Magnetic media for cell culture, and media-manufacturing process

Abstract

The invention relates to a process for forming objects (1) that are intended to be placed in a culture medium, the process comprising: a) forming liquid droplets (12) from a sol-gel solution (2); b) depositing the liquid droplets on a receptacle (10); c) deforming the droplets deposited on the receptacle (10); d) solidifying the droplets by gelling and drying, so as to form solid objects; e) extracting the solidified objects from the receptacle (10); the process being characterized in that the sol-gel solution comprises particles that are sensitive to a magnetic field.

Description

[0001] Description

[0002] Title: Magnetic supports for cell culture and method of manufacturing supports

[0003] TECHNICAL FIELD

[0004] The technical field of the invention is the production of magnetic supports of micrometric or millimetric size.

[0005] PREVIOUS ART

[0006] Some biological cultivation processes use culture media, which carry cells. The media are suspended in a culture medium, the aim being to grow the cells outside their original environment. The culture medium is usually subjected to moderate agitation. This allows the culture medium to be homogenized and the media to be suspended.

[0007] 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 undergone a surface treatment, called surface functionalization, in order to promote cell grafting. This involves promoting cell attachment or adhesion. Supports are frequently used for the culture of adherent cells. Cells can grow and multiply on them.

[0008] W02021140129 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, particularly monolayer cell culture, which is conducive to improved cell viability of the cultured cells and improved collection of these cells.

[0009] 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 method, such as 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, agitation is significant, which can lead to hydrodynamic stress. Generally, the larger the culture volume, the more vigorous the agitation of the medium is required 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 favorable culture conditions.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.

[0010] Additionally, the use of a propeller makes the bioreactor dedicated to a specific application, i.e., a particular culture medium and a predetermined cell type. Indeed, if you want to change the application, you must thoroughly clean the propeller to limit the risk of contamination. This is inconvenient and time-consuming. Some bioreactors have interchangeable propellers, but this makes them more complex.

[0011] After passing through the bioreactor, the cells are separated from the media by detachment, usually using a so-called detachment enzyme. The media resulting from the separation are collected by a filter. However, the filter may become clogged by the media.

[0012] During culture, some cell cultures are performed using perfusion, which involves continuously regenerating the culture medium. This method allows cultures to be performed at high cell concentrations, but requires continuous filtration of the culture medium to replace it with fresh culture medium. Media can clog filters, complicating the implementation of perfusion cultures.

[0013] US20160145600A1 describes the use of magnetic supports in the form of spherical microbeads, produced by encapsulating magnetic particles in a polymer matrix, for example based on alginate. However, the spherical shape of the particles does not confer high sensitivity to a magnetic field, unless intense magnetic fields are available. The use of such supports is limited to small volumes, for example tubes.

[0014] W02021140129 also describes the use of spherical magnetic microparticles. These are colloidal microparticles, with a diameter of less than 10 pm, or even 5 pm. In W02021140129, it is considered that the use of a flat support for cell culture is unfavorable, and that it is preferable to use spherical microparticles allowing the formation of three-dimensional cellular structures. The microparticles are formed from a thermoresponsive polymer. At the end of cell culture, the microparticles disperse in the liquid culture medium. A magnetic field is applied, following cell culture, to recover the microparticles. It is noted that the culture method described in W02021140129 uses a magnetic field only at the end of cell culture, for the purpose of recovering the spherical microparticles.

[0015] The inventor has developed a new type of support, particularly suitable for placement in bioreactors, so as to be exposed to the most homogeneous culture medium possible. The supports allow for simplified cell detachment.

[0016] STATEMENT OF THE INVENTION

[0017] A first aspect of the invention is a method for forming objects, intended to be arranged in a culture medium, the method comprising: a) formation of liquid drops from a sol-gel solution; b) depositing the liquid drops on a receptacle; c) deformation of the drops deposited on the receptacle; d) solidification of the drops by gelling and drying, so as to form solid objects; e) extraction of the solidified objects from the receptacle; the method being characterized in that the sol-gel solution comprises particles sensitive to a magnetic field.

[0018] Magnetically sensitive particles can be metal particles or magnets. They can contain or be made of ferromagnetic or paramagnetic material. The mass fraction of magnetically sensitive particles can be between 1% and 50%.

[0019] In step a), the viscosity of the sol-gel solution can be greater than 5.10 -3 Not.

[0020] The receptacle may be hydrophobic.

[0021] According to one possibility, during step c), the deformation of the drops is a flattening. Following the flattening, each drop then elongates along a longitudinal axis.

[0022] According to one possibility, step c) involves a flattening of the drops on the receptacle, the flattening being obtained spontaneously, during drying, during step d).

[0023] According to one possibility, step c) comprises an application of a plate, preferably hydrophobic, on the drops, at a distance from the receptacle, such that the drops are interposed between the receptacle and the plate, the application of the plate resulting in a flattening of the drops between the plate and the receptacle, the spacing between the receptacle and the plate conditioning the thickness of the objects formed during step d). According to one possibility,

[0024] - the receptacle comprises first parts and at least one second part;

[0025] - the first parts are less hydrophobic than each second part;

[0026] - each first part is bypassed by a second part, the second part forming an outline around said first part;

[0027] - the outline of each first part extends, parallel to a longitudinal axis, along a length, and perpendicular to the longitudinal axis, along a width less than the length;

[0028] - such that during step a), the drops are deposited on each first part, and extend, along each first part, to the outline of said first part.

[0029] According to one possibility:

[0030] - the receptacle comprises first parts and at least one second part;

[0031] - the first parts are hollow in relation to each second part;

[0032] - each first part being circumscribed by a second part, said second part forming an outline, taking the form of an edge, closed around said first part;

[0033] - the contour of each first part extends, parallel to a longitudinal axis, along a length, and perpendicular to the longitudinal axis, along a width less than the length; 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.

[0034] According to one possibility, during step b), drops are arranged close to each other, aligned along a longitudinal axis so that step c) involves coalescence of the drops arranged close to each other.

[0035] A second aspect of the invention is an object, obtained by implementing a method according to the first aspect of the invention. The object can in particular act as a support for cell culture.

[0036] According to one possibility, the object, along the longitudinal axis, extends along a length greater than a thickness defined perpendicular to a plane passing through the longitudinal axis and the lateral axis and / or the object extends, along the longitudinal axis, along a length at least 30% greater than the width. The thickness is preferably less than the width and the length. The thickness is preferably less than 30% or even 50% of the width or the length.

[0037] A third aspect of the invention is a method of cell culture, using at least one object according to the second aspect of the invention for the culture of cells, possibly excluding embryonic stem cells of human origin, the object acting as a cell culture support, comprising:

[0038] - (i) bringing the culture medium into contact with at least one cell;

[0039] - (ii) arrangement of the culture medium in an enclosure containing a culture medium;

[0040] - (iii) applying a magnetic field in the enclosure, the magnetic field forming field lines extending through the enclosure

[0041] - (iv) following step (iii), suspending each culture medium in a culture medium, contained in an enclosure.

[0042] Following step (iv), the method may comprise a displacement of the magnetic field lines in the enclosure, so as to obtain a displacement of the culture supports, in the culture medium.

[0043] According to one possibility, the magnetic field being produced by a magnet,

[0044] - the magnet is moved relative to the enclosure;

[0045] - and / or the magnet is an electromagnet carrying a current, the current being modified so as to generate a variation in the magnetic field in the enclosure.

[0046] According to one possibility, following step (iv), a modification of the culture medium or a variation in the temperature of the culture medium, so as to promote a detachment of cells cultured on each culture support. + induced magneto vibration.

[0047] According to one possibility, the modification of the culture medium includes:

[0048] - addition of an agent promoting a break in bonds between each cell and a support;

[0049] - and / or modification of the pH of the culture medium.

[0050] A fourth aspect of the invention is a method of cell culture, using at least one cell culture support, excluding embryonic stem cells of human origin, the method comprising:

[0051] - (i) bringing the culture medium into contact with at least one cell;

[0052] - (ii) arrangement of the culture support in an enclosure comprising a culture medium; the method being characterized in that the support comprises particles sensitive to a magnetic field, and that the support has, along a longitudinal axis, a dimension greater than the dimensions along another axis, the largest dimension forming a length of the support, the method comprising:

[0053] - (iii) applying a magnetic field in the enclosure, the magnetic field forming field lines extending through the enclosure

[0054] - (iv) following the previous step, suspending each culture medium in a culture medium, contained in an enclosure.

[0055] The culture medium may extend, along a lateral axis, perpendicular to the longitudinal axis, along a width, the longitudinal axis and the lateral axis forming a plane, and, perpendicular to said plane, along a thickness. Preferably, the thickness and the width are less than the length. Preferably, the thickness and / or the width are at least 30% or 50% less than the length. Preferably, the culture medium takes an essentially longitudinal shape, which gives it increased sensitivity to the magnetic field.

[0056] A fifth aspect of the invention is a bioreactor, comprising an enclosure configured to receive a cell culture medium, the bioreactor comprising means for applying a magnetic field through the enclosure, the enclosure comprising supports as described in connection with the aspects of the invention described above. The means for applying the magnetic field are configured to form magnetic field lines through the enclosure. Preferably, the means for applying the magnetic field are configured to allow movement of the field lines in the enclosure. The means for applying the magnetic field may be chosen from: permanent magnet and / or electromagnet.

[0057] Following step (iv), the method may include a modification of the magnetic field so as to induce a vibration of each culture medium inside the enclosure.

[0058] The invention will be better understood by reading the description of the exemplary embodiments presented in the remainder of the description, in conjunction with the figures listed below.

[0059] FIGURES

[0060] Figure 1A represents an example of a support according to the invention.

[0061] Figure 1B shows another example of a support according to the invention.

[0062] Figure 2A illustrates a step of forming drops on a receptacle, preferably hydrophobic.

[0063] Figure 2B shows a diagram of the application of a plate, preferably hydrophobic, facing the receptacle.

[0064] Figure 2C is a diagram illustrating the definition of a contact angle. Figure 3 schematizes the main steps of a support forming process.

[0065] Figure 4 shows a drop formation step, so as to form clusters of adjacent drops in a longitudinal direction.

[0066] Figure 5 shows a deformation of a droplet according to a particular embodiment.

[0067] Figure 6 shows a first substrate structured between hydrophobic parts delimited by highly hydrophobic zones.

[0068] Figure 7 illustrates the formation of field lines across a bioreactor, and the alignment of supports along the field lines.

[0069] Figure 8A shows a module comprising a bioreactor equipped with means for applying a magnetic field through it.

[0070] Figure 8B shows a bioreactor with different adjacent means of applying a magnetic field.

[0071] PRESENTATION OF SPECIAL EMBODIMENTS

[0072] Figure 1A shows an example of a support according to the invention. The support comprises two plane surfaces, or considered as such, opposite each other. Thus, the support 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 each other, or substantially parallel to each other.

[0073] 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°.

[0074] By substantially flat surface is meant flat, admitting surface elements presenting a local flatness defect, within a limit of ± 10° relative to the rest of the surface.

[0075] The first surface Si has or is inscribed in 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 50 pm and 20 mm, and more preferably between 100 pm and 20 mm, or even more. It has been observed that the use of carriers with a diameter or inscribed in a diameter greater than or equal to 1 mm, or even 5 mm, confers greater sensitivity to the magnetic field.

[0076] The first surface Si and the second surface S2 extend parallel to a principal plane

[0077] PXY- The support 1 has 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-

[0078] Whatever the configuration, the thickness e of the support 1 is preferably: less than one fifth of the diameter (or of the largest diagonal)

[0079] - and preferably greater than one tenth, and preferably one twentieth of the diameter (or of the largest diagonal).

[0080] The thickness generally results from a compromise: it is sufficient to ensure a certain solidity to the support, but sufficiently low to confer good floating properties and sensitivity to the magnetic field.

[0081] Figure 1A corresponds to an embodiment, according to which the support 1 has a geometry of revolution. The first surface S1 and the second surface S2 have a circular or elliptical 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.

[0082] In the example shown, the support extends: parallel to a longitudinal axis X, along a length L;

[0083] - parallel to a lateral axis Y, perpendicular to the longitudinal axis, along a width l;

[0084] The length L is preferably strictly greater than the width l. For example, the length to width ratio is preferably greater than 1.2, or even 1.3, or even 1.5. Being longer than it is wide gives a support anisotropic behavior, by promoting alignment with magnetic field lines.

[0085] Thus, whatever the configuration, the support is essentially in the form of two surfaces Si and S2, extending one facing the other, the area of ​​the lateral surface S3 being negligible. The two surfaces Si and S2 can in particular be flat or substantially flat.

[0086] 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: of a molecular precursor of metal or metalloid, for example an organometallic compound or a metal salt; of an organic solvent; of water; of an acid or basic catalyst. of particles sensitive to a magnetic field.

[0087] A particle sensitive to a magnetic field includes, in particular, ferromagnetic particles. These may, for example, be particles containing iron, nickel, cobalt, or a material used in magnets, for example, neodymium. In this example, but not limited to, the particles are iron oxide particles with a diameter between 1 pm and 10 pm.

[0088] The mass fraction of magnetic field-sensitive particles in the solution is preferably between 1 and 50%, or between 5% and 40% or between 5% and 30% or between 5% and 20%.

[0089] In addition to the quantity of magnetic particles, the sensitivity of the particles to a magnetic field is conferred by the shape of the support. As described below, an elongated shape, along the longitudinal axis, increases the sensitivity of the support, which makes it possible to limit the quantity of magnetic particles added.

[0090] Preferably, the solution includes a thickening agent, capable of increasing the viscosity, for example chitosan. This may in particular be a polymer or another thickening agent. The objective of increasing the viscosity is to keep the magnetic or ferromagnetic particles in suspension during the manufacture of the supports. This prevents sedimentation of the particles within the sol solution, during gelation or even drying. The viscosity depends on the size and density of the particles and also on the duration of gelation and drying.

[0091] 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 remove the organic solvent present in the gel. 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 referred to as xerogel, in the form of a monolithic solid.

[0092] 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 may be for example a transition metal, a lanthanide: it may 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.

[0093] The metalloid element can be chosen from Si, Se, Te.

[0094] 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.

[0095] The molecular precursor is placed in an organic solution, for example an alcoholic solution. The organic solvent can be an aliphatic or aromatic monoalcohol, or a diol.

[0096] The sol-gel solution may also contain a catalyst, and / or water, or compounds that can act on porosity, for example a surfactant.

[0097] According to one embodiment, the sol-gel solution comprises a functionalizing 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.

[0098] Alternatively, the sol-gel solution contains a precursor of the 3-aminopropyltriethoxysilane type, usually referred to by the acronym APTES, or 3-aminopropyltrimethoxysilane, usually referred to 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 cells.

[0099] The possibility of adding a functionalization compound to the sol-gel solution constitutes an interesting advantage, because it avoids the need for post-manufacturing functionalization, as in the beads of the prior art. 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.

[0100] The sol-gel supports obtained have a density generally less than 2, and preferably less than 1.8. The density is preferably strictly greater than 1 and advantageously between 1 and 1.4 and even more advantageously between 1.02 and 1.04. Such a density provides good flotation of the supports in aqueous culture media.

[0101] Due to the action of the magnetic field, however, a low density is not essential. Indeed, under the action of the magnetic field, the supports can be kept in suspension in a bioreactor, even if they have a higher density. This is the reason why, although it is advantageous to use sol-gel technology, due to the ease of implementation, low density, or incorporation of active ingredients in the mass, the supports can be the result of another manufacturing process. For example, they can be glass or plastic supports, in which particles sensitive to the magnetic field have been previously incorporated. This type of support can be made by molding. It is preferable that the shape of the supports is not isotropic, so as to define a preferred axis of sensitivity with respect to the magnetic field.

[0102] Thus, regardless of the manufacturing method, the support preferably extends along a longitudinal axis, along a length greater than a width defined along a lateral axis perpendicular to the longitudinal axis. The lateral axis and the longitudinal axis define a plane. Perpendicular to the plane, the support extends along a thickness. The thickness and / or width are preferably less, by at least 10%, or even 20%, or even 30%, or even 40%, or even 50%, or even 60%, or even 70%, or even 80%, or even 90% of the length.

[0103] In addition to a sol-gel type material, the supports can be made of a material chosen, in a non-limiting manner, from synthetic polymer (epoxy) with magnetic particles and functionalization, polycaprolactone, polylactic acid, polyglycolic acid, natural polymer, alginate, chitosan, dextran, or metal oxides (TiO2, SiO2) or other mineral materials. A first example of a method for manufacturing a support, by sol-gel process, is now described, in connection with Figures 2A to 2B. The main steps are shown in Figure 3.

[0104] 100 drop formation

[0105] 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 can be microdrops, the volume of which is between 5.10 10 nl and 15 pl, or even > 1 or a few ml.

[0106] The diameter of the drops formed is preferably between 100 nm and a few centimeters.

[0107] When forming drops, it is preferable that the sol solution has sufficient viscosity, preferably greater than 10' 2 Pa-s (Pascal second) or greater than 10 1 Pa-s. The viscosity of the solution must prevent sedimentation of magnetic particles inside the droplet during gelation or even drying. The formation of drops from a sufficiently viscous sol solution limits or prevents sedimentation of particles sensitive to the magnetic field, contained in the sol, under the effect of gravity. The quantity of thickening agent can be determined experimentally as a function of the volume and geometry of the drops.

[0108] Such viscosity may be obtained by spontaneous gelation of the sol or by the addition of a viscosity promoting agent, for example a thickening agent as previously described.

[0109] The dispenser 3 is arranged at a distance from or in contact with a receptacle 10. The receptacle 10 is preferably flat and preferably hydrophobic. In this example, the receptacle is a flat plate, 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 Figure 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 receptacle. The material forming the receptacle 10 can for example be glass, or polypropylene, or Teflon, or silicon. Preferably, the receptacle 10 is hydrophobic or has previously undergone a hydrophobic treatment. The distance d between the dispenser and the receptacle 10 is preferably less than 10 cm.Drops can be formed upon contact with the receptacle 10. The closer the distance, the better the accuracy in locating the drops.

[0110] During step 100, the drops formed on the receptacle 10 are spaced apart from each other. The spacing between two adjacent drops is sized such that when the drops flatten, resulting from the implementation of step 110, the latter remain spaced apart from each other.

[0111] The fact that the receptacle 10 is hydrophobic makes it possible to avoid excessive spreading of the drops 12. flattening of the drops

[0112] Following the formation of the drops, a plate 20, preferably hydrophobic, is applied parallel to the receptacle 10. The plate 20 is preferably flat and preferably rigid. The plate 20 is applied facing the receptacle 10, being spaced from the latter by a spacing s depending on the thickness e of the supports that it is desired to form. Taking into account a shrinkage occurring during drying (see step 120), the spacing s between the receptacle 10 and the plate 20 is greater than the thickness e of the supports 1 resulting from the implementation of the method. The spacing s between the receptacle 10 and the plate 20 can be ensured by arranging spacers 30 between the receptacle 10 and the plate 20. The spacing s between the receptacle 10 and the plate 20 can be between a few pm, for example 5 pm, and 5 mm or, preferably, between 50 pm and 2 mm or 1 mm.

[0113] The application of the plate 20 leads to a flattening of the drops 12. After their application to the receptacle 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 carried out 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 plate 20. This avoids premature drying and gelling of the drops which would occur before the application of the plate 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 receptacle 10. gelling and drying

[0114] During gelling and drying, the assembly 40 formed by the receptacle 10 and the plate

[0115] 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.

[0116] During drying, the gel resulting from each drop solidifies, so as to form a solid monolithic support 1. Thus, each drop 12 deposited during step 100 results in the formation of a solid monolithic support 1.

[0117] During drying, the supports undergo shrinkage, which is a known phenomenon in the sol-gel field. Thus, during drying, the thickness e of each support 1 decreases, the reduction being for example 50% when the thickness is 100 μm or 50 μm. Also, following drying, the supports 1 are in contact with only one support, for example the receptacle 10 on which they rest by gravity. Some supports may remain attached to the plate 20. When the plate 20 is hydrophobic, these are easily removed.

[0118] The fact that each support is hydrophobic facilitates a loss of contact between each support and the receptacle 10 or the plate 20 facing it, during shrinking. 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 the loss of contact, when the support shrinks under the effect of drying. The hydrophobic treatment of the plates 10, 20 limits the risk of breakage. recovery of the supports

[0119] Following drying, the supports 1 can be easily separated from the receptacle 10 or the plate 20, due to the hydrophobic nature of the latter. During recovery, the supports 1 can be collected on a recovery means, preferably flexible, for example a cloth. This may be a porous nylon filter. The porosity is optimized to retain the supports 1 while allowing the elimination of debris, for example residues of supports 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 supports is equal to 600 μm, the particle size of the recovery means can be 400 μm. When the diameter of the supports is 200 μm, the particle size can be 150 μm. The fact that the receptacle 10 is hydrophobic makes it possible to avoid the formation of OH bonds between the supports, resulting from the implementation of the method, and the receptacle 10.This makes it easier to retrieve media.

[0120] 140 washes

[0121] The supports recovered during step 130, placed on the recovery means, 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.

[0122] 150 post-wash drying

[0123] Following step 140, the supports 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 supports. During drying, the supports may be placed on the recovery means and the assembly is placed in an oven.

[0124] According to a variant, shown in Figure 4, the drops are deposited by forming clusters, each cluster being formed of drops deposited sufficiently close to each other. Preferably, the drops, or certain drops of the same cluster, are aligned parallel to the longitudinal axis X. Under the effect of their flattening (see step 110), the drops coalesce. This results in the formation of a cluster whose length, along the longitudinal axis X, is greater than the width, along a lateral axis Y perpendicular to the longitudinal axis.

[0125] Figure 5 illustrates a variant, according to which the drops 12 are deposited on the receptacle 10, as described in connection with step 100 of the first embodiment. During step 110, the flattening of the drops 12 does not result from the affixing of a plate 20. The flattening of the drops is spontaneous. It is carried out in particular progressively, during drying. This makes it possible to obtain supports 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 supports 1 whose thickness is thin, typically less than 30 μm, or even less than 10 μm. This makes it possible to have supports 1 having good buoyancy. The geometry of the supports 1 obtained according to the second embodiment is not as cylindrical as that of the supports obtained according to the first embodiment.In particular, the face opposite the face resting on the receptacle 10 may be slightly curved.

[0126] According to this embodiment, the drying and gelling step 120 can be carried out in the open air, without passing through an oven.

[0127] The second embodiment is particularly suitable for obtaining supports 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 supports.

[0128] Steps 130 to 150 of the second embodiment are identical to those described in connection with the first embodiment.

[0129] 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 receptacle 10. To lower the contact angle θ, a solvent having a specific tension lower than that of water, for example an alcohol of the ethanol or isopropanol type, can be used. A surfactant can also be added to the sol-gel composition.

[0130] Whatever the embodiment, the receptacle 10 can be structured so that under the effect of flattening, each drop 12 spreads, on the receptacle 10, according to a predetermined shape, according to the structuring of the receptacle. An example of such a receptacle 10 is shown in Figure 6. The receptacle structuring 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.

[0131] Preferably, as shown in Figure 6, each first part 10i extends, parallel to a longitudinal axis X, along a length, and parallel to a lateral axis Y, parallel to the longitudinal axis X, along a width. The length is greater than the width. This makes it possible to obtain supports whose length is greater than the width, as previously described. According to one possibility, the receptacle 10 can be microstructured, so as to comprise hollow first parts 10i of predetermined shapes, for example ellipsoids, 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. Supports such as shown in Figure 1B are thus obtained, the shape of which corresponds to the shape of each first part 10i.

[0132] According to another possibility, the structuring of the receptacle 10 forms first parts 10i that are less hydrophobic than each second part IO2. 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 IO2 is preferably hydrophobic. By slightly hydrophobic, we mean a part in which the contact angle 0 is lower by at least 5° or 10°, compared to the contact angle on the most hydrophobic part.

[0133] 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 support. It also makes it possible to obtain supports whose shape is controlled by the geometry of the first parts.

[0134] Experimental tests.

[0135] A first series of tests was carried out, the experimental conditions being described below: Dispenser: Vermes MDV 3200 A dosing valve equipped with a Vermes Nll- 300 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 receptacle 10.

[0136] Receptacle 10: glass plate 200 mm x 170 mm, thickness 6 mm, previously hydrophobically treated by exposure to dichlorodimethylsilane.

[0137] Plate 20: identical to the receptacle.

[0138] Spacing s between receptacle 10 and plate 20: 140 pm. The spacing is obtained by placing spacers 30 of the adhesive tape type.

[0139] Precursor: Tetramethoxysilane 98% (Alfa Aesar).

[0140] Solvent: Isopropanol Technical (Alfa Aesar).

[0141] Catalyst: HCl 6IVI (Sigma Aldrich). Organic functionalization compound: Bovine collagen type 1: 10mg / ml (Vornia Ltd).

[0142] Iron oxide particles with a diameter between 1 pm and 3 pm - mass fraction l / 10 ième .

[0143] Thickening agent: Chitosan diluted in acetic acid.

[0144] 10 ml of tetramethoxysilane were poured into a 50 ml beaker and 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. 4 ml of collagen solution was added.

[0145] 2 ml of Chitosan solution was added. The Chitosan solution was prepared by pouring 9 ml of deionized water into a beaker and adding, with stirring, 200 mg of chitosan and 1 ml of glacial acetic acid.

[0146] 1 g of iron oxide particles were then added to the solution. The solution was stirred manually for 2 minutes and then allowed to stand for 10 minutes before the droplets were formed as described below.

[0147] For drop formation, the sol-gel solution was introduced into a syringe of the metering valve, the adjustment parameters of which are as follows: rising: 0.55 ms; falling: 0.55 ms; open time: 0 ms; needle lift: 35; delay: 40 ms; air pressure: 0.5 bar. These parameters are adjusted on a case-by-case basis by those skilled in the art.

[0148] A hydrophobic glass plate, acting as a receptacle 10, was placed on the aforementioned robot, the latter being programmed to move parallel to the first glass plate, describing lines spaced 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 2 μl.

[0149] The number of drops formed was 500. After all the drops were formed, each drop spontaneously flattened during drying. The drops were dried at room temperature for 2 hours, and placed in an oven at 120°C for 20 minutes. After drying, the supports were collected on a porous nylon filter, the pores measuring 400 μm in diameter. The filter, retaining the supports, was placed in a crystallizer, comprising isopropanol diluted to 50% (mass fraction) in deionized water, in order to carry out a washing. The washing duration was two hours. The washing was repeated three times. Following the washings, the filter retaining the supports was dried in a study at 100°C for 1h30. (disc-shaped)

[0150] The supports resulting from the implementation of the method had a thickness of 70 μm and a diameter of 1500 μm. They were placed in an enclosure 40 filled with water, as shown in Figure 7. The water here is representative of a culture medium. A magnetic field was applied inside the enclosure, by placing a magnet 50 outside the latter. The magnet was formed: of a first magnet 50i, placed facing an upper part 40 s of the enclosure 40, and segmented between a North polarity N and a South polarity S. of a second magnet 502, arranged facing a lower part 40j of the enclosure 40, forming a bottom, and segmented between a North polarity N and a South polarity S.

[0151] Generally speaking, when using permanent magnets, it is preferable to have a North polarity, facing the upper part of the enclosure, facing a South polarity, at the lower part of the enclosure, or vice versa. This allows the formation of field lines through the enclosure. Other magnetic configurations are possible, which allow the formation of field lines through the enclosure.

[0152] In Figure 7, dotted field lines are shown. Under the effect of the application of the magnetic field, the supports are aligned following the field lines. The magnetic means 50 are magnets, whose North and South polarities (N or S) are indicated. The magnets can be static, or they can be moved relative to the enclosure 40. In this example, the magnets are actuated in rotation around the enclosure. Under the effect of the rotation of the magnet, the field lines rotate. The supports have followed the field lines. This makes it possible to move the supports in the water, without agitating the water.

[0153] Preferably, regardless of the embodiment, it is preferable that the field lines extend parallel, or substantially parallel, to each other. By substantially parallel, we mean parallel to within an angular tolerance, for example up to + / - 10° or + / - 20°. The fact that the field lines are parallel makes it possible to avoid a collision between the supports when the latter are set in motion. The movement can be in translation and / or in rotation.

[0154] The alignment along the field lines is all the more pronounced when the carrier has a higher diameter to thickness ratio or a high length to width ratio. Thus, it is preferable to use planar supports, and / or if possible, longer than wide ones. This is a significant advantage compared to isotropic geometries, for example spherical geometries. Advantage is taken of the process's ability to produce thin supports, whose geometry, in the plane, can be controlled.

[0155] Such a configuration is particularly interesting because it allows the supports to be moved in a culture medium, without having to agitate the culture medium by an intrusive means, such as a propeller. Thus, the supports can be moved collectively at a slow speed, which avoids applying hydrodynamic stress to the cells growing on the surface of the supports. The movement of the supports in the enclosure allows the culture medium to be homogenized and allows homogeneous culture conditions to be obtained: each cell can be considered as having grown in a culture medium identical to another cell having grown in the enclosure.

[0156] An interesting aspect of the invention is that each support acts both as a cell culture support and as a means of stirring, complementary to the magnet, due to the magnetic field-sensitive particles it contains.

[0157] The fact that the supports spontaneously align along the field lines prevents their sedimentation. This avoids the need for agitation to keep the supports suspended in the culture medium. Aligning the supports along the field lines allows a fixed and stable network of supports to be formed in the enclosure. The movement of the magnet 50 allows the network to move, the supports remaining immobile relative to each other, as long as the movement speed is low. The occurrence of shocks between the supports is avoided, due to their alignment along the field lines.

[0158] The arrangement of the supports in a network, distributed in the culture medium, makes it possible to alternate phases during which the supports are moved and phases during which the supports are immobile in the enclosure.

[0159] As the speed of movement of the magnet increases, the agitation in the culture medium also increases. Strong agitation can be used to recover cells that have grown on the surface of the supports. Preferably, the magnetic field can be modulated to induce vibration. Vibration can be produced by inducing rapid and brief movements. This can facilitate detachment of the cells. In this case, the strong agitation is preceded by the addition, to the culture medium, of a detachment agent, for example an enzyme, promoting detachment of the cells from the supports. This can, for example, be an enzyme such as Trypsin or trypLE (registered trademark). The combination of such an agent and strong agitation, or, preferably, jerky agitation, facilitates detachment of the cells from the supports. The cells can be recovered in the culture medium.The use of a detaching agent can be supplemented or replaced by a modification of the parameters of the culture medium, for example a modification of the temperature or a modification of the pH.

[0160] A notable advantage of the supports according to the invention is that after the cells have been detached, the supports can be held along the field lines, while the culture medium is evacuated, and / or renewed, so as to extract the cells. This avoids the need for a filter, to separate the supports from the culture medium, and the associated pitfalls, in particular clogging of the filter by the supports, and / or the passage of debris from the supports through the filter. According to one possibility, the supports are moved into a predetermined part of the bioreactor by means of a magnet. They can for example be arranged in a part remote from the bioreactor discharge.

[0161] The magnet 50 may comprise one or more permanent magnets. In this case, the movement of the magnetic field lines, inside the enclosure, results from the movement of the magnet or a permanent magnet forming the magnet 50. Alternatively, the magnet comprises an electromagnet. The movement of the magnetic field lines may result from the movement of the electromagnet or from a variation of the electric current flowing in the electromagnet.

[0162] In Figure 8A, a modular parallelepiped enclosure is shown, with a volume of 5 liters, a height of 12.5 cm, and a side of 20 cm. Due to the absence of intrusive stirring means, several identical magnetic means can be juxtaposed: in Figure 8B, a bioreactor is shown comprising a large-volume enclosure 40, and a juxtaposition of magnetic means 50i, 502 as described in connection with Figure 7. The juxtaposition of the magnetic means makes it possible to increase the volume of culture medium, and to increase the culture yield, while exposing the cells to the same environment. A difficulty related to the large-volume tanks of the prior art is that by increasing the dimensions, the stirring parameters change, which impacts the homogeneity of the culture medium and the stress to which the cells are subjected.Thus, a change in scale, referred to as a scale-up, between a laboratory bioreactor and a production bioreactor is subject to certain hazards due to the increase in dimensions. The culture conditions in a large-volume production bioreactor can be significantly different from the culture conditions in a laboratory bioreactor.

[0163] The use of magnetic supports can allow a juxtaposition of magnetic means of stirring and holding the supports (magnet or electromagnet). This makes it possible to maintain the cell culture conditions close to or identical to those of a laboratory. According to one possibility, the volume of the tank is increased, for example its section, perpendicular to its height, while duplicating the magnetic stirring means parallel to the section, on either side of the tank according to the height. The configuration of Figure 8B makes it possible to increase the production volume without modifying the culture conditions.

[0164] In the example described above, the term "support" was used. However, the invention can be applied to culture supports of micrometric size, but also of millimetric or even centimetric size. Thus, the invention applies to the production and use of culture supports, the largest dimension of the latter being able to vary between a few tens of μm to a few cm.

[0165] In the example described above, the culture supports are intended for cell culture. However, the invention also applies to the production and use of magnetic objects that do not necessarily have a cell culture support function. The magnetic objects may be dedicated solely to stirring a medium, for example a culture medium, for homogenization purposes.

[0166] The invention may, without limitation, be implemented for applications related to cell culture, preferably excluding embryonic stem cells of human origin. The invention may be implemented for bacterial or fungal culture applications.

[0167] The sol-gel objects described above can be used for the absorption of organic waste in a liquid or gaseous medium. They are held, or even moved, by magnetic means through a liquid or gaseous medium to be purified. The sol-gel, being intrinsically porous, acts as an absorbent.

Claims

CLAIMS 1. Method for forming objects (1), intended to be arranged in a culture medium, the method comprising: a) formation of liquid drops (12) from a sol-gel solution (2); b) depositing the liquid drops on a receptacle (10); c) flattening the drops deposited on the receptacle (10); d) solidifying the drops by gelling and drying, so as to form solid objects e) extracting the solidified objects from the receptacle (10); the method being characterized in that the sol-gel solution comprises particles sensitive to a magnetic field.

2. The method of claim 1, wherein the magnetic field-sensitive particles are particles comprising or consisting of a ferromagnetic or paramagnetic material.

3. Method according to any one of claims 1 and 2, in which the mass fraction of particles sensitive to the magnetic field is between 1% and 50%.

4. Method according to any one of the preceding claims, in which during step a), the viscosity of the sol-gel solution is greater than 5.10 -3 Not.

5. A method according to any preceding claim, wherein the receptacle (10) is hydrophobic.

6. A method according to any preceding claim, wherein following flattening, each object has, or is inscribed in, a diameter greater than 10 pm.

7. A method according to any preceding claim, wherein following flattening, each object has, or is inscribed in, a diameter of between 50 pm and 20 mm.

8. A method according to any preceding claim, wherein following flattening, each object expands to a thickness less than one-fifth of the diameter or largest diagonal of the object.

9. A method according to any preceding claim, wherein following flattening, each drop elongates along a longitudinal axis.

10. Method according to any one of the preceding claims, in which the flattening is obtained spontaneously, during drying, during step d).

11. Method according to any one of claims 1 to 9, in which step c) comprises an application of a plate (20), preferably hydrophobic, on the drops (12), at a distance from the receptacle, such that the drops are interposed between the receptacle and the plate, the application of the plate resulting in a flattening of the drops between the receptacle and the plate, the spacing(s) between the receptacle and the plate conditioning the thickness of the objects formed during step d).

12. Method according to any one of the preceding claims, in which: - the receptacle comprises first parts (10i) and at least one second part (102); - the first parts are less hydrophobic than each second part; - each first part is bypassed by a second part, the second part forming an outline around said first part; - the outline of each first part extends, parallel to a longitudinal axis, along a length, and perpendicular to the longitudinal axis, along a width less than the length; - such that during step a), the drops are deposited on each first part, and extend, along each first part, to the outline of said first part.

13. A method according to any preceding claim, wherein - the receptacle comprises first parts and at least one second part; - the first parts are hollow in relation to each second part; - each first part being circumscribed by a second part, said second part forming an outline, taking the form of an edge, closed around said first part; - the outline of each first part extends, parallel to a longitudinal axis, along a length, and perpendicular to the longitudinal axis, along a width less than the length; such that during step a), the drops are deposited on each first part, and extend, along each first part, to the outline of said first part.

14. Method according to any one of the preceding claims, in which during step b), drops are arranged close to each other, aligned along a longitudinal axis (X), so that step c) comprises a coalescence of the drops arranged close to each other.

15. Object (1), obtained by implementing a method according to any one of claims 1 to 14.

16. Object, obtained by implementing a method according to any one of claims 1 to 14, in which the object, along a longitudinal axis, extends, along a length greater than a thickness defined perpendicular to a plane passing through the longitudinal axis and a lateral axis, perpendicular to the longitudinal axis, and / or in which the object extends, along the longitudinal axis, along a length at least 30% greater than the width.

17. Method of cell culture, using at least one object according to any one of claims 15 or 16 for cell culture, the object acting as a cell culture support, comprising: - (i) bringing the culture medium into contact with at least one cell; - (ii) arrangement of the culture support in an enclosure (40) comprising a culture medium; - (iii) applying a magnetic field in the enclosure, the magnetic field forming field lines extending through the enclosure - (iv) following step (iii), suspending each culture medium in a culture medium, contained in an enclosure.

18. Method according to claim 17, in which, following step (iv), the method comprises a displacement of the magnetic field lines in the enclosure, so as to obtain a displacement of the culture supports, in the culture medium.

19. A method according to claim 17 or claim 18, wherein the magnetic field being produced by a magnet, Tl - the magnet is moved relative to the enclosure; - and / or the magnet is an electromagnet carrying a current, the current being modified so as to generate a variation in the magnetic field in the enclosure.

20. Method according to any one of claims 17 to 19, comprising, following step (iv), a modification of the culture medium or a variation in temperature of the culture medium, so as to promote detachment of cells cultured on each culture support.

21. The method of claim 20, wherein the modification of the culture medium comprises: - addition of an agent promoting a break in bonds between each cell and a support; - and / or modification of the pH of the culture medium.

22. Method according to any one of claims 17 to 21, comprising, following step (iv), a modification of the magnetic field so as to induce a vibration of each culture support inside the enclosure.

Images