Microcarriers for cell culture and microcarrier manufacturing process

Abstract

A process for forming microcarriers (1) comprising: forming liquid droplets (12) from a sol-gel solution (2); depositing the droplets, in liquid form, onto a first support (10), preferably hydrophobic; deforming the droplets deposited on the first support (10); solidifying the droplets by gelation and drying, so as to form solid microcarriers; e) extracting the solidified microcarriers from the first support (10). Figure 1A

Description

Title of the invention: Microcarriers for cell culture and method of manufacturing microcarriers Technical field

[0001] The technical field of the invention is the production of microcarriers of micrometric or millimetric size, conducive to the adhesion and development of cells. PREVIOUS ART

[0002] Some biological culture processes use culture supports, carrying cells. The supports are usually referred to as microcarriers. The microcarriers are placed 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 allows for regular renewal of the culture medium to which the cells are exposed.

[0003] Microcarriers intended to be placed in suspension in a culture medium 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 undergone a surface treatment, called surface functionalization, so as to promote cell grafting. This involves promoting cell attachment or adhesion. Microcarriers are frequently used for the culture of adherent cells. They then act as supports, on which the cells can develop and multiply. Functionalization makes it possible to apply a compound conducive to cell grafting.It can be a biological compound (for example collagen, gelatin, elastin, Poly D-Lysine, fibronectrin), or a molecule allowing the supply of positive or negative charges on the surface (for example cationic trimethyl ammonium or diethylaminoethyl).

[0004] When the cells are suspended in the culture medium, they develop on the surface of the microcarriers. They frequently form, on the surface of the latter, three-dimensional structures, usually called "agglomerates" consisting of a superposition of several layers of cells. Conglomerates then appear, under the effect of a grouping of agglomerates. This results in certain disadvantages: - cell lysis: cells at the center of the agglomerates have reduced access to the culture medium, and in particular to oxygen or nutrients present in the latter. Some may undergo lysis, which reduces the yield of the culture. - cell differentiation: in the case of pluripotent stem cell culture, the formation of agglomerates can promote uncontrolled cell differentiation. However, it is generally desirable to maintain cells in the stem cell state, or to obtain differentiation into a predetermined cell type, in a controlled manner. - contact inhibition: when seeking synthesis of molecules by cell culture, for example proteins, growth factors, antibodies, the formation of agglomerates reduces production. - difficulty in detaching: in certain applications, for example applications related to cell therapy, cell recovery is essential. This involves detaching the cells from the microcarriers, in order to obtain single cells (cells isolated from each other), and viable. The formation of agglomerates makes detachment difficult, as the recovered cells are not single cells.

[0005] Furthermore, the use of such microcarriers can cause other disadvantages. - surface functionalization uses chemical compounds, which can have an effect on cell development; - the need to use relatively significant agitation in the culture medium, in order to keep the microcarriers in suspension, which can lead to cellular stress.

[0006] The inventor has developed a new type of microcarriers, which do not have the disadvantages of ball-shaped microcarriers. The microcarriers designed by the inventor also have significant advantages as described below. Disclosure of the invention

[0007] A first object of the invention is a method of forming microcarriers, comprising: a. formation of liquid drops from a sol-gel solution; b. depositing the liquid drops on a first support, preferably flat, preferably hydrophobic; c. deformation of the drops deposited on the first support; d. solidification of the drops by gelation and drying, so as to form solid microcarriers; e. extraction of the solidified microcarriers from the first support.

[0008] According to one embodiment, the first support is hydrophobic. The first support is preferably flat. The first support is preferably rigid.

[0009] During step b), the liquid drops are preferably spaced apart from each other.

[0010] According to one embodiment, during step c), the deformation of the drops is a flattening. The method may be such that step c) comprises a flattening of the drops on the first support, the flattening being obtained spontaneously, during drying, during step d).

[0011] According to one embodiment, step c) comprises an application of a second support, preferably hydrophobic, on the drops, at a distance from the first support, such that the drops are interposed between the first support and the second support, the application of the second support resulting in a flattening of the drops between the two supports, the spacing between the first support and the second support conditioning the thickness of the microcarriers formed during step d). Preferably, the diameter of the drops formed during step a) and the spacing between the first support and the second support taken into account during step c) are adjusted as a function of a diameter or a larger diagonal, parallel to the first support, of the microcarriers resulting from step e).Step d) may comprise arranging an assembly, formed at least by the first support, the drops in the process of gelling and the second support, in an oven, so as to promote drying. The temperature of the oven may be between 30°C and 70°C. The oven may be placed under partial vacuum. Preferably, during step c), the spacing between the first support and the second support is between 30 μm and 5 mm.

[0012] Preferably, the second support is a planar support. Preferably, the second support is hydrophobic. The second support is preferably rigid.

[0013] During step a), the drops may be formed at a distance from the first support, the distance preferably being less than 10 mm. During step a), the drops may be formed in contact with the first support.

[0014] During step a), the drops formed may have a diameter of between 100 nm and 2 mm.

[0015] According to one embodiment, the first support comprises first parts and at least one second part, the first parts being less hydrophobic than each second part, each first part being bypassed by a second part, the second part forming a closed contour around said first part, such that during step a), the drops are deposited on each first part, and extend, along each first part, to the contour of said first part. According to one possibility, each second part is hydrophobic and each first part is less hydrophobic than each second part which bypasses it. According to one possibility, each second part is hydrophobic and each first part is hydrophilic.

[0016] According to one embodiment, the first support comprises first parts and at least one second part, the first parts being hollow relative to each second part, each first part being surrounded by a second part, said second part forming a contour, taking the form of an edge, closed around said first part, such that during step a), the drops are deposited on each first part, and extend, along each first part, to the contour of said first part.

[0017] The sol-gel solution may comprise a functionalizing compound, conducive to the formation of a grafting agent on the surface of the microcarriers, the grafting agent promoting grafting with a predetermined biological species or a predetermined chemical species. The predetermined chemical species may be a molecule. The predetermined biological species may be a cell, or a protein, or an antibody. The functionalizing compound may be mineral or organic. The sol-gel solution may comprise different functionalizing compounds.

[0018] A second subject of the invention is a microcarrier, in particular obtained by implementing a method according to the first subject of the invention. The microcarrier may comprise a first flat surface and a second surface, the first flat surface and the second surface being connected to each other by a lateral surface forming a border of the microcarrier. The second surface may be flat, and parallel to the first surface. The second surface may be curved, and extend opposite the first surface. The diameter, or the largest diagonal, of the first flat surface and / or the second surface is preferably less than 5 mm or less than 1 mm, and preferably greater than 5 μm. Preferably, the thickness of the microcarrier, corresponding to the distance between the first flat surface and the second surface, is less than 1 mm.

[0019] A third object of the invention is a use of a microcarrier according to the second object of the invention for cell culture, the microcarrier being intended to be placed in suspension in a biological medium, for example a culture medium.

[0020] 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

[0021] [Fig.lA] [Fig.lA] represents an example of a microcarrier according to the invention.

[0022] [Fig.lB] [Fig.lB] shows another example of a microcarrier according to the invention.

[0023] [Fig.lC] [Fig.lC] shows another example of a microcarrier according to the invention.

[0024] [Fig.2A] [Fig.2A] illustrates a step of forming drops on a first support, preferably hydrophobic.

[0025] [Fig.2B] [Fig.2B] shows schematically an apposition of a second support, preferably hydrophobic, facing the first support.

[0026] [Fig.2C] [Fig.2C] is a diagram illustrating the definition of a contact angle.

[0027] [Fig.2D] [Fig.2D] shows schematically a flattening of a drop during a drying.

[0028] [Fig.3] [Fig.3] shows schematically the main stages of a process for forming microcarriers.

[0029] [Fig.4A]

[0030] [Fig.4B]

[0031] [Fig.4C] Figures 4A to 4C show a deformation of a drop according to a particular embodiment.

[0032] [Fig.4D] [Fig.4D] shows examples of microcarriers obtained by the embodiment shown schematically in Figures 4A to 4C.

[0033] [Fig.4E] [Fig.4E] shows a diagram of a first substrate structured between hydrophobic parts delimited by highly hydrophobic zones.

[0034] [Fig.5] [Fig.5] shows an example of cells having developed on microcarriers. PRESENTATION OF SPECIAL EMBODIMENTS

[0035] [Fig. 1A] shows an example of a microcarrier according to the invention. The microcarrier comprises two plane surfaces, or considered as such, opposite one another. Thus, the microcarrier has a first plane surface, or substantially plane surface S1 and a second plane surface, or substantially plane surface S2. The first plane surface and the second plane surface are parallel to one another, or substantially parallel to one another.

[0036] 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°.

[0037] By substantially flat surface is meant flat, admitting surface elements having a local flatness defect, within a limit of ± 10° relative to the rest of the surface.

[0038] The first surface Si has a diameter ¢, or a larger diagonal, less than or equal to 20 mm, and preferably less than or equal to 10 mm, and preferably less than or equal to 1 mm. The same applies to the second surface S2. The diameter <e>is preferably greater than 5 pm or 10 pm. The diameter <e>is preferably between 50 pm and 1 mm, and more preferably between 100 pm and 1 mm. The first surface Si and the second surface S2 extend parallel to a main plane PXY. The microcarrier 1 comprises a lateral surface S3, extending between the first surface Si and the second surface S2. The lateral surface S3 extends with a thickness e around a transverse axis Z perpendicular to the main plane PXy-

[0039] [Fig. 1 A] corresponds to a preferred embodiment, according to which the microcarrier 1 has a cylindrical geometry of revolution. The first surface Si and the second surface S2 have a circular shape, of revolution, the lateral surface S3 being an annular surface. Other cylindrical configurations are possible, for example a cylinder with a polygonal base, for example in the shape of a quadrilateral, as shown in [Fig.lB].

[0040] Whatever the configuration, the thickness e of the microcarrier 1 is preferably: - less than one fifth of the diameter (or of the largest diagonal) - and preferably greater than one tenth, and preferably one twentieth of the diameter (or of the largest diagonal) ¢.

[0041] When the microcarrier is cylindrical in revolution, due to this small thickness, such a microcarrier takes the form of a thin disc. The thickness and the diameter are adjusted so as to avoid breakage of the microcarriers.

[0042] Thus, whatever the configuration, the microcarrier is essentially in the form of two surfaces Si and S2, extending one facing the other, making the lateral surface S3 negligible. The two surfaces Si and S2 may in particular be flat or substantially flat.

[0043] Preferably, the microcarrier is transparent.

[0044] The inventor chose such a geometry because of the advantages it provides: - an increased specific surface area, compared to the ball microcarriers usually used in the prior art. The specific surface area corresponds to the surface area available per unit of mass: • For a spherical glass microcarrier with a diameter between 160 pm and 180 pm, the specific surface area varies between 340 cm2 / g and 360 cm2 / g. • For a cylindrical microcarrier of revolution, made of glass, the specific surface area depends on the thickness e. For thicknesses of 5 pm, 10 pm and 20 pm, the specific surface area reaches 3338 cm2 / g, 1722 cm2 / g and 888 cm2 / g respectively. On this type of microcarrier, the available surface area corresponds to the cumulative total of the flat surfaces Si and S2. The higher the specific surface area, the higher the quantity of cells that can be cultivated, per unit mass of microcarrier. - better flotation ability, due to the high specific surface area, providing high lift. A consequence is that with microcarriers according to the invention, obtaining a suspension in the culture medium does not require as much agitation as using bead microcarriers. This significantly reduces the stress of the cells growing on the surface of the microcarriers, due to increased lift. Indeed, the impact of agitation on cell development has already been described, for example in the publication Wang. Y "Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation, J. Cell. Mol. Med 17(7), pp 823-832 or in the publication Yeats, A. "Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture Systems", Biochim Biophys Acta, 1830(2), pp 2470-2480. The inventor considered that slower agitation allows for better stability of the cellular environment.Reducing agitation also limits the risk of collision between multiple microcarriers suspended in the same culture medium. This also limits the hydrodynamic stress to which the cells are subjected.

[0045] Other advantages will be described in connection with the experimental tests carried out and described subsequently.

[0046] The microcarriers 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] In the presence of water, a network of oxides is formed, by means of hydrolysis-condensation reactions, trapping the organic solvent, so as to form a gel. The latter then undergoes drying, to remove the organic solvent present in the gel. The drying can be of the evaporative type, at a pressure lower than or equal to atmospheric pressure, so as to form a dry gel, usually designated by the term xerogel, in the form of a monolithic solid.

[0048] 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 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 can be an alkyl group, for example containing 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.

[0049] 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.

[0050] The sol-gel solution may also comprise a catalyst, and / or water, or compounds making it possible to act on the porosity, for example a surfactant.

[0051] 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 promotes grafting of a cell, of a predetermined type. The grafting agent may then be collagen, or polylysine, or a milk protein. However, due to regulatory or quality control constraints, it is sometimes preferable to avoid molecules of animal origin.A functionalization compound containing an epoxy function can then be used, the latter being 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. An amine function can also be integrated into the sol-gel, by means of a compound of the APTES type (3-aminopropyl-triethoxysilane). Such a compound allows the formation of positive charges on the surface of the microcarriers, which promotes the attachment of cells with negative surface charges. An amine function is conducive to the formation of peptide bonds with the amino acids of the cell wall.

[0052] The possibility of adding a functionalization compound in the sol-gel solution constitutes an interesting advantage, because this avoids carrying out post-manufacturing functionalization, as in the beads of the prior art. This makes it possible to manufacture microcarriers specific to a predefined application, taking into account the element chemical or biological intended to attach to the microcarriers, and / or the environment in which the microcarrier is intended to be placed.

[0053] A first example of a method for manufacturing a microcarrier, using a sol-gel process, is now described, in connection with Figures 2A to 2B. The main steps are shown in [Fig. 3].

[0054] Step 100: formation of drops

[0055] A sol-gel solution 2, as previously described, is introduced into a dispenser 3, making it possible to form drops 12, and preferably calibrated drops. The drops are preferably microdrops, the volume of which is between 5.10 10 nl and 15 μl.

[0056] The diameter of the drops formed is preferably between 100 nm and 5 mm, or between 100 nm and 1 mm.

[0057] The distributor 3 is arranged at a distance from or in contact with a first support. The first support 10 is preferably flat and preferably hydrophobic. In this example, the first support is a flat support, which corresponds to a preferred embodiment: it is a rigid plate. The hydrophobic nature of a material can be characterized by a contact angle θ, as shown in [Fig.2C]. The contact angle θ is measured in the presence of a drop of the sol-gel solution used. Preferably, the contact angle θ is between 70° and 150°. This makes it possible to avoid excessive spreading of the drops on the first support. The material forming the first support 10 can for example be glass, or polypropylene, or Teflon, or silicon. Preferably, the first support 10 is hydrophobic or has previously undergone a hydrophobic treatment.

[0058] The distance d between the dispenser and the first support 10 is preferably less than 10 cm. The drops can be formed in contact with the first support 10. The closer the distance, the better the accuracy in locating the drops.

[0059] During step 100, the drops formed on the first support 10 are spaced apart from each other. The spacing between two adjacent drops is sized such that during the flattening of the drops, resulting from the implementation of step 110, the latter remain spaced apart from each other.

[0060] The fact that the first support 10 is hydrophobic makes it possible to avoid excessive spreading of the drops 12.

[0061] Step 110: Flattening the drops

[0062] Following the formation of the drops, a second support 20, preferably hydrophobic, is applied parallel to the first support 10. The second support 20 is preferably flat and preferably rigid. The second support 20 is applied opposite the first support 10, being spaced from the latter by a spacing e depending on the thickness e of the microcarriers that it is desired to form. Taking into account a shrinkage occurring during drying (see step 120), the spacing e between the first support 10 and the second support 20 is greater than the thickness e of the microcarriers 1 resulting from the implementation of the method. The spacing e between the first support 10 and the second support 20 can be ensured by placing spacers 30 between the first support 10 and the second support 20. The spacing e between the first support 10 and the second support 20 can be between a few μm, for example 5 μm, and 5 mm or, preferably, between 50 μm and 2 mm or 1 mm.

[0063] The application of the second support 20 leads to a flattening of the drops 12. After their application to the first support 10, the drops 12 quickly form a gel. It is therefore preferable for the flattening to be carried out before the gelling is too advanced, as soon as all the drops 12 have been formed. Also, step 110 is implemented as quickly as possible after step 100. The solvent used in the sol-gel composition can be chosen to be of low volatility, so as to be able to delay the application of the second support 20. This avoids drying and premature gelling of the drops which would occur before the application of the second support 20. It is also possible to lower the temperature or increase the pressure to be able to delay the gelling of the drops deposited on the first support 10.

[0064] Step 120: gelation and drying

[0065] During gelling and drying, the assembly 40 formed by the first support 10 and the second support 20 is preferably placed in an oven, so as to finalize the gelling and facilitate drying of the gel. The temperature of the oven may be, for example, between 30°C and 80°C. The oven may be placed at a pressure lower than atmospheric pressure, for example at a pressure between -200 mbar and -970 mbar relative to atmospheric pressure. This significantly accelerates drying. The duration of the passage in the oven may be between 10 minutes and 24 hours.

[0066] During drying, the gel resulting from each drop solidifies, so as to form a solid monolithic microcarrier 1. Thus, each drop 12 deposited during step 100 results in the formation of a solid monolithic microcarrier 1.

[0067] During drying, the microcarriers undergo shrinkage, which is a known phenomenon in the sol-gel field. Thus, during drying, the thickness e of each microcarrier 1 decreases, the reduction being for example 50% when the thickness is 100 μm or 50 μm. Also, following drying, the microcarriers 1 are in contact with only one support, for example the first support 10 on which they rest by gravity. Some microcarriers may remain attached to the second support 20. When the second support 20 is hydrophobic, the latter are easily removed.

[0068] The fact that each support is hydrophobic facilitates a loss of contact between each microcarrier and the support facing it, during shrinking. The facing support is the support opposite the support supporting, by gravity, the microcarriers in formation. Indeed, in the absence of hydrophobic treatment, bonds, for example OH bonds, can form between the gel and the supports. This leads to a risk of breakage during loss of contact, when the microcarrier shrinks under the effect of drying. The hydrophobic treatment of each support limits the risk of breakage.

[0069] Step 130: recovery of microcarriers

[0070] Following drying, the microcarriers 1 can be easily separated from the first support 10 or from the second support 20, due to the hydrophobic nature of the latter. During recovery, the microcarriers 1 can be collected on a recovery support, preferably flexible, for example a cloth. This may be a porous nylon filter. The porosity is optimized to retain the microcarriers 1 while allowing the elimination of debris, for example residues of microcarriers having broken during the implementation of the method, the latter passing through the recovery support. For example, when the diameter (or the largest diagonal) of the microcarriers is equal to 600 μm, the particle size of the recovery support can be 400 μm. When the diameter of the microcarriers is 200 μm, the particle size can be 150 μm.

[0071] The fact that the first support 10 is hydrophobic makes it possible to avoid the formation of OH bonds between the microcarriers, resulting from the implementation of the method, and the first support 10. This facilitates the recovery of the microcarriers.

[0072] Step 140: washing

[0073] The microcarriers recovered during step 130, arranged on the recovery support, are washed, for example by a bath in a washing solution making it possible to eliminate residual acids present in the sol-gel solution or any unreacted precursors. The washing solution may be an aqueous solution, for example an aqueous solution comprising 50% by mass of isopropanol. The method may successively comprise several baths, for example two or three successive baths.

[0074] Step 150: Post-wash drying

[0075] Following step 140, the microcarriers are dried. Drying may be carried out at room temperature or at a higher temperature, for example up to 100°C or above. The drying temperature may be lowered if a partial vacuum is formed around the microcarriers. During drying, the microcarriers may be placed on the recovery support and the assembly is placed in an oven.

[0076] According to one possibility, the drying can be carried out by applying different successive pressure levels in the oven, for example by gradually lowering the pressure. This helps reduce drying time. The temperature is, for example, 50°. Drying time depends on the temperature and pressure conditions. It can be between 10 minutes and 5 hours, or even longer.

[0077] According to a variant of the first embodiment, the spacers 30 arranged between the two supports during step 110 are compressible. The spacers are sized such that when the second support 20 is affixed facing the first support 10, each drop 12 is not in contact with the second support 20. See [Fig.4A]. During step 110, pressure is exerted on the second support 20, so as to temporarily bring it closer to the first support 10. The second support 20 then comes into contact with each drop 12. See [Fig.4B]. When the pressure is released, the second support 20 moves away from the first support 10. See [Fig.4C]. According to this variant, step 110 corresponds to a deformation of the drop 12. When the pressure is released, the drop 12 remains in contact with the second support 20, by surface tension of the liquid. Thus, when the pressure is released, the drop 12 stretches along the transverse axis Z.This results in a particular microcarrier shape, comprising a thinning li extending between two ends 12. When the microcarriers are cylindrical in revolution, the shape resembles that of an hourglass.

[0078] [Fig.4D] is a photograph of hourglass-shaped microcarriers 1 obtained by implementing such a variant.

[0079] According to a second embodiment, the drops 12 are deposited on the first support 10, as described in connection with step 100 of the first embodiment. Cf. [Fig.2A].

[0080] Unlike the first embodiment, during step 110, the flattening of the drops 12 does not result from the application of a second support 20. The flattening of the drops is spontaneous. It is carried out in particular progressively, during drying. See [Fig.2D]. This makes it possible to obtain microcarriers 1 whose thickness is between a few μm, for example 2 μm or 3 μm, up to several μm, or even hundreds of μm. This embodiment is particularly suitable for obtaining microcarriers 1 whose thickness is thin, typically less than 30 μm, or even less than 10 μm. This makes it possible to have microcarriers 1 having good buoyancy.

[0081] The geometry of the microcarriers 1 obtained according to the second embodiment is not as cylindrical as that of the microcarriers obtained according to the first embodiment. In particular, the face opposite the face resting on the support 10 may be slightly curved.

[0082] According to this embodiment, the drying and gelling step 120 can be carried out in the open air, without passing through an oven.

[0083] The second embodiment is particularly suitable for obtaining microcarriers of low thickness, for example less than 10 μm. Generally speaking, the second embodiment makes it possible to obtain a very high diameter / thickness ratio, which is conducive to good flotation. The first embodiment allows better control of the flatness of the microcarriers. It is particularly suitable for microcarriers of large diameters, typically greater than 100 μm.

[0084] Steps 130 to 150 of the second embodiment are identical to those described in connection with the first embodiment.

[0085] The second embodiment is preferably implemented by lowering the concentration of precursors in the sol-gel solution compared to the first embodiment. Preferably, in the second embodiment, the contact angle θ of each drop is lower than in the first embodiment, this to promote spreading of the drop on the first support 10. To lower the contact angle θ, it is possible to use a solvent having a specific tension lower than that of water, for example an alcohol of the ethanol or isopropanol type. It is also possible to add a surfactant to the sol-gel composition.

[0086] Whatever the embodiment, the first support 10 can be structured so that under the effect of the flattening, each drop 12 spreads, on the first support 10, according to a predetermined shape, according to the structuring of the first support. An example of such a support is shown in [Fig.4E]. The structuring of the support 10 makes it possible to form first parts 10i, intended to receive the drops, and second parts 102. Each first part is delimited by a second part, such that a second part forms a closed contour around each first part.

[0087] According to one possibility, the first support 10 may be microstructured, so as to comprise first hollow parts 10i of predetermined shapes, for example quadrilateral-type polygons or hexagons, and delimited by an edge, formed by a second part 102. The drops are deposited on each first part 10i, and extend as far as the edge delimiting said first part. Microcarriers such as those shown in FIGS. 1B or 1C are thus obtained, the shape of which corresponds to the shape of each first part 10,.

[0088] According to another possibility, the structuring of the first support 10 forms first parts 10i less hydrophobic than each second part 102. The hydrophobicity of a material is a concept known to those skilled in the art and can be determined by measuring the contact angle. The higher the latter, the more hydrophobic the material is with respect to the solution forming the drop. The first parts 10i may be slightly hydrophobic or hydrophilic. Each second part 102 is preferably hydrophobic. By slightly hydrophobic is meant a part in which the contact angle θ is lower by at least 5° or 10°, compared to the contact angle on the most hydrophobic part.

[0089] According to this embodiment, each first part 10i is delimited by a second part 102, such that during step 110, the drop spreads along the first part on which it was deposited. This variant allows better control of the shape of the microcarrier. It also makes it possible to obtain microcarriers whose shape is controlled by the structuring of the first support 10. Experimental tests.

[0090] A first series of tests was carried out, the experimental conditions being described below: - Dispenser: Vermes MDV 3200 A dosing valve equipped with a Vernies NI 1-150 nozzle to form sol-gel microdrops. The dispenser was mounted on a Janome 200 mm / 200 mm tri-axis robot, allowing the dispenser to move parallel to a first support 10. - First support 10: glass plate 200 mm x 170 mm, thickness 6 mm, previously hydrophobically treated by exposure to dichlorodimethylsilane. - Second support 20: identical to the first support. - Spacing e between the first support 10 and the second support: 140 pm. The spacing is obtained by placing spacers 30 of the adhesive tape type. - Precursor: Tetramethoxysilane 98% (Alfa Aesar). - Solvent: Isopropanol Technical (Alfa Aesar). - Catalyst: 6M HCl (Sigma Aldrich). - Mineral functionalization compound: Hydroxyapatite Ca5(OH)(PO4)3 (Sigma Aldrich). - Organic functionalization compound: Bovine collagen type 1: 10 mg / ml (Vornia Ltd).

[0091] 10 ml of tetramethoxysilane was poured into a 50 ml beaker, kept stirring at room temperature. A solution of 5 ml of deionized water was prepared to which 0.1 ml of HCl was added. The solution was slowly poured into the beaker containing the tetramethoxysilane (10 ml). Since the hydrolysis of tetramethoxysilane is exothermic, the water + HCl mixture is poured at a rate of 2.5 ml / min. 20 ml of water was then added to the beaker. 3 ml of a hydroxyapatite solution was added, as well as 4 ml of collagen solution. The hydroxyapatite solution was obtained by dissolving 200 mg of hydroxyapatite powder in 2.5 ml of deionized water and 0.5 ml of HCl, HCl facilitating dissolution of the hydroxyapatite powder.

[0092] The sol-gel solution, the preparation of which is described in the previous paragraph, was introduced into a syringe of the metering valve, the adjustment parameters of which are as follows: rising: 0.25 ms; falling: 0.35 ms; open time: 2 ms; needle lift: 30; delay: 7 ms; air pressure: 0.5 bar. These parameters are adjusted on a case-by-case basis by those skilled in the art.

[0093] A first hydrophobic glass plate, acting as the first support 10, was placed on the aforementioned robot, the latter being programmed to move parallel to the first glass plate, describing lines spaced apart from each other by a distance of 3 mm. The drops are formed at a distance of 2 mm from the hydrophobic plate. The spacing between two adjacent drops, placed on the same line, was 2 mm. The volume of each drop was 500 μl.

[0094] An adhesive film, acting as a spacer 30, was placed at the four corners of the first glass plate. The thickness of the adhesive thread was 0.14 mm.

[0095] After all the drops have been formed. The number of drops formed was 5000. A second area of ​​hydrophobic glass, acting as a second support 20, was gently placed above the first glass plate, in contact with the adhesive film, so as to flatten the drops, as described in connection with step 110.

[0096] The two glass plates, confining the gelling / drying drops, were then placed in an oven at 50°C. The temperature was then maintained at 50°C and the pressure was gradually lowered according to 8 pressure levels, respectively -200 mBar, -400 mBar, -500 mBar, -600 mBar, -700 mBar, -800 mBar, -900 mBar and -970 mBar relative to atmospheric pressure. The duration of each level was 15 minutes. Solid microcarriers were thus formed between the two glass plates, resulting from the gelling and drying of the beads.

[0097] After drying, the plates were separated and the microcarriers were recovered on a porous nylon filter, the pores measuring 400 μm in diameter. The filter, retaining the microcarriers, was placed in a crystallizer, comprising isopropanol diluted to 50% (mass fraction) in deionized water, in order to carry out a wash. The washing duration was 2 hours. The washing was repeated three times. Following the washes, the filter retaining the microcarriers was dried in a study at 100°C for 1 hour 30 minutes.

[0098] The microcarriers resulting from the implementation of the method had a thickness of 70 μm and a diameter of 700 μm.

[0099] Microcarriers have been used for cell culture applications. For example, a culture of human adipose-derived stem cells (ASCs) was carried out. [Fig. 5] shows a photograph of cultured microcarriers after 72 hours of static culture in a culture medium. of the "mesenchymal stem cell growth medium 2" type (Promocell). The development of cells, on the microcarriers, in a monolayer was confirmed by microscopy.

[0100] During these tests, other advantages conferred by the microcarriers according to the invention were observed: - a flat culture surface, conducive to the development of cells under the same conditions as in culture supports such as culture flasks or Petri dishes commonly used. These are different culture conditions from those resulting from bead-shaped microcarriers. Indeed, on the latter, the culture surface is highly curved. On bead-shaped microcarriers, cells develop by forming aggregates. It is considered that cells tend to favor the establishment of bonds between cells to the detriment of bonds with the surface of the bead. On microcarriers with flat surfaces, cells develop by forming a monolayer, because they favor bonds with the flat surface of the microcarrier. - easy recovery of cells cultured on each microcarrier. The cells grow by forming a monolayer, which facilitates their detachment from the microcarriers. The separation of cells from each microcarrier can be facilitated by an enzyme, for example TrypLE (Microfischer). - recovery of individual (or unitary) cells, which can be used to be reseeded for a new culture. - behavior, after culture, close to cells grown in culture flasks or Petri dishes. Tests have shown that after recovery of the cells and their reseeding in a culture flask, the cells extend in a fibroblastic form, characteristic of mesenchymal stem cells. Their capacity for osteogenic, chondrogenic and adipogenic differentiation remains intact. The cells remain pluripotent stem cells, unlike what can be observed when the cells form agglomerates. In addition, a rate of sensescent cells similar to that obtained during 2D culture on conventional flasks was observed; - a higher rate of viable cells than that obtained using bead-shaped microcarriers, in particular due to better access to nutrients and oxygen from the culture medium; - better cell productivity, due to the fact that the cells are cultured in the form of monolayers, without the formation of agglomerates. thus avoids the disadvantage of contact inhibition described in connection with art prior.

[0101] The invention may, without limitation, be implemented for applications related to cell culture.< / e> < / e>

Claims

Claims

1. A method of forming microcarriers (1), comprising: a. forming liquid drops (12) from a sol-gel solution (2); b. depositing the liquid drops on a first support (10); c. deforming the drops deposited on the first support (10); d. solidifying the drops by gelling and drying, so as to form solid microcarriers; e. extracting the solidified microcarriers from the first support (10).

2. The method of claim 1, wherein the first support (10) is hydrophobic.

3. A method according to claim 1 or claim 2, wherein in step c), the deformation of the drops is a flattening.

4. Method according to claim 3, in which step c) comprises a flattening of the drops (12) on the first support (10), the flattening being obtained spontaneously, during drying, during step d).

5. Method according to any one of claims 1 to 3, in which step c) comprises an application of a second support (20), preferably hydrophobic, on the drops (12), at a distance from the first support, such that the drops are interposed between the first support and the second support, the application of the second support resulting in a flattening of the drops between the two supports, the spacing (e) between the first support and the second support conditioning the thickness of the microcarriers formed during step d).

6. Method according to claim 5, wherein the diameter of the drops formed during step a) and the spacing between the first support (10) and the second support (20) taken into account during step c) are adjusted according to a diameter or a larger diagonal, parallel to the first support, of the microcarriers resulting from step e).

7. A method according to claim 5 or claim 6, wherein step d) comprises arranging an assembly (40), formed at least by the first support (10), the drops (12) being

8.

9.

10.

11.

12.

13.

14.

15.

16. gelation and the second support (20), in an oven, so as to promote drying. The method of claim 7, wherein the oven temperature is between 30°C and 70°C. A method according to claim 7 or claim 8, wherein the oven is placed under partial vacuum. Method according to any one of claims 5 to 9, wherein during step c), the spacing between the first support (10) and the second support (20) is between 30 pm and 5 mm. Method according to any one of the preceding claims, wherein during step a), the drops (12) are formed at a distance (d) from the first support (10), the distance being less than 10 mm. Method according to any one of claims 1 to 10 in which during step a), the drops are formed in contact with the first support (10). Method according to any one of the preceding claims, in which during step a), the drops (12) formed have a diameter of between 100 nm and 2 mm. Method according to any one of the preceding claims, in which the first support (10) comprises first parts (10i) and at least one second part (102), the first parts being less hydrophobic than each second part, each first part being bypassed by a second part, the second part forming a closed contour around said first part, such that during step a), the drops are deposited on each first part (10i), and extend, along each first part, to the contour of said first part. The method of claim 14, wherein: - each second part (102) is hydrophobic and each first part (100) is less hydrophobic than the second part which surrounds it; - or each second part is hydrophobic (102) and each first part (10i) is hydrophilic. A method according to any one of claims 1 to 13, wherein the first support (10) comprises first parts (10i) and at least one second part (102), the first parts being hollow relative to each second part, each first part being bypassed by a second part, said second part forming a contour, taking the form of an edge, closed around said first part, such that during step a), the drops are deposited on each first part (10i), and extend, along each first part, to the contour of said first part.

17. Method according to any one of the preceding claims, in which the sol-gel solution comprises a functionalizing compound, conducive to the formation of a grafting agent on the surface of the microcarriers, the grafting agent promoting grafting with a predetermined biological species or a predetermined chemical species.

18. The method of claim 17, wherein the functionalizing compound is inorganic or organic.

19. A method according to claim 17 or claim 18, wherein the sol-gel solution comprises different functionalizing compounds.

20. Microcarrier (1), obtained by implementing a method according to any one of claims 1 to 19.

21. Microcarrier (1), according to claim 20, comprising a first planar surface (Si) and a second surface (S2), the first planar surface and the second surface being connected to each other by a lateral surface (S3) forming a border of the microcarrier.

22. Microcarrier (1) according to claim 20, comprising a first flat surface (Si) and a second surface (S2) curved relative to the first flat surface, the first flat surface and the second surface being connected to each other by a lateral surface (S3) forming a border of the microcarrier.

23. Microcarrier according to any one of claims 20 to 22, wherein: - the diameter, or the largest diagonal, of the first planar surface is less than 5 mm or less than 1 mm, and greater than 5 μm, - and / or the diameter, or the largest diagonal of the second planar surface is less than 5 mm or less than 1 mm, and greater than 5 μm.

24. A microcarrier according to claim 23, wherein the thickness of the microcarrier, corresponding to the distance between the first planar surface and the second surface, is less than 1 mm.

25. Use of a microcarrier according to any one of claims 20 to 24 for the culture of cells, excluding embryonic stem cells of human origin, the microcarrier being intended to be placed in suspension in a culture medium.

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