cell culture method

Abstract

A cell culture method comprising: arranging an initial quantity of cells, a culture medium, and supports in an enclosure, the enclosure extending from a bottom to a height; incubation, during which the cells grow on the supports; extraction of cells from the supports and recovery of the extracted cells; the method being characterized in that: each support extends between two opposite faces, with a characteristic dimension of less than 10 mm, the characteristic dimension corresponding to the largest diameter or the largest diagonal of one of the two faces; each support extends to a thickness of less than half the characteristic dimension; during at least 50% of the incubation time, at least 50% of the supports are arranged at the bottom of the enclosure.

Description

Title of the invention: Method for culturing cells technical field

[0001] The technical field of the invention is the culture of cells with supports suitable for cell culture. EARLIER ART

[0002] Certain biological culture processes utilize culture supports bearing cells. The supports are suspended in a culture medium, the objective being to grow the cells outside their original environment. The culture medium is usually subjected to moderate agitation. This homogenizes the culture medium and suspends the supports.

[0003] The supports are intended to be suspended in a culture medium. They often take the form of microbeads made of glass, plastic, or an organic compound, for example, a polymer (e.g., polystyrene or a polysaccharide). Generally, the microbeads have undergone a surface treatment, known as surface functionalization, to promote cell grafting. This involves facilitating cell attachment or adhesion. The supports are frequently used for culturing adherent cells. The cells can grow and multiply on them.

[0004] Application WO2021140129 describes a method for manufacturing sol-gel supports having a flat or flattened surface relative to a spherical surface. Such supports have proven suitable for cell culture, in particular monolayer cell culture, which promotes improved cell viability and cell collection.

[0005] Culture media are usually agitated to prevent sedimentation of the supports and to homogenize the culture medium. This often involves the use of a hydrodynamic agitation device, for example, a propeller or wave bioreactor. However, using a propeller in a bioreactor has certain drawbacks: the agitation of the culture medium may not be homogeneous. Furthermore, near the propeller, the agitation is significant, which can lead to hydrodynamic and mechanical stress. Wave bioreactors also induce hydrodynamic stress.

[0006] Generally, the larger the culture volume, the stronger the agitation of the medium must be, so as to suspend the supports throughout the entire culture volume. This necessarily increases the shear forces to which the cells are exposed, which do not constitute culture conditions favorable. Thus, for large volume bioreactors, it is necessary to find a compromise between the resuspension of the supports and the hydrodynamic stress to which the cells are subjected due to the means implemented to maintain the supports in suspension.

[0007] Furthermore, the use of a propeller makes the bioreactor dedicated to a specific application, that is, to a particular culture medium and a predetermined cell type. Indeed, if it is necessary to change the application, the propeller must undergo thorough cleaning to limit the risk of contamination. This is impractical and time-consuming. Some bioreactors have interchangeable propellers, but this makes them more complex. Some bioreactors are also for single use only.

[0008] After passing through the bioreactor, the cells are separated from the supports by detachment, generally using a so-called detachment enzyme. The supports resulting from the separation are collected by a filter. However, the filter can become clogged by the supports.

[0009] During cell culture, some cell cultures are performed by perfusion, which involves continuously regenerating the culture medium. This method allows for cultures at high cell concentrations but requires filtration and renewal of the culture medium. The growth media can clog the filters, complicating the implementation of cultures under perfusion.

[0010] The inventor has developed a process, making it possible to avoid the need for agitation, while allowing for efficient and good quality cell culture. Description of the invention

[0011] A first aspect of the invention is a method for culturing cells, the method comprising: a. arrangement, in an enclosure, of an initial quantity of cells, a culture medium, and supports, the enclosure extending between a bottom and an upper end; b. incubation, during an incubation period, during which the cells develop on the culture media; c. extraction of cells from the supports and recovery of the extracted cells;

[0012] the process being characterized in that: - each support extends between two opposite faces, according to a characteristic dimension, preferably less than 20 mm or 10 mm, the characteristic dimension corresponding to the largest diameter or the largest diagonal of one of the two faces; - each support extends by a thickness, between opposite faces, less than half of the characteristic dimension; - during at least 50% of the incubation period, at least 50% of the supports are placed, by gravity, in contact with the bottom of the enclosure.

[0013] The process may exclude the culture of human embryonic stem cells,

[0014] Preferably, during at least 80% or 90%, or even 95% or 99% of the incubation period, at least 80%, or even at least 90% of the supports are arranged, by gravity, in contact with the bottom of the enclosure.

[0015] Preferably, for at least 50% or 80% or 90% of the incubation time, the culture medium is static.

[0016] During step b), supports can be moved spontaneously and progressively under the effect of cell development, straightening towards an upper end of the enclosure, opposite the bottom.

[0017] Preferably, each support is such that a characteristic dimension-to-thickness ratio is between 5 and 100 and preferably between 5 and 50.

[0018] According to one possibility, one of said opposite faces is a planar face. According to another possibility, each of said opposite faces is planar.

[0019] According to one possibility, at least one of said opposite faces is convex.

[0020] According to one possibility, each support is formed of at least one mineral material or organic material chosen from: sol-gel material, glass, polymer, plastic, ceramic, silicon, metal.

[0021] For each support, the characteristic dimension can be between 10 pm and 20 mm or between 10 pm and 500 pm.

[0022] The density of each support may be less than 3 g / cm3 or less than 2.5 g / cm2.

[0023] Preferably, during incubation, the enclosure is configured so that the bottom is below the upper end, relative to a vertical axis.

[0024] The invention will be better understood upon reading the description of the exemplary embodiments presented later in this description, in connection with the figures listed below. FIGURES

[0025] Fig. 1A represents an example of a support according to the invention.

[0026] Fig. 1B shows another example of a support according to the invention.

[0027] Fig. 2A schematically illustrates supports introduced into an enclosure.

[0028] Fig. 2B schematically represents the supports after cells have developed on their respective flat faces.

[0029] Fig. 3 is a photograph of an enclosure, comprising supports on which VERO type cells have been developed.

[0030] Fig. 4A shows, for different culture conditions (diameter and concentration of supports - x-axis), a quantity of cells recovered following incubation (y-axis).

[0031] Fig. 4B shows, for the different culture conditions (diameter and concentration of the supports - x-axis), a percentage of live cells recovered (y-axis).

[0032] Figures 5A and 5B are photographs of an enclosure, comprising supports on which MSC (Mesenchymal Stem Cells) type cells have developed.

[0033] Figure 6 summarizes the main steps of a process according to the invention. PRESENTATION OF SPECIFIC IMPLEMENTATION METHODS

[0034] Figure 1A represents a growing medium 1 enabling an implementation of the invention. In this example, the medium 1 has two opposite faces that are flat, or considered as such. Thus, the medium 1 has a first flat, or substantially flat, face S1 and a second flat, or substantially flat, face S2. The first flat face and the second flat face are parallel to each other, or substantially parallel to each other.

[0035] By substantially parallel, we mean parallel taking into account an angular tolerance, for example less than or equal to ± 20°, and preferably less than or equal to ± 10°.

[0036] By substantially flat face, we mean flat, admitting surface elements having a local flatness defect, within a limit of ± 10° with respect to the rest of the surface.

[0037] The first face Si has a diameter ¢, preferably less than or equal to 20 mm, or less than or equal to 10 mm, or less than or equal to 1 mm. The same applies to the second surface S2. The diameter is preferably greater than 5 pm or 10 pm. The diameter is preferably between 10 pm and 10 mm or 20 mm, and even more preferably between 10 pm and 1 mm, or preferably between 10 pm and 500 pm.

[0038] By diameter of a face, we mean the diameter along which the face extends when the face is of circular geometry, or the largest diameter when it is an oval face.

[0039] The first face Si and the second face S2 extend parallel to a principal plane PXY. The support 1 has a lateral face S3, extending between the first face Si and the second face S2. Hereafter, the lateral face of the supports is referred to as the edge. The opposite faces Si and S2 are separated by a thickness e.

[0040] Regardless of the configuration, the thickness e of the support 1 is preferably less than half the diameter or a quarter of the diameter, and preferably less to one-tenth of the diameter, or even to one-twentieth of the diameter. An optimal diameter-to-thickness ratio is considered to be between 100 and 5, and preferably between 50 and 5.

[0041] The thickness e generally results from a compromise: it is sufficient to ensure a certain solidity to the support, but low enough to allow movement of the support under the effect of cell development, as described later.

[0042] Figure 1A illustrates one embodiment in which the support 1 has a geometry of revolution. The first face S1 and the second face S2 have a circular or elliptical shape of revolution, the lateral face S3 being an annular edge. Other cylindrical configurations are possible, for example, a cylinder with a polygonal base, for example in the shape of a quadrilateral or a hexagon, or a cylinder with an oval base. Annular cylinders can also be considered, the base being of the crown or ring type.

[0043] Figure 1B represents a cylindrical support 1 with a polygonal base, in this case a quadrilateral. In this case, the longest diagonal A is preferably less than or equal to 50 mm, or less than or equal to 20 mm or 10 mm, or less than or equal to 1 mm. The longest diagonal A is preferably between 10 pm and 10 mm, and even more preferably between 10 pm and 1 mm, or preferably between 10 pm and 500 pm.

[0044] Generally, each substrate has a characteristic dimension which is either a larger diameter, when the substrate is circular, or a larger diagonal, when the substrate has a polygonal face. The characteristic dimension can range from 10 µm to 10 mm, or 20 mm, or even 50 mm. The preferred range for the characteristic dimension is 100 µm - 300 µm. An optimal characteristic dimension-to-thickness ratio is between 100 and 5, and preferably between 50 and 5.

[0045] Generally, regardless of the configuration, the support consists of two opposing faces Si and S2, extending one towards the other, making the lateral edge S3 negligible. The two opposing faces Si and S2 may, in particular, be flat or substantially flat, or convex, and preferably slightly convex. By slightly convex, we mean convex with a diameter of curvature smaller than the characteristic dimension, and preferably less than 2 times, or even 5 or 10 times, the characteristic dimension. Both opposing faces may be convex. One of the opposing faces may be convex, while the other is flat.

[0046] The supports according to the invention can be obtained by implementing a sol-gel process, short for solution-gelation. This is a chemical process known to those skilled in the art, allowing the manufacture, at low temperature, of glasses or ceramics. Such a process involves the use of a sol-gel solution, formed: - of a molecular precursor of metal or metalloid, for example an organometallic compound or a metallic salt; - of an organic solvent; - water; - of an acidic or basic catalyst.

[0047] Thus, the supports according to the invention can be obtained by implementing the process described in WO2021140129.

[0048] In the presence of water, a network of oxides forms through hydrolysis-condensation reactions, trapping the organic solvent to form a gel. This gel is then dried to remove the organic solvent present within it.

[0049] The molecular precursor can for example be an organometallic compound of metal or metalloid, for example a metal alkoxide of formula M(0R)n, where M is a metal or a metalloid, and R is an organic group. - Metal M can be, for example, a transition metal, a lanthanide: it can be Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, Hf, Ra, W, Re, Os, Ir, Pt, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Er, Yb, Al, Ga, In, Ge, Sn, Pb. - The metalloid element can be chosen from Si, Se, Te. - R can be an alkyl group, for example with between 1 and 10 carbon atoms, or a phenyl group. - n is a natural number corresponding to the number of ligands bound to M, which corresponds to the valence of M.

[0050] The molecular precursor is placed in an organic solution, for example an alcoholic solution. The organic solvent may be an aliphatic or aromatic monoalcohol, or a diol.

[0051] The sol-gel solution comprises water, and preferably a catalyst, and possibly compounds enabling action on porosity, for example a surfactant.

[0052] According to one embodiment, the sol-gel solution comprises a functionalizing compound, in particular an organic compound, whose function is to form a grafting agent. By grafting agent, we mean a molecule or a functional group capable of promoting the attachment, by grafting, of a chemical or biological element to the surface of the xerogel resulting from the implementation of the sol-gel process. The chemical or biological element is predetermined. It may be a molecule, a cell, a protein, or another organic compound, for example, a growth factor or an antibody. For applications related to cell culture, the grafting agent Grafting facilitates the grafting of a cell of a predetermined type. The grafting agent can be collagen, polylysine, or a milk protein. The grafting agent may contain an epoxy group, which is conducive to the formation of chemical bonds with amine groups, the latter being present in most cell membranes. The incorporation of an epoxy group can be achieved using a compound such as glycidoxypropyltrimethoxysilane, commonly referred to by the acronym GPTM.

[0053] According to one possibility, the sol-gel solution comprises a precursor of the 3-aminopropyltrimethoxysilane type, usually designated by the acronym APTES, or 3-aminopropyltrimethoxysilane, usually designated by the acronym APTMS. The amine function of the precursor of such precursors brings positive charges to the surface of the support. This improves cell adsorption and adhesion.

[0054] The possibility of adding a functionalizing compound to the sol-gel solution is a significant advantage, as it eliminates the need for post-manufacturing functionalization. This allows for the production of supports specific to a predefined application, taking into account the chemical or biological element intended to bind to the supports, and / or the environment in which the support is intended to be placed.

[0055] The supports 1 used preferably have a density less than 2.5, and even more preferably less than 2, or even 1.8. An advantage of supports obtained by the sol-gel method is that it is possible to obtain supports of low density, in particular compared to glass supports obtained by molding.

[0056] Alternatively, the supports 1 comprise or are made of materials, preferably biocompatible, selected from: glass, metal, synthetic polymer, polycaprolactone, polylactic acid, polyglycolic acid, natural polymer, alginate, chitosan, dextran, or metal oxides (TiO2, SiO2).

[0057] According to one possibility, the supports comprise particles sensitive to a magnetic field, for example, particles of a ferromagnetic material. These could be supports such as those described in patent application FR2307600 filed on 16 / 07 / 2023. This facilitates the retrieval of the supports using magnets.

[0058] The supports 1 are intended to be placed in contact with a culture medium and cells, so that the cells adhere to the flat faces of the supports, and then develop at the level of said flat faces.

[0059] The method of the invention involves the use of a chamber 10, into which the supports 1 are introduced, as well as the culture medium 13 and an initial quantity of cells to be cultured. The chamber is placed in an incubator, the temperature of which is generally controlled, and possibly other environmental parameters such as humidity, ambient gas... Incubation corresponds to the phase during which the enclosure is placed in the incubator.

[0060] The enclosure 10 extends between a lower wall 11, forming a bottom, and an upper end 12, which may be open or closed. The distance h between the bottom 11 and the upper end 12 corresponds to a height h of the enclosure. The upper end 12 forms a top wall, generally permeable to certain gases. The enclosure may be of the Petri dish type, the well of a well plate, or a flange.

[0061] It has been found that by using such supports, having at least one flat or slightly convex face, and preferably two opposing flat or slightly convex faces, it is possible to carry out cell culture without requiring continuous agitation. It has been found that agitation is not necessary for at least 50% of the incubation time, or even at least 80%, or at least 90%, or even 95% or 99% of the incubation time.

[0062] Indeed, during incubation, given the geometry of the supports 1, in particular the characteristic thickness-to-dimension ratio, and advantageously the low density, cell development is accompanied by a spontaneous and progressive arrangement of the supports, so as to form a three-dimensional structure from the bottom of the chamber. Figure 2A shows a cross-sectional view of a chamber 10 at an initial stage of incubation. The supports 1 are introduced and tend to settle against the bottom 11 of the chamber 10.

[0063] During incubation, cell development is accompanied by a 3D structuring of the supports 1: these are progressively and spontaneously arranged to form 3D structures along and against the bottom 11 of the enclosure. Figure 2B schematically illustrates the formation of such structures. Spontaneously, this is understood to occur in the absence of any means of agitation applied to the enclosure. 3D structuring refers to the fact that the supports are displaced and / or oriented in three dimensions. In particular, some supports spontaneously straighten, so that their opposite faces move towards the upper end. The opposite faces of some supports extend parallel, or substantially parallel, to the height of the enclosure. Substantially parallel to the height of the enclosure means parallel, taking into account an angular tolerance of ±10° or ±20°.

[0064] When the bottom of the enclosure has been treated with an agent conducive to the adhesion and proliferation of cells, cells develop at the bottom of the enclosure, and contribute to forming bonds between the bottom of the enclosure and the supports.

[0065] The inventors attribute this progressive and spontaneous arrangement to the development of cells at the level of the flat faces, combined with the specific shape of the supports, as well as their dimensions, giving them a particularly low mass and a good aptitude for movement in a liquid.

[0066] This arrangement makes it possible to obtain a three-dimensional cell culture: this increases the exchange surface between the flat faces of the supports 1 and the culture medium 13. It thus spontaneously creates conditions that are particularly favorable to the development of cells, and this without requiring agitation of the culture medium, with the disadvantages that this entails.

[0067] Unlike processes involving agitation, during incubation, most of the supports—that is, more than 50%, or even 80% or 90%—are held against the bottom of the chamber 11 by gravity. However, contact with the bottom of the chamber can be maintained at the edge due to the spontaneous arrangement described above, and in particular the straightening of supports towards the upper end of the chamber. Thus, during incubation, the supports 1 are not distributed throughout the volume of the chamber by agitation. They are held against the bottom in various orientations. It cannot be ruled out that some supports may be displaced slightly above the bottom by resting on another support placed in contact with the bottom.Generally speaking, during incubation, the supports form a 3D structure, comprising supports placed in contact with each other, in different orientations, and resting on the bottom of the enclosure.

[0068] The formation of the 3D structure allows for a significant increase in the culture surface area, i.e., the surface area of ​​the supports exposed to the culture medium. It is estimated that the developed surface area can be between 2 and 30 times the surface area of ​​the enclosure exposed to the culture medium. Experimental trials.

[0069] A series of tests was carried out, the experimental conditions being described below: - Enclosure: Nunc™ 24-well plate treated for cell culture: surface treatment to promote cell adhesion and development. - Cells: epithelial cells from kidney extracted from the African green monkey, usually referred to as Vero cells - Culture medium: DMEM (Dulbecco Modified Eagle Medium), supplier GIBCO, supplemented with 10% fetal bovine serum (GIBCO) and 100 units / mL and 100 pg / mL of penicillin and streptomycin (GIBCO), respectively. - Supports: flat discs of different diameters (70, 100, 140, 180 pm) and approximately 5 pm thick, produced by sol-gel process, according to the principles described in patent application WO2021140129. - Cell detachment agent: TrypLE™ select enzyme (Thermofisher) - Culture hood: ESCO AC2 microbiological safety cabinet. - Cell culture environment: CellXpert® Cl70 incubator (Eppendorf) set at 37 °C in the presence of 5% industrial CO2 (Air Liquide). - Cell counting agent: Trypan Blue 0.4% (PAN Biotech). - Cell counting equipment: LUNA II automated counter (Logos Biosystems).

[0070] Under a fume hood, carriers of the same diameter were sterilely placed in each well of a 24-well plate treated for cell culture. For each carrier diameter, carrier concentrations of 0, 1, 2, 4, and 8 mg / cm² were used, respectively. The surface area expressed in cm² corresponds to the surface area of ​​the base of the chamber. Since the surface area of ​​the base of each well was 2 cm², carrier concentrations of 0, 2, 4, 8, and 16 mg per well were used, respectively.

[0071] Subsequently, 10,000 cells were carefully added to each well, diluted in 1 ml of culture medium. To ensure homogeneous culture, the multi-well plate containing the supports was gently agitated along one or more horizontal axes X, Y. The bottom 11 of each well was horizontal.

[0072] The well plate was incubated for 14 days. Throughout the culture period, and to ensure a sufficient supply of nutrients for the cells, the culture medium was regularly changed. For this purpose, under a fume hood, 500 µL of culture medium was carefully withdrawn from each well, and 500 µL of fresh culture medium was carefully added to each well. During the incubation period, the cumulative time during which the well plate was handled was considered to be less than 1 hour over the total duration of 14 days. The culture was therefore carried out in an essentially static manner.

[0073] After incubation, the culture medium was carefully removed, and the cells were detached by incubating for 10 minutes in the incubator with 500 µL per well of TrypLE™ select. 500 µL of fresh culture medium was then added to each well, and the detached cells were carefully pipetted from each well into 1.5 mL tubes, taking care not to recover the supports. After centrifuging the cells for 5 minutes at 0.2 RCF (Relative Centrifuge Force), the culture medium was carefully removed, and the cells were suspended in 50 µL of Trypan Blue diluted 2:1 in fresh culture medium.

[0074] The quantity and viability of the cells present in each well were quantified. This made it possible to compare the different culture conditions, in particular the size and concentration of the supports.

[0075] During incubation, the displacement and modification of the supports were observed. Figure 3 shows a photograph of a well, taken from the upper opening of the well.

[0076] Figure 4A shows, for different support diameters and different support concentrations in a well (x-axis), the number of cells counted after incubation (y-axis). The x-axis "2D" corresponds to a cell culture performed directly without support added to the well: this is a prior art configuration, in which cell development occurs along the inner wall of the chamber exposed to the culture medium.

[0077] It is observed that, in this example: - the presence of supports systematically allows for cell development; - An optimal concentration of carriers is less than 4 mg / cm². The optimal concentration range is estimated to be between 0.5 mg / cm² and 3 mg / cm² or 4 mg / cm². Within this range, regardless of their size, the carriers allow for a greater number of cells than the prior art configuration.

[0078] The observation of an optimal concentration range is due to the fact that when the concentration of supports is too high, their arrangement, as described in relation to Figures 2B and 3, becomes more difficult. Culture performance decreases. The optimal concentration range may vary depending on the culture conditions or cell types.

[0079] Figure 4B shows, for different support diameters and different support concentrations in a well (x-axis), the percentage of live cells (y-axis). Viability performance is comparable to the prior art, or even slightly superior, in the concentration range of 0.5 mg / cm² - 3 mg / cm² or 4 mg / cm².

[0080] The optimal concentration can be defined on a case-by-case basis, depending on the size of the media and the dimensions of the enclosure. However, it is considered that, as a first approximation, the concentration range of 0.5 mg / cm² - 3 mg / cm² or 4 mg / cm² may be suitable when the media size is between 10 µm and 500 µm, or even 1 mm.

[0081] Similar tests were carried out using MSC (mesenchymal stem cell) type cells. Figures 5A and 5B are photographs, taken from the top opening of a well, showing the spontaneous and progressive arrangement of the supports, as described in connection with Figures 2B and 3.

[0082] Figure 6 schematically illustrates the main steps of a process according to the invention.

[0083] Step 100: arranging, in an enclosure, an initial quantity of cells, a culture medium, and supports as previously described

[0084] Step 110: Possible handling of the enclosure, for example by gentle agitation, so as to homogenize the initial distribution of the supports and cells within the enclosure. Unlike the prior art, the aim is not to keep the supports dispersed within the enclosure by taking advantage of their buoyancy.

[0085] Step 120: Incubation, for a specified duration, preferably under controlled environmental conditions, for example, temperature and / or humidity and / or ambient gas. During incubation, the cells grow on the supports. For at least 50%, or even at least 80%, or at least 90%, 95%, or 99% of the incubation time, at least 50%, or even at least 80%, or at least 90% of the supports are placed against the bottom of the chamber. Preferably, for at least 50%, or even at least 80%, or even at least 90%, 95%, or 99% of the incubation time, the culture medium is static. During step 120, the culture medium may be renewed. Thus, the incubation time may be interspersed with phases of changes to the culture medium. However, the duration of such phases is preferably limited to a few percent of the static incubation time.

[0086] Step 130: extraction of cells from supports and recovery of extracted cells;

[0087] The invention makes it possible to improve the performance of cell culture compared to configurations in which cell culture is carried out on the walls of the enclosure, while conferring an equivalent, or even slightly higher, viability rate.

[0088] The invention is particularly suitable for use with speakers having a large base. This allows for greater use of the 3D arrangement of the supports against the base, some of which straighten as they move away from the base.

[0089] An advantage of the invention is that incubation can be carried out without stirring the culture medium, thus avoiding the drawbacks associated with stirring, as previously described, while allowing the use of simple enclosures without any means of stirring. The invention therefore makes it possible to use enclosures with a high surface area to height ratio. Several low-height enclosures, typically less than 10 cm or 5 cm high, can be stacked, with a 3D structuring of the supports occurring at the base of each one.

Claims

Demands

1. A method for culturing cells, excluding human embryonic stem cells, the method comprising: a. arranging an initial quantity of cells, a culture medium, and supports (1) in a chamber (10), the chamber extending from a bottom to a top end; b. incubation, during an incubation period, during which the cells grow on the supports; c.cell extraction from supports and recovery of extracted cells; the process being characterized in that: - each support extends between two opposite faces, according to a characteristic dimension less than 20 mm, the characteristic dimension corresponding to the largest diameter or the largest diagonal of one of the two faces; - each support extends according to a thickness, between the opposite faces, less than half of the characteristic dimension; - during at least 50% of the incubation time, at least 50% of the supports are arranged, by gravity, in contact with the bottom of the chamber.

2. A method according to claim 1, wherein during at least 80% or 90% of the incubation time, at least 80% of the supports are arranged, by gravity, in contact with the bottom of the enclosure.

3. A method according to any one of the preceding claims, wherein for at least 50% or 80% or 90% of the incubation time, the culture medium is static.

4. A method according to any one of the preceding claims, wherein during step b), supports are moved spontaneously and progressively under the effect of cell development, straightening towards the upper end of the enclosure, opposite the bottom.

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12. A method according to any one of the preceding claims, wherein each support is such that a characteristic dimension-to-thickness ratio is between 5 and 100 and preferably between 5 and 50. A method according to any one of the preceding claims, wherein one of said opposite faces is a flat face. Method according to claim 6, wherein each of said opposite faces is planar. A method according to any one of claims 1 to 6, wherein at least one of said opposite faces is convex. A method according to any one of the preceding claims, wherein each support is formed of at least one mineral or organic material selected from: sol-gel material, glass, polymer, plastic, ceramic, silicon, metal. A method according to any one of the preceding claims, wherein for each support, the characteristic dimension is between 10 pm and 20 mm or between 10 pm and 500 pm. A method according to any one of the preceding claims, wherein the density of each support is less than 3 g / cm3 or less than 2.5 g / cm2. A method according to any one of the preceding claims, wherein during incubation, the enclosure is configured so that the bottom is below the upper end, relative to a vertical axis (Z).

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