS filtration modules are known comprising a body comprising a filter membrane intended to retain the OCMS but for example capable of allowing the solution in which the OCMS are bathed to pass through.

[0022] Several filtration techniques exist, in particular frontal filtration, where the OCMS are injected normally at the plane of the filter membrane. This technique can cause the accumulation and compaction of OCMS on the membrane which can prevent the proper filtration of the OCMS, damage them and clog the filter.

[0023] Another filtration technique is tangential flow filtration, where the OCMS in solution are injected through a tube porous to the solution but not to the OCMS, in which the solution is withdrawn through the tube wall, while the OCMS pass through the tube. This tangential flow filtration technique minimizes the risk of accumulation and compaction. The disadvantage of this technique is that it relies on filters that are complex to manufacture and it is difficult to completely rinse the OCMS. Indeed, there is always a residual proportion of soaking solution after rinsing due to the fact that the OCMS are not confined in a compartment and multiple rinsing steps are required to achieve the desired rinse solution concentration.

[0024] Another objective of the invention is to overcome these drawbacks.

[0025] Summary of the invention

[0026] To meet this need, the inventors have developed a process for preparing a particular cellular microcompartment which makes it possible to optimize cell culture in three dimensions.

[0027] Thus, the subject of the invention is a new method for preparing a three-dimensional cellular microcompartment comprising the implementation of the following steps:

[0028] - a step of encapsulating at least one cell in hydrogel, so as to form at least one cellular microcompartment,

[0029] - a step of suspending said cellular microcompartment in a solution, preferably a soaking solution; and

[0030] - at least one step applying a centrifugal acceleration of at least 10 g on at least one cell and / or at least one cellular microcompartment. The step of encapsulating at least one cell in hydrogel can be carried out simultaneously with suspending the cellular microcompartment(s) in the solution.

[0031] Advantageously, the method according to the invention advantageously uses the application of gravity, in particular via the centrifugal effect, to optimize the conditions for culturing cells in a 3D culture system.

[0032] Preferably, the method according to the invention comprises a step applying a centrifugal acceleration of between 10 and 3000 g.

[0033] The application of at least one step applying a centrifugal acceleration of at least 10g on at least one cell and / or at least one microcompartment is particularly advantageous, and this, at any time during the process of preparing a three-dimensional cellular microcompartment.

[0034] According to a preferred subject, the invention relates to a method for preparing a three-dimensional cellular microcompartment comprising the implementation of the following steps: a) Encapsulation of at least one cell in hydrogel, so as to form at least one cellular microcompartment, b) Suspending said at least one cellular microcompartment in a solution, preferably a solution, preferably a soaking solution; c) Applying a centrifugal acceleration of at least 10g to the suspension of step b); d) Rinsing at least one cellular microcompartment obtained in step c) using a rinsing solution; and e) Culturing at least one microcompartment from step d).

[0035] Steps a) and b) can be carried out simultaneously, the formation of the microcompartments can take place directly in the solution in which they are suspended.

[0036] Step d) of rinsing can be carried out so as to form a suspension comprising at least one cellular microcompartment in a rinsing solution.

[0037] Advantageously, step c) applying a centrifugal acceleration of at least 10g after encapsulation makes it possible to promote the appearance of cell clusters within the cell microcompartments before culturing the microcompartments.

[0038] Preferably, step c) is carried out by applying a centrifugal acceleration of between 10 and 3000g, even more preferably between 50 and 1000g, in particular between 200 and 400g. Advantageously, step c) makes it possible to group the cells in order to form clusters without disturbing the structural integrity of the cellular microcompartments, nor the viability of the cells present within said microcompartments.

[0039] The invention thus relates to the use of centrifugal acceleration, in a method of preparing a cellular microcompartment, by any means, in particular to group the cells within the cellular microcompartments.

[0040] Surprisingly, the method according to the invention makes it possible to resolve the drawbacks of the prior art, by improving the survival and cellular amplification of cells within the cellular microcompartments.

[0041] According to a preferred object, steps c) and d) are carried out using at least one device for filtering suspended mesoscopic objects. Preferably, steps c) and d) are carried out using at least one device comprising a body comprising at least a first port and a second port and a filter membrane extending along a plane P in the body thus defining two compartments and capable of preventing the passage of OCMS from one compartment to the other, the first port being intended for the injection and withdrawal of OCMS bathed in a soaking solution and / or a rinsing solution and the second port being intended for the withdrawal and injection of the soaking solution and / or the rinsing solution.

[0042] According to a particularly suitable embodiment, steps c) and d) are carried out using at least one device comprising a body comprising at least a first port and a second port and a filter membrane extending along a plane P in the body, thus defining two compartments and capable of preventing the passage of the cellular microcompartments from one compartment to the other, the first port being intended for the injection and withdrawal of the cellular microcompartments bathed in a soaking solution and / or a rinsing solution and the second port 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 is carried out along a direction D not substantially orthogonal to the plane P of the filter membrane.

[0043] According to another aspect, the invention also relates to a device for filtering suspended mesoscopic cellular objects (OCMS) comprising a body comprising at least a first port and a second port and a filter membrane extending along a plane P in the body, thus defining two compartments and capable of preventing the passage of the OCMS from one compartment to the other, the first port being intended for the injection and withdrawal of the OCMS bathed in a soaking solution and / or a rinsing solution and the second port being intended for the withdrawal and injection of the soaking solution and / or the rinsing solution.According to a particular embodiment, the invention relates to a device comprising a body comprising at least a first port and a second port and a filter membrane extending along a plane P in the body, thus defining two compartments and capable of preventing the passage of the cellular microcompartments from one compartment to the other, the first port being intended for the injection and withdrawal of the cellular microcompartments bathed in a soaking solution and / or a rinsing solution and the second port 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 is carried out along a direction D not substantially orthogonal to the plane P of the filter membrane.

[0044] Advantageously, the device according to the invention is suitable for implementing steps c) and d) of the method according to the invention. In other words, the device according to the invention is therefore suitable for applying a centrifugal acceleration of at least 10g to a suspension comprising at least one cellular microcompartment and rinsing said at least one cellular microcompartment using a rinsing solution so as to change the suspension medium of said at least one cellular microcompartment. The device thus places the cellular microcompartments in optimal conditions for starting the culturing of the cells.

[0045] According to the invention, the injection into the first port is carried out in a direction D not substantially orthogonal to the plane P of the filter membrane.

[0046] Advantageously, the injection into the first port is a confined tangential of the OCMS, preferentially of the cellular microcompartments, and thus makes it possible not to create accumulation or structural damage of the OCMS, preferentially of the cellular microcompartments.

[0047] The ability of the device according to the invention to form a tangential flow or a vortex within the device makes it possible not to damage the OCMS, preferably cellular microcompartments, by keeping them in motion, in particular during the injection of the rinsing solution.

[0048] In one embodiment, the body is designed such that the shortest path of an OCMS, preferably a cellular microcompartment in the body between its injection and its withdrawal is a curved trajectory, passing through only one of the two compartments. Preferably, only one of the compartments is designed to receive the OCMS, preferably the cellular microcompartments injected through the first port, both compartments being designed to be crossed by a soaking solution and / or a rinsing solution.

[0049] According to one embodiment, the device for filtering suspended mesoscopic cellular objects (OCMS), preferably cellular microcompartments, comprises a body comprising a first port and a second port and a filter membrane extending along a plane P in the body, thus defining two compartments and capable of preventing the passage of OCMS, preferably cellular microcompartments, from one compartment to the other, the first port being intended for the injection and withdrawal of OCMS bathed in a soaking solution and / or a rinsing solution and the second port being intended for the withdrawal and injection of the soaking solution and / or the rinsing solution, the first port being a single port in the first compartment.

[0050] In this example, the OCMS, preferably the cellular microcompartments, injected through the first port into a first compartment are stored in this first compartment until they are withdrawn through the first port.

[0051] The implementation of a single port in the first compartment of the filtration device according to the invention allows the injection and withdrawal of OCMS bathed in a soaking solution through the same port, thus reducing the number of channels and the use of tubes and also making it possible to obtain a more compact filtration device.

[0052] Advantageously, the use of a single port for the injection and withdrawal of OCMS, preferably cellular microcompartments, bathed in a soaking solution and / or the rinsing solution makes it possible to reduce the number of constituent elements of the filtration device, thus limiting the impact of the environment such as risks of external contamination at the ports.

[0053] According to a characteristic of the invention, the direction D is parallel to the plane P.

[0054] According to one embodiment of the invention, the body comprises an inner casing with edges having a rounded shape and the useful section of the filter membrane has the shape of a circle or an ellipse. The implementation of rounded edges makes it possible to reduce the nooks inside the body of the device, thus limiting the accumulation of OCMS, preferably cellular microcompartments, in the nooks and making it possible to avoid the loss of OCMS, preferably cellular microcompartments, stuck in the device during the phases of recovery of the cellular microcompartments. This also makes it possible to limit the dead volume and therefore the volume necessary to rinse the OCMS, preferably the cellular microcompartments.

[0055] In one embodiment of the invention, the body has a perforated shape or a cavity in its envelope. It may in particular be a toric shape, the envelope of which is a surface generated by a closed generating curve, in particular a circle or an ellipse, circulating on another closed directrix curve, in particular a circle. This cavity or this hole formed in the envelope makes it possible to create or reinforce a circular circulation of the OCMS, preferably cellular microcompartments, in the body, and therefore to avoid a phenomenon of clogging of the filtering device. According to an exemplary embodiment of the invention, the body has a toric shape and the useful section of the filtering membrane has the shape of a circle or an ellipse.Advantageously, the toric shape makes it possible to increase the swirl effect, therefore also the centrifugal force and therefore the gravity for the OCMS, preferentially the cellular microcompartments, while greatly reducing the dead volume inside the body, thus limiting the volume necessary to rinse the OCMS, preferentially the cellular microcompartments.

[0056] According to a characteristic of the invention, any section perpendicular to the plane P of the compartment intended to receive the OCMS, preferably the cellular microcompartments, has a surface area strictly less than the useful surface area of ​​the filter membrane.

[0057] According to another characteristic of the invention, any section perpendicular to the plane P of the compartment intended to receive the OCMS, preferably the cellular microcompartments, has a surface area twice, five times and preferably ten times smaller than the useful surface area of ​​the filter membrane.

[0058] Tangential injection associated with such a difference in dimension makes it possible to inject the OCMS, preferentially the cellular microcompartments with a certain flow rate, promoting the vortex and therefore the centrifugal force, while allowing the concentration of a certain number of cellular microcompartments inside the body of the device and minimizing the dead volume.

[0059] According to a characteristic of the invention, the injection into the second port is also carried out in a non-orthogonal direction, and preferably parallel, to the plane P of the filter membrane. The second tangential injection makes it possible to promote swirling flows, and therefore centrifugal force, inside the body of the device.

[0060] According to another characteristic of the invention, the soaking solution is a culture medium suitable for OCMS, preferably a culture medium suitable for cellular microcompartments. Preferably, the soaking solution allows the stiffening of at least one hydrogel so as to form cellular microcompartments. The cellular microcompartments are thus maintained in a favorable environment and are not degraded.

[0061] According to a characteristic of the invention, the soaking solution contains at least one bivalent cation, preferably chosen from the following list: calcium, iron, barium, strontium, copper, lead, aluminum, magnesium and their combinations, even more preferably calcium, iron, barium.

[0062] According to another characteristic of the invention, the filter membrane comprises a microfabric covered with a fiber membrane. The implementation of such a filter membrane makes it possible to maintain control over the type of OCMS, preferably over the type of cellular microcompartment to be filtered.

[0063] According to one embodiment of the invention, the filter membrane is capable of allowing other OCMS to pass through, preferably other cellular microcompartments of a different type contained in the soaking solution. This characteristic allows in particular filtering of a certain type of OCMS, preferably a certain type of microcompartment according to the invention.

[0064] According to another embodiment of the invention, the filter membrane is capable of filtering cellular microcompartments having a largest dimension of between 120 and 700 micrometers, preferably between 200 pm and 400 pm, preferably between 200 pm and 350 pm, even more preferably 200 pm and 300 pm, in particular between 200 pm and 250 pm. Where appropriate, the filter membrane comprises porosities of less than 100 micrometers, or even less than 50 micrometers, or even less than 10 micrometers. Preferably, the filter membrane is capable of filtering cellular microcompartments having a largest dimension of less than 600 micrometers, the filter membrane comprises porosities of less than 100 micrometers.

[0065] According to another characteristic of the invention, the device comprises two compartments having different volumes. Preferably, the compartment intended to receive OCMS, preferably cellular microcompartments, bathed in a soaking solution and / or a rinsing solution has a volume greater than the compartment intended to receive the soaking solution and / or the rinsing solution. The difference in volume advantageously makes it possible to improve the efficiency and the quantity of microcompartments harvested. Preferably, the compartment intended to receive cellular microcompartments bathed in a soaking solution and / or a rinsing solution has a volume at least 50% greater than the microcompartment intended to receive the soaking solution and / or the rinsing solution.

[0066] According to one embodiment of the invention, the filter membrane is capable of allowing other OCMS to pass through, preferably other cellular microcompartments, of a different type contained in the soaking solution. This characteristic in particular allows filtering of a certain type of OCMS, preferably cellular microcompartments.

[0067] The invention also relates to a method for accumulating OCMS, preferably cellular microcompartments, comprising the following steps:

[0068] - Injection of OCMS, preferably cellular microcompartments bathed in the soaking solution into a first port of a device according to the invention, filtration of the cellular microcompartments by the filter membrane and recovery of a portion of the soaking solution through the second port, - Injection of the soaking solution into the second port and recovery of the OCMS, preferably cellular microcompartments, accumulated through the first port.

[0069] According to one embodiment of the invention, the process of accumulating OCMS, preferably cellular microcompartments, is carried out continuously. The OCMS, preferably cellular microcompartments, can thus be collected as quickly as they are produced, thus minimizing the residence time in the soaking solution.

[0070] The invention also relates to a method for rinsing OCMS, preferably cellular microcompartments, comprising the following steps:

[0071] - Injection of cellular microcompartments bathed in a soaking solution into a first port of a device according to the invention, filtration of the cellular microcompartments by the filter membrane and recovery of the soaking solution and the rinsing solution by the second port,

[0072] - Injection of a rinsing solution into the first given port intended to replace the soaking solution and recovery of both AA and rinsing solutions through the second port.

[0073] Advantageously, the tangential flow reduces damage to OCMS, preferentially to cellular microcompartments, by minimizing contact with the filter membrane during the rinsing process.

[0074] According to a characteristic of the invention, the rinsing method further comprises a step of injecting the rinsing solution into the second port and recovering the cellular microcompartments rinsed through the first port.

[0075] The invention also relates to a cell encapsulation system, the system comprising at least two containers, one of the containers comprising a cell solution and the other of the containers comprising a solution capable of gelling, an encapsulation device comprising a fluidic chip comprising main and secondary inlets each being connected to one of the containers via a distributor and capable of forming a concentric jet from the solutions supplied by the distributors, the encapsulation device being arranged so that the jet splits, at the outlet of the encapsulation device, into drops of which the outer layer is the solution capable of gelling and the core the cell solution, a gelling bath arranged downstream of the encapsulation device to collect the drops formed by the encapsulation device and arranged to cause gelling of the outer layer of each drop during its immersion in the bath,and a filtration device according to the invention.,

[0076] Advantageously, the gelling bath and a single-use closed harvesting system are connected to the first port of the filtration device, in particular by a flow valve. In an exemplary embodiment of the invention, the encapsulation system according to the invention comprises a control unit arranged to control a solution displacement device for injecting OCMS, preferably cellular microcompartments, bathing in the soaking solution of the gelling bath into the first compartment of the filtration device via the first port, the OCMS, preferably the cellular microcompartments, being stopped by the filter membrane and accumulated in a first compartment of the filter device, and said soaking solution being evacuated from the filter device, passing from the first compartment to the second compartment via the filter membrane and then via the second port.A waste container may be connected to the second port of the filtration device to receive the drained soaking solution.

[0077] Advantageously, the control unit is arranged to control a solution displacement device for injecting a soaking solution into the second compartment of the filtration device through the second port, said solution being discharged from the filtration device by passing from the second compartment to the first compartment via the filter membrane and then through the first port, recovering the OCMs, preferably the cellular microcompartments, accumulated in the first compartment during the previous step. A container of a soaking solution may be connected to the second port of the filtration device to inject the soaking solution. The control unit may be arranged to control the flow valve to direct the OCMs recovered by the soaking solution to the single-use closed collection system.

[0078] Alternatively or cumulatively, in particular prior to the step of recovering the accumulated OCMS, preferably the cellular microcompartments, the control unit is arranged to control a solution displacement device to inject a rinsing solution into the first compartment of the filtration device via the first port, said rinsing solution being evacuated from the filtration device, passing from the first compartment, to rinse the accumulated OCMS, preferably the cellular microcompartments, to the second compartment via the filter membrane and then via the second port.

[0079] According to a variant of the invention, the device can also be used as a filtration device, preferably as a device for filtering mesoscopic cellular objects in suspension (OCMS), preferably cellular microcompartments.

[0080] According to another object, the invention relates to a device for filtering suspended mesoscopic cellular objects (OCMS) comprising a body comprising at least a first port and a second port and a filter membrane extending along a plane P in the body, thus defining two compartments and capable of preventing the passage of OCMS from one compartment to the other, the first port being intended for the injection and withdrawal of OCMS bathed in a soaking solution and / or a rinsing solution and the second port being intended for the withdrawal and injection of the soaking solution and / or the rinsing solution.

[0081] Of course, the various features, variants and embodiments of the invention may be combined with each other in various combinations to the extent that they are not incompatible or mutually exclusive.

[0082] In addition, various other characteristics of the invention emerge from the appended description given with reference to the drawings which illustrate non-limiting forms of embodiment of the invention.

[0083] Brief descriptions of the Figures:

[0084] [Fig.l] Figure 1 is a perspective view of an exemplary embodiment of a device according to the invention,

[0085] [Fig.2] Figure 2 is a schematic view of an exemplary embodiment of a device according to the invention,

[0086] [Fig.3] Figure 3 is a perspective view of an exemplary embodiment of a device according to the invention,

[0087] [Fig.4] Figure 4 is a half-sectional view of Figure 3 along the section plane AA,

[0088] [Fig.5] Figure 5 is a half-sectional view of another exemplary embodiment of a device according to the invention, and

[0089] [Fig.6] Figure 6 is a schematic view of an exemplary embodiment with two devices according to the invention.

[0090] [Fig 7] Figure 7 groups together photographs taken under a microscope of the evolution of cellular microcompartments having undergone a centrifugal acceleration of 400g vs. control cellular microcompartments as a function of time.

[0091] Detailed description of the invention

[0092] Definitions

[0093] For the purposes of the invention, "centrifugal acceleration" means 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. 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 centrifugal force.

[0094] For the purposes of the invention, the term "alginate" means linear polysaccharides formed from pD-mannuronate and aL-guluronate, salts and derivatives thereof.

[0095] For the purposes of the invention, “differentiated” cells are understood to mean cells which have a particular phenotype, as opposed to pluripotent stem cells which are not differentiated or progenitor cells which are in the process of differentiating.

[0096] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention consist of or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0097] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in cell differentiation but not yet differentiated.

[0098] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. In particular, they may be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0099] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106).

[0100] By "cell layer" or "cell base" within the meaning of the invention, is meant several cells forming a layer or base that can be structured around a lumen, it can be for example a tissue or a cellular micro-tissue or a culture grouped in three dimensions. The thickness of the cell layer can be variable. This layer is organized in three dimensions in the microcompartment. By "Feret diameter" of an OCMS according to the invention, preferably of a cellular microcompartment according to the invention, is meant the distance "d" between two tangents to said OCMS, preferably to said microcompartment, these two tangents being parallel, such that the entire projection of said OCMS, preferably to said microcompartment, is between these two parallel tangents.

[0101] For the purposes of the invention, the term "drop" also means a three-dimensional structure formed from at least one liquid solution comprising the constituents of a non-rigidified hydrogel (polymerization precursors, non- or partially crosslinked polymer chains, etc.), hydrogel precursor elements. Also, the drop constitutes a transitional state between the co-injection of the different constituents and the microcompartment according to the invention.

[0102] For the purposes of the invention, the term "microcompartment" or "capsule" also means a partially or totally enclosed three-dimensional structure. This is formed from a matrix of polymer chains, for example alginate, containing several cells. The structure is thus made up of a rigidified external hydrogel layer and an internal part comprising at least one cell and a hydrogel layer or mesh suitable for cell culture and growth of said cells. The encapsulated cells can take various forms, including single cells, cell aggregates, cellular microtissues, multicellular aggregates, cellular tissues, or any other configuration allowing the encapsulation, confinement, or culture of cells. Furthermore, said microcompartments can be designed to be full of one or more biocompatible materials, such as alginate, thus forming solid beads.Alternatively, said microcompartments may be hollow, thus forming an internal cavity in which cells may be contained or cultured.

[0103] By "suspended mesoscopic cellular objects", hereinafter referred to as OCMS, within the meaning of the invention, is meant the set of objects whose dimension varies between 5 pm and 5 mm, which are in suspension and which comprise at least one eukaryotic cell, encapsulated or not. These may in particular be single cells, cellular microtissues, aggregates of cells, cellular tissues, these elements being or not encapsulated in a hollow capsule or in a solid capsule. Preferably these objects are cellular microcompartments.

[0104] By "largest dimension" of an OCMS, preferably of a cellular microcompartment within the meaning of the invention, is meant the value of the largest Feret diameter of said OCMS, preferably of said microcompartment.

[0105] By "tissue" or "biological tissue" within the meaning of the invention, we mean the common meaning of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a set of similar cells of the same origin (most often from a common cell lineage, although they can find their origin by association of distinct cell lineages), grouped in clusters, networks or bundles (fibers). A tissue forms a functional whole, that is to say that its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs.

[0106] Method for preparing a cellular microcompartment

[0107] The subject of the present invention is a method for preparing a three-dimensional cellular microcompartment, characterized in that it comprises the implementation of the following steps:

[0108] - a step of encapsulating at least one cell in hydrogel, so as to form at least one cellular microcompartment,

[0109] - a step of suspending said at least one cellular microcompartment in a solution, preferably a soaking solution; and

[0110] - at least one step applying a centrifugal acceleration of at least 10 g on at least one cell and / or at least one cellular microcompartment.

[0111] The step of encapsulating at least one cell in hydrogel can be carried out simultaneously with suspending the cellular microcompartment(s) in the solution.

[0112] Advantageously, the method according to the invention makes it possible to use the centrifugal effect, by any means, to optimize the culture conditions in a three-dimensional system.

[0113] In the context of the invention, the centrifugal acceleration exerted on at least one cell and / or at least one cellular microcompartment can be calculated by the following formula:

[0114] [Math 1]

[0115] 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 ms-2

[0116] Preferably, the centrifugal acceleration applied to at least one cell and / or at least one microcompartment is between 10 and 3000g, even more preferably between 50 and 1000, in particular between 200 and 400g.

[0117] According to one embodiment, the centrifugal acceleration applied to at least one cell and / or at least one microcompartment is between 10 and 500g, preferably between 10 and 400g. According to one embodiment, the application of the centrifugal acceleration is carried out by centrifugation, in particular using a centrifuge.

[0118] According to another embodiment, the application of centrifugal acceleration is carried out using a filtration device.

[0119] According to one embodiment, the method according to the invention comprises the implementation of the following steps: a) Encapsulation of at least one cell in hydrogel, so as to form at least one cellular microcompartment, b) Suspending said at least one cellular microcompartment in a solution, preferably a solution, preferably a soaking solution; c) Applying a centrifugal acceleration of at least 10g to the suspension of step b); d) Rinsing at least one cellular microcompartment obtained in step c) using a rinsing solution; and e) Culturing at least one microcompartment from step d).

[0120] Steps a) and b) can be carried out simultaneously, the formation of the microcompartments can take place directly in the solution in which they are suspended.

[0121] Step d) of rinsing can be carried out so as to form a suspension comprising at least one cellular microcompartment in a rinsing solution.

[0122] Preferably, at least one three-dimensional cellular microcompartment obtained at the end of step a) is hollow in the sense that it has an external layer and a hollow internal part which can contain elements such as, for example, a solution, extracellular matrix or an extracellular matrix substitute and / or one or more cells. Preferably, at least one microcompartment obtained at the end of step a) has an external hydrogel layer and an internal part comprising at least one cell.

[0123] According to one embodiment, the microcompartment obtained at the end of step a) has an external hydrogel layer and an internal part comprising an internal matrix and at least one cell. Preferably, the internal matrix is ​​an extracellular matrix or extracellular matrix substitute.

[0124] The cellular microcompartment resulting from step a) comprises at least one cell encapsulated in hydrogel, said hydrogel being biocompatible, that is to say it is not toxic for the cells. The hydrogel must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival. According to a particularly preferred embodiment, the external hydrogel layer of the microcompartment resulting from step a) comprises at least alginate. According to a particular embodiment, the external hydrogel layer of the microcompartment resulting from step a) may consist exclusively of alginate.

[0125] 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 10 kPa, preferably greater than 60 kPa, more preferably greater than 100 kPa.

[0126] According to one embodiment, the external hydrogel layer of the microcompartment resulting from step a) comprises alginate, said alginate advantageously having an average molecular weight of 100 to 400 kDa, more preferably the external layer having a molecular weight of between 150 and 250 kDa. When the hydrogel of the external layer of the microcompartment resulting from step a) is alginate, the concentration of the alginate solution intended to form said external 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.

[0127] When the concentration of the alginate solution intended to form the external layer of the microcompartment of step a) is equal to 2%, the viscosity of the alginate is preferably equal to 144mPa / s.

[0128] Advantageously, the external hydrogel layer of the microcompartment resulting from step a) is devoid of cells.

[0129] The external hydrogel layer of the microcompartment resulting from step a) thus makes it possible to protect the cells from the external environment, to limit the uncontrolled proliferation of the cells, and their differentiation in the event of differentiation.

[0130] In the context of the invention, the cells present in the microcompartment resulting from step a) may be any type of cell, in particular the cells are eukaryotic cells. More preferably, the cells are human, plant or animal cells.

[0131] In a particular embodiment, the microcompartment resulting from step a) comprises pluripotent stem cells. A pluripotent stem cell, or pluripotent cell, is understood to mean a cell which has the capacity to form all the tissues present in the entire organism of origin, without being able to form an entire organism as such. The pluripotent stem cells may in particular be induced pluripotent stem (iPS) cells, MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells. According to one embodiment, the microcompartment according to the invention does not comprise embryonic stem (ES) cells. According to a particularly suitable variant of the invention, the microcompartment resulting from step a) comprises human or animal induced pluripotent stem cells.

[0132] 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 and / or progenitor cells have preferably been obtained from pluripotent stem cells, in particular human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile has been 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, the 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.

[0133] According to another variant, the microcompartment resulting from step a) comprises differentiated human or animal cells. The differentiated cells have preferably been 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 have been obtained from pluripotent or multipotent stem cells or progenitor cells after contact with a solution capable of initiating the differentiation of said stem cells.Alternatively, the cellular contents of the microcompartment comprise homogeneous or mixed cellular identities.

[0134] In particular, differentiated cells may 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 in the microcompartment. It may be a compacted or non-compacted tissue or microtissue, with or without a lumen.

[0135] The microcompartment resulting from step a) may therefore comprise several types of cells. In particular, the microcompartment resulting from step a) may comprise, for example, stem cells induced to pluripotency and / or multipotent cells and / or progenitor cells and / or cells in the process of differentiation and / or differentiated cells.

[0136] According to one variant, the microcompartment resulting from step a) comprises:

[0137] - an external hydrogel layer, and - an internal part comprising at least:

[0138] * at least one layer of cells, and

[0139] * a hydrogel layer or mesh arranged between the outer layer and the cell layer.

[0140] According to this variant, the layer or mesh of the internal hydrogel part, preferably alginate, may comprise other constituents, thus said layer or mesh of the internal hydrogel part preferably comprises at least one peptide sequence, more preferably a peptide sequence of interest capable of interacting with the cells constituting in particular the layer of cells present in the microcompartment resulting from step a). By way of example, the peptide sequence may be a peptide, or a protein. According to a particularly preferred subject, 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 internal hydrogel part.The RGD motif is a peptide with the sequence Arginine-Glycine-Asparagine which also facilitates cell adhesion to the mesh of the internal hydrogel part.

[0141] According to the same variant, the layer or mesh of the internal hydrogel part may comprise at least one second hydrogel distinct from the first hydrogel of the layer or mesh of the internal part, more preferably this is chosen from fibrin, laminin, fibronectin, entactin, hyaluronic acid, and collagen.

[0142] The microcompartment obtained at the end of step a) is then a three-dimensional microcompartment, delimited by the external hydrogel layer and inside said external layer, an internal part comprises at least one cell and the hydrogel layer or mesh.

[0143] The microcompartment 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-shaped shape.

[0144] On the one hand, the external layer of hydrogel makes it possible to protect the cells from the external environment, to limit the uncontrolled proliferation of the cells, as well as their differentiation in the event of differentiation; on the other hand, the possible presence of a layer or mesh in the internal part of hydrogel and / or the extracellular matrix and / or extracellular matrix substitute, makes it possible to provide an environment suitable for the growth of the cells and their multiplication.

[0145] According to one embodiment, the microcompartment resulting from step a) has a larger dimension of between 120 and 700 micrometers, preferably between 200 pm and 400 pm, preferably between 200 pm and 350 pm, even more preferably 200 pm and 300 pm, in particular between 200 pm and 250 pm.

[0146] According to one embodiment, step a) and / or b) comprises the stiffening of the hydrogel. Preferably, the stiffening is carried out in step b) in a stiffening solution or bath or soaking solution.

[0147] Preferably, the method according to the invention comprises the following steps:

[0148] -a) bringing into contact at least one cell and a hydrogel solution intended to form an external layer to form at least one drop, and

[0149] -b) collecting the drop obtained in a soaking solution capable of stiffening said hydrogel solution to form the external layer of each microcompartment.

[0150] Once the outer hydrogel layer is stiffened by the soaking solution, the microcompartment is formed.

[0151] According to one embodiment, step a) of encapsulation is carried out by co-injection of two or three solutions:

[0152] -a hydrogel solution;

[0153] - possibly an isotonic intermediate solution;

[0154] - a solution comprising cells, culture medium and possibly extracellular matrix and / or an extracellular matrix substitute, concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of solutions, said jet breaking up into drops, said drops being cellular microcompartments within the meaning of the invention.

[0155] In this embodiment, in step b) the drops formed in step a) are collected in a soaking solution which stiffens the hydrogel solution to form the outer layer of each microcompartment, the inner part of each drop being constituted by the solution comprising the cells, culture medium and the extracellular matrix.

[0156] Thus, according to a preferred embodiment, the method according to the invention comprises the implementation of the following steps: a) Encapsulation of at least one cell in hydrogel so as to form a suspension comprising at least one cellular microcompartment in a soaking solution, said encapsulation is carried out by co-injection of two or three solutions:

[0157] -a hydrogel solution;

[0158] - possibly an isotonic intermediate solution;

[0159] -a solution comprising cells, culture medium and optionally extracellular matrix and / or an extracellular matrix substitute, concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of solutions, said jet breaking up into drops, b) collecting said drops in a soaking solution which stiffens the hydrogel solution to form the outer layer of each microcompartment, the inner part of each drop being constituted by the solution comprising the cells, culture medium and optionally extracellular matrix; c) Applying a centrifugal acceleration of at least 10g to the suspension comprising the cellular microcompartments obtained in step b); d) Rinsing at least one cellular microcompartment obtained in step c) using a rinsing solution; and e) Culturing at least one microcompartment resulting from step d).

[0160] In this embodiment, the isotonic intermediate solution of encapsulation step a) is preferably a sorbitol solution.

[0161] In the same embodiment, the final opening diameter of the microfluidic injector of the encapsulation step a) 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.

[0162] According to one embodiment, the solution of step b) comprises at least one divalent cation capable of stiffening the hydrogel of the external layer, preferably a cation chosen from calcium, iron, barium, strontium, copper, lead, aluminum, magnesium, and their combinations, preferably calcium, iron, barium, even more preferably calcium.

[0163] Step c) consists of applying a centrifugal acceleration of at least 10g to the suspension obtained in step b). Preferably, step c) is carried out by applying a centrifugal acceleration of between 10 and 3000g, even more preferably between 200 and 400g. According to another embodiment, step c) consists of applying a centrifugal acceleration of between 10 and 500g, preferably between 10 and 400g.

[0164] Advantageously, step c) makes it possible to group the cells present within the microcompartment in order to make their distribution homogeneous, reducing the number of isolated cells without degrading the structure of the microcompartments, or reducing the viability of the cells within the microcompartments.

[0165] According to one embodiment, the application of centrifugal acceleration is carried out by centrifugation, in particular using a centrifuge.

[0166] Preferably, when the centrifugal acceleration is carried out by centrifugation, step c) is carried out for a duration of between 2 and 20 minutes, even more preferably between 5 and 10 minutes.

[0167] Step c) is carried out at a temperature adapted to the type of hydrogel used and the intended use. A person skilled in the art can adapt the temperature depending on the hydrogel. For example, when the hydrogel is heat-sensitive, the centrifugation is carried out at 4 degrees Celsius.

[0168] According to another embodiment, the application of centrifugal acceleration is carried out using a filtration device.

[0169] The application of a step applying a centrifugal acceleration of at least 10g on at least one cell and / or at least one microcompartment such as step b) is particularly advantageous, and this, at any time of the process for preparing a three-dimensional cellular microcompartment.

[0170] Advantageously, step c) located after encapsulation and before culturing makes it possible to promote the formation of cell clusters or aggregates within the microcompartments before culturing in order to improve cell survival and growth as well as the appearance of better structuring of the cells for the initiation of 3D amplification.

[0171] Preferably, the cellular microcompartments obtained at the end of step c) comprise less than 5% of single cells in number of all the encapsulated cells, preferably less than 1%.

[0172] Indeed, in the context of the invention, it is preferable that the encapsulated cells are in the form of clusters in order to optimize amplification and improve cell survival.

[0173] Step c) advantageously forces the cells to organize into clusters before the culturing step. Thus, step c) makes it possible to standardize the structure of the cells within the microcompartment, drastically reducing the percentage of isolated cells. Advantageously, the centrifugal acceleration of at least 10g in step c) is suitable for promoting the appearance of cell clusters without affecting cell viability.

[0174] Step d) allows the soaking solution to be replaced by the rinsing solution. The rinsing step can be carried out by any means suitable for cell survival.

[0175] The rinsing solution of step d) is a medium adapted to the cells present in the microcompartment according to the knowledge of a person skilled in the art.

[0176] According to another embodiment, steps c) and d) are carried out using at least one device comprising a body comprising at least a first port and a second port and a filter membrane extending along a plane P in the body thus defining two compartments and capable of preventing the passage of the cellular microcompartments from one compartment to the other, the first port being intended for the injection and withdrawal of the cellular microcompartments bathed in a soaking solution and / or a rinsing solution and the second port 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 is carried out along a direction D not substantially orthogonal to the plane P of the filter membrane.

[0177] According to one embodiment, steps c) and d) are carried out continuously using at least 2 devices according to any of the preceding embodiments. Preferably, when steps c) and d) are carried out continuously using at least 2 devices according to any of the preceding embodiments, said devices are arranged in series.

[0178] According to one embodiment, the method for preparing a three-dimensional cellular microcompartment comprises the implementation of the following steps: a') Application of a centrifugal acceleration of at least 10g on a suspension comprising at least one cell; a) Encapsulation of at least one cell from step a') in hydrogel so as to form at least one cellular microcompartment; b) Suspending said at least one cellular microcompartment in a solution, preferably in a soaking solution; c) Optionally, application of a centrifugal acceleration of at least 10g on the suspension from step b); d) Rinsing of at least one cellular microcompartment obtained in step b) or c) using a rinsing solution; and d) Culturing of at least one microcompartment from step d).Advantageously, depending on the characteristics of the hydrogel and the specific constraints of the cells, the method may comprise a step a') upstream of the encapsulation step a). Thus, the cells will form clusters upstream of the encapsulation.

[0179] According to one embodiment, step a') is carried out on a suspension comprising at least one cell and a culture medium.

[0180] Preferably, the culture medium of step a') is a culture medium is a medium adapted to the cells present in the microcompartment according to the knowledge of a person skilled in the art.

[0181] According to a particular embodiment of the invention, the method comprises the implementation of the following steps: a') Application of a centrifugal acceleration of at least 10g on a suspension comprising at least one cell; a) Encapsulation of at least one cell from step a') in hydrogel so as to form at least one cellular microcompartment, at least one microcompartment being delimited by an external hydrogel layer and inside said external layer, an internal part comprises at least one cell and a hydrogel layer or mesh comprising a hydrogel chosen from fibrin, laminin, fibronectin, entactin, hyaluronic acid, and collagen; b) Suspending said at least one cellular microcompartment in a solution, preferably in a soaking solution; c) Optionally, application of a centrifugal acceleration of at least 10g on the suspension from step b);d) Rinsing at least one cellular microcompartment obtained in step b) or c) using a rinsing solution; and e) Culturing at least one microcompartment from step d).;

[0182] Advantageously, this embodiment of the method according to the invention makes it possible to facilitate the grouping of the cells before gelling of the hydrogel of the internal part.

[0183] According to one embodiment, when at least one microcompartment resulting from step a) comprises an internal matrix, the method according to the invention comprises a step x) of gelling the internal matrix, carried out after step b) or c). Preferably, step x) of gelling the internal matrix is ​​carried out using at least one polymerizing factor chosen from temperature, an enzyme, a chemical reagent such as thrombin and combinations thereof. Thus, according to one embodiment, the method according to the invention comprises the implementation of the following steps: a) Encapsulation of at least one cell in hydrogel so as to form at least one cellular microcompartment, said at least one cellular microcompartment comprising an external hydrogel layer and an internal part comprising an internal matrix and at least one cell;b) Suspending said at least one cellular microcompartment in a solution, preferably in a soaking solution; c) Applying a centrifugal acceleration of at least 10g to the suspension of step b); x) Gelling the internal part of at least one cellular microcompartment resulting from step c) using at least one polymerizing factor chosen from temperature, an enzyme, a chemical reagent and combinations thereof; d) Rinsing at least one cellular microcompartment obtained in step x) using a rinsing solution so as to form a suspension comprising at least one cellular microcompartment in a rinsing solution; and e) Culturing at least one microcompartment resulting from step d).

[0184] According to a particular embodiment, the internal matrix is ​​exposed to at least one polymerizing factor before step c) and the step of gelling the internal matrix is ​​carried out after step c).

[0185] Advantageously, the gelling process of the internal matrix may be sufficiently long or delayed to allow step c) to be carried out without the internal matrix being gelled, although exposure to at least one polymerising factor has been carried out before step c).

[0186] According to another preferred embodiment, the method comprises implementing the following steps: a) Encapsulation of at least one cell in hydrogel so as to form at least one cellular microcompartment, said at least one microcompartment being delimited by the external hydrogel layer, and inside said external layer, an internal part comprises at least one cell and a hydrogel layer or mesh comprising fibrin; b) Suspending said at least one cellular microcompartment in a solution, preferably in a soaking solution; c) Applying a centrifugal acceleration of at least 10g to the suspension resulting from step b); x) Adding thrombin to the suspension obtained at the end of step c) so as to gel at least a part of the internal part of at least one cellular microcompartment;d) Rinsing at least one cellular microcompartment obtained in step x) using a rinsing solution so as to form a suspension comprising at least one cellular microcompartment in a rinsing solution; and e) Culturing at least one microcompartment resulting from step d).;

[0187] Thrombin is a polymerizing agent capable of crossing the outer hydrogel layer of the cellular microcompartment to polymerize fibrin.

[0188] According to another embodiment, the method according to the invention comprises the implementation of the following steps: a) Encapsulation of at least one cell in hydrogel so as to form at least one cellular microcompartment; b) Suspending said at least one cellular microcompartment in a solution, preferably in a soaking solution; c) Optionally, applying a centrifugal acceleration of at least 10g to the suspension obtained at the end of step b); d) Rinsing at least one cellular microcompartment obtained in step b) or c) using a rinsing solution so as to form a suspension comprising at least one cellular microcompartment in a rinsing solution; and e) Culturing at least one microcompartment from step d) in a solution; and e') Centrifugation of the solution from step e) comprising a microcompartment or centrifugation of at least one microcompartment from step e).

[0189] Advantageously, the method according to the invention may comprise a centrifugation step after culturing. Step e') is particularly advantageous insofar as it makes it possible to promote the dissociation of the 3D cellular complexes formed during step e) of culturing.

[0190] According to a particular embodiment of the invention, the method according to the invention comprises the implementation of the following steps: a') Application of a centrifugal acceleration of at least 10g on a suspension comprising at least one cell; a) Encapsulation of at least one cell resulting from step a') in hydrogel so as to form a suspension comprising at least one cellular microcompartment in a soaking solution; Tl b) Suspending said at least one cellular microcompartment in a solution, preferably in a soaking solution; c) Application of a centrifugal acceleration of at least 10g on the suspension resulting from step b); d) Rinsing of at least one cellular microcompartment obtained in step c) using a rinsing solution; and e) Culturing of at least one microcompartment resulting from step d); and e') Centrifugation of at least one microcompartment from step e).

[0191] Preferably, in the embodiments of the method according to the invention, steps a') and / or c) and / or d) and / or e') are implemented using a device according to the invention as described below.

[0192] Device

[0193] It should be noted that in the figures the structural and / or functional elements common to the different variants may have the same references.

[0194] The device according to the invention is capable of carrying out at least part of the method according to the invention. In particular, the device is capable of applying a centrifugal acceleration of at least 10g to at least one cell and / or at least one microcompartment. Particularly advantageously, the device according to the invention is suitable for implementing steps a') and / or c) and / or d) and / or e') of the method according to the invention.

[0195] According to one variant, the device aims to filter suspended mesoscopic cellular objects, known as OCMS.

[0196] For these purposes, the invention designated by the reference 1 as a whole and illustrated in FIG. 1 relates to a filtration device comprising a body 2 comprising a filter membrane 3 which extends in the body 2 along a plane P.

[0197] According to the invention, the body 2 of the filtration device 1 comprises an inner casing 4 having rounded edges.

[0198] According to one embodiment of the invention illustrated in particular in Figures 2 and 5, the inner casing 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.

[0199] 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.

[0200] According to the embodiment illustrated in Figure 2, the filtration 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 part intended to receive OCMS, preferably cellular microcompartments, may comprise larger volume dimensions than the part on the other side of the filter membrane 3 or vice versa.

[0201] The filter membrane 3 extends along a plane P in the body 2 of the filtration device 1. The filter membrane 3 extends to the edges of the inner casing 4 of the body 2 of the filtration device 1. According to the illustrated embodiments, the useful section of the filter membrane 3 has the shape of a circle. According to other embodiments not illustrated, the useful section of the filter membrane 3 has the shape of an ellipse.

[0202] In order to retain the OCMS, preferably the cellular microcompartments, the filter membrane 3 comprises a fibered membrane capable of preventing the passage of the OCMS, preferably the OCMS, preferably the cellular microcompartments.

[0203] The filter membrane 3 is therefore adapted according to the type of OCMS, preferably according to the type of cellular microcompartments, implemented.

[0204] The OCMS, preferably the cellular microcompartments, are preferably immersed in a soaking solution which may be a culture medium. Advantageously, the soaking solution comprises elements allowing the stiffening of the hydrogel. For example, if the OCMS are cellular microcompartments constituted in particular by an external layer of alginate or forming solid alginate beads, the soaking solution preferably comprises calcium ions in the form of calcium chloride which allow the stiffening of the alginate.

[0205] According to one embodiment of the invention, the filter membrane 3 comprises a microfabric covered with a fiber membrane. The fiber membrane contains a material of the wide mesh textile type (mesh), nylon, or PET. Other filtering materials, OCMS, preferably cellular microcompartments, are used in the context of the invention.

[0206] According to one embodiment of the invention, for OCMS, and more particularly for cellular microcompartments, having a largest dimension of between 120 and 700 micrometers, the filter membrane comprises porosities of less than 100 micrometers.

[0207] According to one embodiment of the invention, for OCMS, and more particularly for cellular microcompartments, having a largest dimension less than 600 micrometers, the filter membrane comprises porosities less than 100 micrometers.

[0208] According to one embodiment of the invention, the filter membrane 3 is capable of filtering at least one type of OCMS, preferably at least one type of cellular microcompartments, and is also capable of allowing another type of OCMS to pass, preferably at least one other type of cellular microcompartment. The mesh can thus be adapted according to the dimensions of the cellular microcompartments to be filtered.

[0209] The body 2 of the filtration device 1 comprises a first port 5 intended for a tangential injection of the OCMS, preferably cellular microcompartments, bathed in a soaking solution.

[0210] 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.

[0211] 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 filtration 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 filtration device 1.

[0212] According to the embodiment illustrated in Figure 1, the first port 5 comprises an inlet 7 and an injection channel 8.

[0213] The injection channel 8 allows tangential injection into the inner casing 4 of the body 2 of the filtration device 1.

[0214] According to other embodiments not illustrated, the body 2 of the filtration device 1 comprises more than two inlet and outlet ports thus allowing the injection of different types of solution or OCMS, preferably cellular microcompartments, on each side of the filter membrane 3.

[0215] The filtration device 1 according to the invention is used for different functions.

[0216] The filtration device 1 is notably used for various processes such as the accumulation and / or rinsing of OCMS, preferably of cellular microcompartments.

[0217] A first use of the filtration device 1 relates to a process for accumulating OCMS, preferably cellular microcompartments.

[0218] A first step of the method for accumulating cellular microcompartments consists of injecting OCMS bathed in the soaking solution into the first port 5 of the device according to the invention. The injection is carried out according to a flow rate Q1 for a time T1. For example, the flow rate Q1 is 130 mL / min and the time T1 is 45 s. The OCMS, preferably of cellular microcompartments, are stopped by the filter membrane 3. A portion of the soaking solution is evacuated through the second port 6 of the filtration device 1. A second step of the method for accumulating OCMS, preferably of cellular microcompartments, consists of a reversal of the flow. To do this, the injection of the previously evacuated soaking solution is carried out into the second port 6.

[0219] The OCMS, preferably the cellular microcompartments accumulated in the filtration device 1 during the first step of the OCMS accumulation process, preferably cellular microcompartments, are then recovered via the first port 5.

[0220] Such a method of accumulating OCMS, preferably cellular microcompartments, using the filtration device 1 according to the invention can advantageously be continuous by repeating the two steps described.

[0221] A second use of the filtration device 1 according to the invention relates to a method of rinsing OCMS, preferably cellular microcompartments.

[0222] A first step of the rinsing process of the OCMS, preferably of the cellular microcompartments, consists of the injection of cellular microcompartments bathed in a soaking solution into the first port 5 of the filtration device 1 according to a flow rate Q2, and the filtration of the OCMS, preferably of the cellular microcompartments by the filter membrane 3 and the recovery of a part of the soaking solution by the second port 6 for a time T2. For example, the flow rate Q2 is 260 mL / min and the time T2 is 15 s.

[0223] A second step in the rinsing process for OCMS, preferably cellular microcompartments, consists of injecting a rinsing solution into the first port 5 at a flow rate Q3 for a given time T3, the rinsing solution then replacing the soaking solution and recovering the soaking solution mixed with the rinsing solution and then the rinsing solution alone via the second port 6. For example, the flow rate Q3 is 250 ml / min and the time T3 is 20 s.

[0224] A third step consists of reversing the flow with the injection into the second port 6 of the rinsing solution at a flow rate Q4 and the recovery of the OCMS, preferably cellular microcompartments, rinsed by the first port 5 for a time T4. For example, the flow rate Q4 is 250 mL / min and the time T4 is 10 s.

[0225] Such a method of rinsing OCMS, preferably cellular microcompartments, using the filtration device 1 according to the invention can advantageously be continuous by repeating the three steps described.

[0226] Figure 6 illustrates the implementation of the two methods described above in series. A first filtration device 1 is used for a process of accumulating OCMS, preferably cellular microcompartments, and a second filtration device is used for a process of rinsing OCMS, preferably cellular microcompartments.

[0227] Two filtration devices 1 are used to accumulate the OCMS, preferably the cellular microcompartments, produced and transfer them, and to wash the OCMS, preferably the cellular microcompartments with a rinsing solution and to collect the OCMS, preferably the cellular microcompartments, in a closed single-use system. To do this, a flow valve 10 is interposed between the first two respective ports of the two filtration devices 1. And the steps described above can take place continuously.

[0228] The first port 5 of a first filtration device 1 is connected to the first port 5 of a second filtration device 1 in order to be able to directly inject OCMS, preferably cellular microcompartments, accumulated by the first filtration device 1 and to rinse them using the second filtration device 1.

[0229] Other architectural forms may be shaped according to the use of the invention.

[0230] Of course, various other modifications may be made to the invention within the scope of the appended claims. comparative effect of the a of a centrifugal acceleration of at least 10g on cell amplification within a microcom cellular

[0231] The objective of this example is to demonstrate the advantageous effect of the method for preparing a microcompartment according to the invention on the amplification of the cells included within at least one microcompartment.

[0232] For this purpose, cellular microcompartments, within the meaning of the invention, were produced so that the microcompartments have the following characteristics:

[0233] - an outer hydrogel layer surrounding an inner part;

[0234] - at least one cell in the internal part; and

[0235] - a larger dimension of 600pm.

[0236] From this set of microcompartments, two groups of microcompartments were formed, namely:

[0237] (a) a group of cellular microcompartments undergoing centrifugal acceleration of 400g for 4 min; and

[0238] (b) a control group. On the first day after application of centrifugal acceleration, it can be observed in Figure 7 that the cells present within the microcompartments of group (a) are agglomerated at the periphery of the intracapsular space. Furthermore, it can also be observed that the integrity of the cellular microcompartments of group (a) is preserved.

[0239] On the sixth day, it can be observed in Figure 7 that the cell colonies of group (a) are larger than those of group (b).

[0240] This visual observation is confirmed by the measurement of the amplification factor.

[0241] The measurement of the amplification factor is carried out according to the following method:

[0242] The volume of the microcompartments is measured at D0 and D6 after amplification using the same method.

[0243] Method :

[0244] 1) Measurement of the volume of the capsules;

[0245] 2) Dissociation of the outer hydrogel layer;

[0246] 3) Cell counting according to the aggregated cell counting protocol

[0247] The number of cells present within the cellular microcompartment at D0 is known and the number of cells recovered after 6 days of amplification is known by counting.

[0248] The number of viable cells in the total suspension is calculated as follows:

[0249] ((Viable cells in sample at D6 x Vol of total suspension) / Vol collected for analysis) xlOO / % of suspension that was dissociated.

[0250] Thus, the amplification factor is calculated by the following formula: Number of viable cells in the sample at D0 / Number of cells in the sample at D6.

[0251] The results of the amplification factor measurement are shown below:

[0252] - The amplification factor of group (a) after 6 days of culture is 14.8;

[0253] - The amplification factor of group (b) after 6 days of culture is 11.7.

[0254] Thus, applying centrifugal acceleration to cellular microcompartments before culturing makes it possible to increase the amplification factor.

Claims

CLAIMS

1. Method for preparing a three-dimensional cellular microcompartment, characterized in that it comprises the implementation of the following steps: - a step of encapsulating at least one cell in hydrogel, so as to form at least one cellular microcompartment, - a step of suspending said cellular microcompartment in a solution, preferably a soaking solution; and - at least one step applying a centrifugal acceleration of at least 10 g on at least one cell and / or at least one cellular microcompartment.

2. Method according to the preceding claim, characterized in that the step of encapsulating at least one cell in hydrogel is carried out simultaneously with the step of suspending the cellular microcompartment(s) in the solution.

3. Method according to one of the preceding claims, characterized in that it comprises at least one step applying a centrifugal acceleration of between 10g and 3000g on at least one cell and / or at least one cellular microcompartment.

4. Method according to one of the preceding claims, characterized in that it comprises the implementation of the following steps: a) Encapsulation of at least one cell in hydrogel, so as to form at least one cellular microcompartment; b) Suspending said cellular microcompartment in a solution, preferably a soaking solution; c) Applying a centrifugal acceleration of at least 10g to the suspension of step b); d) Rinsing at least one cellular microcompartment obtained in step c) using a rinsing solution, preferably so as to form a suspension comprising at least one cellular microcompartment in a rinsing solution; and e) Culturing at least one microcompartment resulting from step d).

5. Method according to the preceding claim, characterized in that steps c) and / or d) are carried out using at least one device comprising a body (2) comprising 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 cellular microcompartments from one compartment to the other, the first port (5) being intended for the injection and withdrawal of the cellular 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).

6. Method according to one of claims 4 or 5, characterized in that the microcompartment obtained at the end of step a) has an external hydrogel layer and a hollow internal part comprising at least one cell.

7. Method according to one of claims 4 to 6, characterized in that step a) of encapsulation is carried out by co-injection of two or three solutions: - a hydrogel solution; - optionally an isotonic intermediate solution; - a solution comprising cells, culture medium and optionally extracellular matrix and / or extracellular matrix substitute, concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of solutions, said jet splitting into drops, said drops being collected in step b) in a solution which stiffens the hydrogel solution to form the external layer of each microcompartment, the internal part of each drop being constituted by the solution comprising the cells, culture medium and optionally extracellular matrix and / or extracellular matrix substitute.

8. Method according to one of claims 4 to 8, characterized in that the soaking solution of step b) comprises at least one cation chosen from calcium, iron, barium, strontium, copper, lead, aluminum, magnesium and their combinations.

9. Method according to one of claims 4 to 9, characterized in that the microcompartment obtained at the end of step a) comprises an external hydrogel layer and an internal part comprising an internal matrix and at least one cell.

10. Method according to the preceding claim, characterized in that it comprises a step x), carried out after step b), of gelling the internal matrix present in the internal part of at least one cellular microcompartment.

11. Device (1) suitable for implementing steps c) and / or d) according to one of claims 4 to 10, comprising a body (2) comprising 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 cellular microcompartments from one compartment to the other, the first port (5) being intended for the injection and withdrawal of the cellular 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).

12. Device (1) according to the preceding claim, in which the direction D is parallel to the plane P.

13. Device (1) according to one of claims 11 or 12, wherein the body (2) comprises an inner envelope with edges having a rounded shape and the useful section of the filter membrane (3) has the shape of a circle or an ellipse.

14. Device (1) according to one of claims 11 to 13, in which any section perpendicular to the plane P of the compartment intended to receive the cellular microcompartments has a surface area strictly less than the useful surface area of ​​the filter membrane (3).

15. Device (1) according to the preceding claim in which any section perpendicular to the plane P of the compartment intended to receive the cellular microcompartments has a surface area twice, five times and preferably ten times smaller than the useful surface area of ​​the filter membrane.

16. Device (1) according to one of claims 11 to 15, in which the body (2) has a toric shape and the useful section of the filter membrane (3) has the shape of a circle or an ellipse.

17. Device (1) according to one of claims 11 to 16, in which the injection into the second port (6) is also carried out in a direction which is non-orthogonal, and preferably parallel, to the plane P of the filter membrane (3).

18. Device (1) according to one of claims 11 to 17, in which the two compartments have different volumes.

19. Device (1) according to the preceding claim, characterized in that the compartment intended to receive cellular microcompartments bathed in a soaking solution and / or a rinsing solution has a volume greater than the compartment intended to receive the soaking solution and / or the rinsing solution.

20. Device (1) according to the preceding claim, characterized in that the compartment intended to receive cellular microcompartments bathed in a soaking solution and / or a rinsing solution has a volume at least 50% greater than the microcompartment intended to receive the soaking solution and / or the rinsing solution.

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