cell culture process

Abstract

Method for culturing cells, comprising: arranging, in an enclosure, an initial quantity of cells, a culture medium, and supports, the enclosure extending, from a bottom, along a height; incubation, during an incubation period, during which the cells develop on the supports; extraction of the cells from the supports and recovery of the extracted cells; the method being characterized in that: each support extends between two opposite faces, along a characteristic dimension 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 along a thickness, less than half of the characteristic dimension; during at least 50% of the incubation period, 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. PREVIOUS ART

[0002] Some biological culture processes use culture supports, carrying cells. The supports are arranged in suspension, 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 makes it possible to homogenize the culture medium and suspend the supports.

[0003] The supports are intended to be placed in suspension in a culture medium. They often take the form of microbeads, made of glass, or plastic or an organic compound, for example a polymer (for example polystyrene or a polysaccharide). Generally, the microbeads have previously been subjected to a surface treatment, called surface functionalization, so as to promote cell grafting. This involves promoting cell attachment or adhesion. The supports are frequently used for the culture of adherent cells. 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 been found to be suitable for cell culture, in particular monolayer cell culture, conducive to improved cell viability of the cultured cells and improved collection of these cells.

[0005] Culture media are usually agitated to avoid sedimentation of the supports and to homogenize the culture medium. This often involves the use of a hydrodynamic agitation means, for example a propeller or wave bioreactor. However, the use of a propeller in a bioreactor has certain disadvantages: the agitation of the culture medium may not be homogeneous. In addition, 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 more vigorous the agitation of the medium is required, so as to suspend the supports throughout the entire culture volume. This necessarily increases the shear forces to which the cells are exposed, which does 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] In addition, the use of a propeller makes the bioreactor dedicated to a specific application, i.e. to a particular culture medium and a predetermined cell type. Indeed, if one wishes to change the application, it is necessary to carry out a thorough cleaning of the propeller, in order to limit the risks of contamination. This is not practical and takes time. Some bioreactors have interchangeable propellers, but this makes them more complex. Some bioreactors are also single-use.

[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 may become clogged by the supports.

[0009] During culture, some cell cultures are performed by perfusion, which consists of continuously regenerating the culture medium. Such a method allows cultures to be performed at high cell concentrations, but requires filtration and renewal of the culture medium. The supports can clog the filters, which complicates the implementation of cultures under perfusion.

[0010] The inventor has developed a process which makes it possible to avoid the need for stirring, whilst still allowing efficient and good quality cell culture. Statement of the invention

[0011] A first aspect of the invention is a method of 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 grow on the culture media; c. extraction of cells from the supports and recovery of the extracted cells;

[0012] the method 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 with a thickness, between the opposite faces, less than half of the characteristic dimension; - for 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 method may exclude the culture of embryonic stem cells of human origin,

[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 the development of the cells, by straightening up 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 one possibility, each of said opposite faces is planar.

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

[0020] According to one possibility, each support is formed from 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 in the remainder of the description, in conjunction 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] shows a diagram of supports introduced into an enclosure.

[0028] [Fig.2B] schematizes 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 developed.

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

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

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

[0033] [Fig.6] summarizes the main steps of a method according to the invention. PRESENTATION OF SPECIAL METHODS OF IMPLEMENTATION

[0034] [Fig. 1A] represents a culture support 1 allowing an implementation of the invention. In this example, the support 1 comprises two opposite faces which are flat, or considered as such. Thus, the support 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 is meant 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 is meant flat, admitting surface elements having a local flatness defect, within a limit of ± 10° relative 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 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 of the largest diameter when it is an oval face.

[0039] The first face Si and the second face S2 extend parallel to a main plane PXY. The support 1 has a lateral face S3, extending between the first face Si and the second face S2. Subsequently, the lateral face of the supports is designated the edge. The opposite faces Si and S2 are spaced apart by a thickness e.

[0040] Whatever 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 be able to ensure a certain solidity to the support, but sufficiently weak to allow a displacement of the support under the effect of the development of cells, as described below.

[0042] [Fig.1A] corresponds to an embodiment, according to 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 base cylinders can be envisaged, the base being of the crown or ring type.

[0043] [Fig. 1B] represents a cylindrical support 1 with a polygonal base, in this case a quadrilateral. In this case, the largest 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 largest diagonal A is preferably between 10 μm and 10 mm, and more preferably between 10 μm and 1 mm, or preferably between 10 μm and 500 μm.

[0044] Generally, each support has a characteristic dimension which is either a larger diameter, when the support is circular, or a larger diagonal, when the support has a polygonal face. The characteristic dimension can extend between 10 μm and 10 mm, or 20 mm, or even 50 mm. The preferred range of 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 speaking, whatever the configuration, the support is in the form of two opposite faces S1 and S2, extending one facing the other, making the lateral edge S3 negligible. The two opposite faces S1 and S2 may in particular be flat or substantially flat, or curved, and preferably slightly curved. By slightly curved is meant curved according to a diameter of curvature less than the characteristic dimension, and preferably less than 2 times or even 5 times or 10 times the characteristic dimension. The two opposite faces may be curved. One of the opposite faces may be curved, while the other is flat.

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

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

[0048] In the presence of water, a network of oxides is formed, through hydrolysis-condensation reactions, trapping the organic solvent, so as to form a gel. The latter then undergoes drying, to eliminate the organic solvent present in the gel.

[0049] The molecular precursor may for example be an organometallic compound of metal or metalloid, for example a metal alkoxide of formula M(OR)n, where M is a metal or a metalloid, and R is an organic group. - The 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 may be an alkyl group, for example having between 1 and 10 carbon atoms, or a phenyl group. - n is a natural integer 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 making it possible to act on the porosity, for example a surfactant.

[0052] According to one embodiment, the sol-gel solution comprises a functionalization compound, in particular an organic compound, the function of which is to form a grafting agent. By grafting agent is meant a molecule or a functional group capable of promoting 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, or a protein or another organic compound, for example a growth factor or an antibody. For applications related to cell culture, the grafting agent Grafting promotes grafting of a cell of a predetermined type. The grafting agent can then be collagen, or polylysine, or a milk protein. The grafting agent can include an epoxy function, conducive to the formation of chemical bonds with amine functions, the latter being present in most cell membranes. The incorporation of an epoxy function can be carried out by a compound of the glycidoxypropyltrimethoxysilane type, usually designated by the acronym GPTM.

[0053] According to one possibility, the sol-gel solution comprises a precursor of the 3-aminopropyltriethoxysilane 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 the adsorption and attachment of the cells.

[0054] The possibility of adding a functionalization compound to the sol-gel solution constitutes an interesting advantage, because this avoids the need for post-manufacturing functionalization. This makes it possible to manufacture supports specific to a predefined application, taking into account the chemical or biological element intended to be fixed on the supports, and / or the environment in which the support is intended to be placed.

[0055] The supports 1 used have a density preferably less than 2.5, and even more preferably less than 2, or even 1.8. An advantage of supports obtained by 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, chosen 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 may be supports as described in patent application FR2307600 filed on 07 / 16 / 2023. This makes it easier to recover 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, then develop at the level of said flat faces.

[0059] The method which is the subject of the invention involves the use of an enclosure 10, into which the supports 1 are introduced, as well as the culture medium 13 and an initial quantity of cells which it is desired to cultivate. The enclosure is placed in an incubator, the temperature of which is generally controlled, and possibly other environmental parameters such as humidity, ambient gas, etc. 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, 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 an upper wall, generally permeable to certain gases. The enclosure may be of the Petri dish type, the well of a well plate, or a flask.

[0061] It has been found that by using such supports, comprising at least one flat or slightly curved face, and preferably two opposite flat or slightly curved faces, it is possible to carry out cell culture without requiring continuous stirring. It has been found that stirring was 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, taking into account the geometry of the supports 1, in particular the characteristic thickness to dimension ratio, and advantageously the low density, the cellular 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 enclosure. In [Fig.2A], a sectional view of an enclosure 10 is shown, at an initial moment of incubation. The supports 1 are introduced and tend to sediment against the bottom 11 of the enclosure 10.

[0063] During incubation, cell development is accompanied by a 3D structuring of the supports 1: the latter are progressively and spontaneously arranged, so as to form 3D buildings, along and against the bottom 11 of the enclosure. [Fig.2B] schematizes the formation of such buildings. By spontaneously, we mean in the absence of a means of agitation applied to the enclosure. The 3D structuring designates the fact that the supports are moved and / or oriented according to the 3 dimensions of space. In particular, we witness a spontaneous straightening of certain supports, so that their opposite faces approach the upper end. The opposite faces of certain supports extend parallel, or substantially parallel, to the height of the enclosure. By substantially parallel to the height of the enclosure, we mean 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 the 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 ability to move 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. This 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 methods using stirring, during incubation, most of the supports, i.e. more than 50%, or even 80% or 90% of the supports, are held against the bottom of the enclosure 11, under the effect of gravity. However, contact with the bottom of the enclosure can be ensured at the edge, due to the spontaneous arrangement described above, and in particular the straightening of supports towards the upper end of the enclosure. Thus, during incubation, the supports 1 are not distributed in the volume of the enclosure, by stirring. They are held against the bottom, according to different orientations. It cannot be excluded that certain supports are moved slightly above the bottom, by resting on another support arranged in contact with the bottom.Generally speaking, during incubation, the supports form a 3D structure, comprising supports arranged in contact with each other, in different orientations, and resting on the bottom of the enclosure.

[0068] The formation of the 3D building allows a significant increase in the culture surface, that is to say the surface of the supports exposed to the culture medium. It is estimated that the developed surface can be between 2 times and 30 times the surface of the enclosure exposed to the culture medium. Experimental tests.

[0069] A series of tests were 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: kidney epithelial cells extracted from the African green monkey, usually referred to as Vero cells - Culture medium: DMEM (Dulbecco's Modified Eagle's 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 method, according to the principles described in patent application WO2021140129. - Cell detaching 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 culture hood, supports of the same diameter were integrated in a sterile manner into each well of a 24-well well plate treated for cell culture. For each support diameter, a support concentration of 0, 1, 2, 4 and 8 mg / cm2 was respectively integrated. The surface area expressed in cm2 corresponds to the surface area of ​​the bottom of the enclosure. Since the surface area of ​​the bottom of each well is 2 cm2, concentrations of 0, 2, 4, 8, 16 mg of supports were respectively placed per well.

[0071] 10,000 cells per well diluted in 1 ml of culture medium were then gently added. In order to ensure homogeneous culture, the multi-well plate containing the supports was gently shaken along a horizontal X axis or several horizontal X, Y axes. The bottom 11 of each well was horizontal.

[0072] The well plate was incubated for 14 days. Throughout the duration of the culture, and in order to ensure sufficient nutrients for the cells, the culture medium was changed regularly. For this, under a culture hood, 500 pL of culture medium was carefully removed per well, and 500 pL of fresh culture medium was gently added to each well. During the incubation period, the cumulative time during which the well plate was handled is 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 gently removed, and the cells were detached by incubation for 10 minutes in the incubator in the presence of 500 pL per well of TrypLE™ select. 500 pL of fresh culture medium was then added per well, and the detached cells from each well were gently pipetted into 1.5 milliliter tubes respectively, taking care not to recover the media. After centrifugation of the cells for 5 minutes at 0.2 RCF (Relative Centrifuge Force), the culture medium was gently removed, and the cells were suspended in 50 pL of Trypan Blue diluted 2 times in fresh culture medium.

[0074] The quantity and viability of cells present in each well was 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 was observed. [Fig. 3] shows a photograph of a well, taken from the upper opening of the well.

[0076] [Fig.4A] shows, for different diameters of supports, and for different concentrations of supports in a well (x-axis), a quantity of cells counted after incubation (y-axis). The “2D” abscissa corresponds to a cell culture directly carried out without support added to the well: this is a configuration according to the prior art, according to which cell development takes place along the internal wall of the enclosure exposed to the culture medium.

[0077] We observe that, in this example: - the presence of supports systematically allows cellular development; - an optimal carrier concentration 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 make it possible to obtain a quantity of cells greater than the configuration according to the prior art.

[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 connection with Figures 2B and 3, is made more difficult. Culture performance decreases. The optimal concentration range may vary depending on the culture conditions or cell types.

[0079] [Fig.4B] shows, for different diameters of supports, and for different concentrations of supports in a well (x-axis), a percentage of living cells (y-axis). A performance, in terms of viability, comparable to the prior art, or even slightly superior in the concentration range 0.5 mg / cm2 - 3 mg / cm2 or 4 mg / cm2 is observed.

[0080] The optimal concentration can be defined on a case-by-case basis, depending on the size of the supports and the dimensions of the enclosure. However, it is considered that as a first approximation, the concentration range 0.5 mg / cm2 - 3 mg / cm2 or 4 mg / cm2 may be suitable when the size of the supports is between 10 pm and 500 pm, 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 upper opening of a well, showing the spontaneous and progressive arrangement of the supports, as described in connection with Figures 2B and 3.

[0082] [Fig.6] shows schematically the main steps of a method according to the invention

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

[0084] Step 110: possible handling of the enclosure, for example by slight agitation, so as to homogenize the initial distribution of the supports and cells in the enclosure. Unlike the prior art, it is not a question of keeping the supports dispersed in the enclosure, taking advantage of their flotation.

[0085] Step 120: incubation, during an incubation period, preferably under controlled environmental conditions, for example temperature and / or humidity level and / or ambient gaseous medium. During the incubation, the cells grow on the supports. During at least 50%, or even at least 80%, or even at least 90% or 95% or 99% of the incubation period, at least 50%, or even at least 80% or at least 90% of the supports are placed against the bottom of the enclosure. Preferably, during at least 50%, or even at least 80%, or even at least 90% or 95% or 99% of the incubation period, the culture medium is static. During step 120, the culture medium can be renewed. Thus, the incubation period can be interspersed with phases of modification of the culture medium. However, the duration of such phases is preferably limited to a few % of the static incubation time.

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

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

[0088] The invention is particularly suitable for the use of enclosures having a bottom with a large surface area. This makes it possible to take greater advantage of the 3D arrangement of the supports against the bottom, some of them straightening up as they move away from the bottom.

[0089] An advantage of the invention is that the incubation can be carried out without stirring the culture medium, which avoids the drawbacks linked to stirring, previously described, while allowing the use of simple enclosures, without stirring means. The invention thus makes it possible to use enclosures having a high surface area to height ratio. Different enclosures, of low height, typically less than 10 cm or 5 cm, can be stacked, a 3D structuring of the supports occurring at the bottom of each of them.

Claims

Claims

1. A method of culturing cells, excluding embryonic stem cells of human origin, the method comprising: a. arranging, in an enclosure (10), an initial quantity of cells, a culture medium, and supports (1), the enclosure extending between a bottom and an upper end; b. incubation, for an incubation period, during which the cells grow on the supports; c.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, according to a characteristic dimension less than 20 mm, the characteristic dimension corresponding to the largest diameter or to 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 duration of the incubation, at least 50% of the supports are arranged, by gravity, in contact with the bottom of the enclosure.

2. Method according to claim 1, in which during at least 80% or 90% of the duration of the incubation, at least 80% of the supports are arranged, by gravity, in contact with the bottom of the enclosure.

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

4. Method according to any one of the preceding claims, in which during step b), supports are moved spontaneously and progressively under the effect of the development of the cells, straightening up 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. The method of 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 curved. Method according to any one of the preceding claims, in which each support is formed from at least one mineral or organic material chosen from: sol-gel material, glass, polymer, plastic, ceramic, silicon, metal. Method according to any one of the preceding claims, in which 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 preceding claim, wherein the density of each support is less than 3 g / cm3 or less than 2.5 g / cm2. A method according to any preceding claim, wherein during incubation the enclosure is configured such that the bottom is below the upper end, relative to a vertical axis (Z).

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