Magnetic supports for cell culture and support manufacturing process

Abstract

A cell culture method, using a cell culture medium, excluding human embryonic stem cells, the method comprising: (i) contacting the culture medium with at least one cell; (ii) placing the culture medium in a chamber (40) containing a culture medium; the method being characterized in that the medium comprises particles sensitive to a magnetic field, and that the medium has, along a longitudinal axis, a dimension greater than the dimensions along another axis, the larger dimension forming a length of the medium, the method comprising: (iii) applying a magnetic field in the chamber, the magnetic field forming field lines extending through the chamber; (iv) following (iii) suspending each culture medium in a culture medium contained in a chamber. Figure 6

Description

Title of the invention: Magnetic supports for cell culture and method for manufacturing supports technical field

[0001] The technical field of the invention is the production of magnetic supports of micrometric or millimetric size EARLIER ART

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

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

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

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

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

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

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

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

[0010] US patent 20160145600A1 describes the use of magnetic carriers in the form of spherical microbeads, produced by encapsulating magnetic particles in a polymer matrix, for example, an alginate-based matrix. However, the spherical shape of the particles does not provide high sensitivity to a magnetic field, except with intense magnetic fields. The use of such carriers is limited to small volumes, for example, tubes.

[0011] The inventor has developed a new type of support, particularly suited for placement in bioreactors, so as to be exposed to the most homogeneous culture medium possible. The supports simplify cell detachment. Description of the invention

[0012] A first aspect of the invention is a method for forming objects (1), intended to be placed in a culture medium, the method comprising: a. formation of liquid drops (12) from a sol-gel solution (2); b. deposition of liquid drops onto a receptacle (10); c. deformation of the drops deposited on the receptacle (10); d. solidification of the drops by gelation and drying, so as to form solid objects e. extraction of solidified objects from the receptacle (10);

[0013] the process being characterized in that the sol-gel solution comprises particles sensitive to a magnetic field.

[0014] Particles sensitive to a magnetic field can be metallic particles or magnets. They may contain or be composed of a ferromagnetic or paramagnetic material. The mass fraction of particles sensitive to the magnetic field can be between 1% and 50%.

[0015] During step a), the viscosity of the sol-gel solution may be greater than 5.103 Pa s.

[0016] The receptacle may be hydrophobic.

[0017] 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.

[0018] According to one possibility, step c) involves flattening the drops on the receptacle, the flattening being obtained spontaneously during drying in step d).

[0019] According to one possibility, step c) involves applying a plate, preferably hydrophobic, to 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).

[0020] According to one possibility, - the receptacle comprises first parts and at least one second part; - the first parts are less hydrophobic than each second part; - each first part is bypassed by a second part, the second part forming a contour around said first part; - 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.

[0021] According to one possibility: - the receptacle has first parts and at least one second part; - the first parts are hollow compared 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; - 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.

[0022] According to one possibility, in step b), drops are arranged close to each other, aligned along a longitudinal axis so that step c) involves a coalescence of the drops arranged close to each other.

[0023] A second aspect of the invention is an object, obtained by implementing a process according to the first aspect of the invention. The object can, in particular, serve as a support for cell culture.

[0024] According to one possibility, the object, along the longitudinal axis, extends over a length greater than a defined thickness perpendicular to a plane passing through the longitudinal axis and the lateral axis, and / or the object extends, along the longitudinal axis, over a length at least 30% greater than its width. The thickness is preferably less than both the width and the length. The thickness is preferably less than 30% or even 50% of the width or length.

[0025] A third aspect of the invention is a cell culture method, using at least one object according to the second aspect of the invention for culturing cells, excluding human embryonic stem cells, the object serving as a cell culture support, comprising: - (i) bringing the culture medium into contact with at least one cell; - (ii) arrangement of the culture medium in an enclosure (40) comprising a culture medium; - (iii) application of a magnetic field within the enclosure, the magnetic field forming field lines extending through the enclosure - (iv) following step (iii), suspension of each culture support in a culture medium, contained in an enclosure.

[0026] Following step (iv), the process may include a displacement of the magnetic field lines in the enclosure, so as to obtain a displacement of the culture supports, in the culture medium.

[0027] According to one possibility, the magnetic field being produced by a magnet, - the magnet is displaced relative to the enclosure; - and / or the magnet is an electromagnet carrying a current, the current being modified so as to generate a variation of the magnetic field in the enclosure.

[0028] 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 the detachment of cultured cells on each culture support. + magneto-induced vibration.

[0029] According to one possibility, the modification of the culture medium involves: - addition of an agent promoting a break in the bonds between each cell and a support; - and / or modification of the pH of the culture medium.

[0030] A fourth aspect of the invention is a cell culture method, using at least one cell culture medium, excluding human embryonic stem cells, the method comprising: - (i) bringing the culture medium into contact with at least one cell; - (ii) arrangement of the culture medium in an enclosure comprising a culture medium;

[0031] The process 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 larger dimension forming a length of the support, the process comprising: - (iii) application of a magnetic field within the enclosure, the magnetic field forming field lines extending through the enclosure - (iv) following the previous step, suspension of each culture support in a culture medium, contained in an enclosure.

[0032] The culture medium can extend, along a lateral axis perpendicular to the longitudinal axis, in width, the longitudinal and lateral axes forming a plane, and, perpendicular to said plane, in thickness. Preferably, the thickness and width are less than the length. Preferably, the thickness and / or width are at least 30% or 50% less than the length. Preferably, the culture medium has a predominantly longitudinal shape, which gives it increased sensitivity to the magnetic field.

[0033] A fifth aspect of the invention is a bioreactor comprising a chamber configured to receive a cell culture medium, the bioreactor comprising means for applying a magnetic field through the chamber, the chamber 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 chamber. Preferably, The magnetic field application means are configured to allow movement of the field lines within the enclosure. The magnetic field application means can be chosen from: permanent magnet and / or electromagnet.

[0034] Following step (iv), the process may include a modification of the magnetic field so as to induce a vibration of each culture support inside the enclosure.

[0035] The invention will be better understood upon reading the description of the exemplary embodiments presented later in this description, in connection with the figures listed below. FIGURES

[0036] Fig. 1A represents an example of a support according to the invention.

[0037] Fig. 1B shows another example of a support according to the invention.

[0038] Figure 2A illustrates a step in the formation of drops on a receptacle, hydrophobic preference.

[0039] Fig. 2B schematically illustrates the application of a plate, preferably hydrophobic, to the receptacle.

[0040] Fig. 2C is a diagram illustrating the definition of a contact angle.

[0041] Fig. 3 schematically illustrates the main steps of a support formation process.

[0042] Fig. 4 shows a drop formation step, so as to form clusters of adjacent drops in a longitudinal direction.

[0043] The [Fig.5] a deformation of a drop according to a particular embodiment.

[0044] Figure 6 schematically represents a first substrate structured between hydrophobic parts delimited by strongly hydrophobic zones.

[0045] Fig. 7 illustrates the formation of field lines through a bioreactor, and the alignment of supports along the field lines.

[0046] Fig. 8A shows a module comprising a bioreactor equipped with means for applying a magnetic field through the latter.

[0047] Fig. 8B shows a bioreactor comprising various adjacent means for applying a magnetic field. PRESENTATION OF SPECIFIC IMPLEMENTATION METHODS

[0048] Figure 1A shows an example of a support according to the invention. The support comprises two flat surfaces, or surfaces considered as such, opposite each other. Thus, the support has a first flat, or substantially flat, surface S1 and a second flat, or substantially flat, surface S2. The first flat surface and the second flat surface are parallel to each other, or substantially parallel to each other.

[0049] By substantially parallel, we mean parallel taking into account an angular tolerance, for example less than or equal to ± 20°, and preferably less than or equal to ± 10°.

[0050] By substantially flat surface, we mean flat, admitting surface elements having a local flatness defect, within a limit of ± 10° with respect to the rest of the surface.

[0051] The first surface S1 is present or inscribed within 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 even more preferably between 100 pm and 20 mm, or even larger. It has been observed that using carriers with a diameter greater than or equal to 1 mm, or even 5 mm, confers greater sensitivity to the magnetic field.

[0052] The first surface Si and the second surface S2 extend parallel to a principal plane 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 principal plane PXY.

[0053] Whatever the configuration, the thickness e of the support 1 is preferably: - less than one fifth of the diameter (or of the largest diagonal) - and preferably greater than one tenth, and preferably more than one twentieth of the diameter (or of the largest diagonal) ¢.

[0054] The thickness generally results from a compromise: it is sufficient to ensure a certain solidity to the support, but low enough to confer good buoyancy properties and sensitivity to the magnetic field.

[0055] Figure 1A represents an embodiment in 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.

[0056] In the example shown, the support extends: - parallel to a longitudinal axis X, along a length L; - parallel to a lateral axis Y, perpendicular to the longitudinal axis, according a width1;

[0057] The length L is preferably strictly greater than the width { For example, the length-to-width ratio is preferably greater than 1.2, way 1.3, or even 1.5. Being longer than it is wide gives a support anisotropic behavior, favoring alignment with magnetic field lines.

[0058] Thus, regardless of the configuration, the support essentially takes the form of two surfaces Si and S2, extending one face to the other, making the lateral surface S3 negligible. The two surfaces Si and S2 can, in particular, be planar or substantially planar.

[0059] The supports according to the invention can be obtained by implementing a sol-gel process, short for solution-gelation. This is a chemical process known to those skilled in the art, allowing the manufacture of glasses or ceramics at low temperatures. Such a process involves the use of a sol-gel solution, formed: - of a molecular precursor of metal or metalloid, for example an organometallic compound or a metallic salt; - of an organic solvent; - water; - of an acidic or basic catalyst. - of particles sensitive to a magnetic field.

[0060] By particle sensitive to a magnetic field, we mean in particular ferromagnetic particles. These may, for example, be particles containing iron, nickel, cobalt, or a material used in magnets, such as neodymium. In this example, without limitation, the particles are iron oxide particles with a diameter between 1 pm and 10 pm.

[0061] 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%.

[0062] 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.

[0063] Preferably, the solution comprises a thickening agent capable of increasing viscosity, for example, chitosan. This may, in particular, be a polymer or another thickening agent. The purpose of increasing viscosity is to keep the magnetic or ferromagnetic particles in suspension during the manufacturing of the supports. This prevents the particles from settling within the sol solution during gelation and even drying. The viscosity depends on the size and density of the particles, as well as the duration of gelation and drying.

[0064] In the presence of water, a network of oxides forms, through hydrolysis-condensation reactions, trapping the organic solvent to form a gel. It then undergoes drying to remove the organic solvent present in the gel. The drying can be evaporative, at a pressure less than or equal to atmospheric pressure, so as to form a dry gel, usually referred to as a xerogel, in the form of a monolithic solid.

[0065] The molecular precursor can for example be an organometallic compound of metal or metalloid, for example a metal alkoxide of formula M(0R)n, where M is a metal or a metalloid, and R is an organic group. - Metal M can be, for example, a transition metal, a lanthanide: it can be Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, Hf, Ra, W, Re, Os, Ir, Pt, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Er, Yb, Al, Ga, In, Ge, Sn, Pb. - The metalloid element can be chosen from Si, Se, Te. - R can be an alkyl group, for example with between 1 and 10 carbon atoms, or a phenyl group. - n is a natural number corresponding to the number of ligands bound to M, which corresponds to the valence of M.

[0066] 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.

[0067] The sol-gel solution may also include a catalyst, and / or water, or compounds that act on porosity, for example a surfactant.

[0068] According to one embodiment, the sol-gel solution comprises a functionalizing compound, in particular an organic compound, whose function is to form a grafting agent. A grafting agent is understood to be a molecule or functional group capable of promoting the attachment, by grafting, of a chemical or biological element to the surface of the xerogel resulting from the implementation of the sol-gel process. The chemical or biological element is predetermined. It may be a molecule, a cell, a protein, or another organic compound, for example, a growth factor or an antibody. For cell culture applications, the grafting agent promotes the grafting of a cell of a predetermined type. The grafting agent may then be collagen, polylysine, or a milk protein. However, due to regulatory or quality control constraints, it is sometimes preferable to avoid molecules of animal origin.A functionalizing compound containing an epoxy group can then be used, as this group is conducive to the formation of chemical bonds with amine groups, which are present in most cell membranes. The incorporation of an epoxy group can be carried out by a glycidoxypropyltrimethoxysilane type compound, usually designated by the acronym GPTM.

[0069] According to one possibility, the sol-gel solution comprises a precursor of the 3-aminopropyltrimethoxysilane type, usually designated by the acronym APTES, or 3-aminopropyltrimethoxysilane, usually designated by the acronym APTMS. The amine function of the precursor of such precursors brings positive charges to the surface of the support. This improves cell adsorption and adhesion.

[0070] The possibility of adding a functionalizing compound to the sol-gel solution is a significant advantage, as it avoids the need for post-fabrication functionalization, as in prior art beads. This allows for the production of supports specific to a predefined application, taking into account the chemical or biological element intended to bind to the supports, and / or the environment in which the support is intended to be placed.

[0071] The sol-gel supports obtained generally have a density 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 gives good flotation of the supports in aqueous culture media.

[0072] Due to the action of the magnetic field, a low density is not essential. Indeed, under the influence of the magnetic field, the supports can be kept in suspension in a bioreactor, even if they have a higher density. This is why, although it is advantageous to use sol-gel technology due to its ease of implementation, low density, or the incorporation of active ingredients into the mass, the supports can be produced by another manufacturing process. For example, they can be glass or plastic supports into which magnetically sensitive particles have been previously incorporated. This type of support can be produced by molding. It is preferable that the shape of the supports not be isotropic, so as to define a preferred axis of sensitivity with respect to the magnetic field.

[0073] Thus, regardless of the manufacturing method, the support extends, along a longitudinal axis, to 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 to a thickness. The thickness and / or the width are preferably less than, by at least 10%, or even 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the length.

[0074] In addition to a sol-gel type material, the supports can be made of a material chosen, without limitation, 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.

[0075] 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 [Fig.3].

[0076] Step 100: Droplet formation

[0077] A sol-gel solution 2, as previously described, is introduced into a dispenser 3, enabling the formation of drops 12, and preferably calibrated drops. The drops may be microdroplets, the volume of which is between 5 x 10⁻¹⁰ n₀ and 15 µl, or even > 1 or a few ml.

[0078] The diameter of the drops formed is preferably between 100 nm and a few centimeters.

[0079] During droplet formation, it is preferable for the sol solution to have sufficient viscosity, preferably greater than 10² Pa s or greater than 10¹ Pa s. The viscosity of the solution must prevent the magnetic particles from settling within the droplet during gelation and even drying. Forming drops from a sufficiently viscous sol solution limits or prevents the sedimentation of magnetically sensitive particles contained in the soil under the effect of gravity. The amount of thickening agent can be determined experimentally based on the volume and geometry of the drops.

[0080] Such viscosity can be obtained by spontaneous gelation of the soil or by the addition of a viscosity-enhancing agent, for example a thickening agent as previously described.

[0081] The dispenser 3 is arranged at a distance from, or in contact with, a receptacle 10. The receptacle 10 is preferably flat and 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 [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 prevents excessive spreading of the drops on the receptacle. The material forming the receptacle 10 can, for example, be glass, polypropylene, Teflon, or silicon. Preferably, the receptacle 10 is hydrophobic or has previously undergone a hydrophobic treatment.

[0082] The distance d between the distributor and the receptacle 10 is preferably less than 10 cm. The drops can be formed upon contact with the receptacle 10. The closer the distance, the better the accuracy in locating the drops.

[0083] During step 100, the drops formed on the receptacle 10 are spaced apart. The spacing between two adjacent drops is dimensioned such that when the drops are flattened, resulting from the implementation of step 110, they remain spaced apart.

[0084] The fact that the receptacle 10 is hydrophobic prevents the drops 12 from spreading too much.

[0085] Step 110: flattening the drops

[0086] Following the formation of the droplets, a plate 20, preferably hydrophobic, is applied parallel to the receptacle 10. The plate 20 is preferably flat and rigid. The plate 20 is applied facing the receptacle 10, spaced from it by a distance e that depends on the thickness e of the substrates to be formed. Taking into account shrinkage occurring during drying (see step 120), the distance e between the receptacle 10 and the plate 20 is greater than the thickness e of the substrates 1 resulting from the implementation of the process. The spacing e between the receptacle 10 and the plate 20 can be ensured by placing spacers 30 between the receptacle 10 and the plate 20. The spacing e 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.

[0087] Applying the plate 20 flattens the drops 12. After being applied to the receptacle 10, the drops 12 rapidly form a gel. It is therefore preferable to flatten them before gelation is too advanced, as soon as all the drops 12 have formed. Therefore, 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 low-volatility, so that the application of the plate 20 can be delayed. This prevents premature drying and gelation of the drops that would occur before the application of the plate 20. It is also possible to lower the temperature or increase the pressure to delay the gelation of the drops deposited on the receptacle 10.

[0088] Step 120: Gelation and drying

[0089] During gelation and drying, the assembly 40 formed by the receptacle 10 and the plate 20 is preferably placed in an oven to complete gelation and facilitate drying of the gel. The oven temperature can, for example, be between 30°C and 80°C. The oven can be placed at a pressure lower than atmospheric pressure, for example, between -200 mbar and -970 mbar relative to atmospheric pressure. This significantly accelerates drying. The time spent in the oven can be between 10 minutes and 24 hours.

[0090] 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.

[0091] During drying, the supports undergo shrinkage, a phenomenon known in the field of sol-gel. Thus, during drying, the thickness e of each support 1 decreases, for example, by 50% when the thickness is 100 µm or 50 µm. Also, after 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.

[0092] 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 shrinkage. 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 when contact is lost, as the support shrinks due to drying. The hydrophobic treatment of the plates 10, 20 limits the risk of breakage.

[0093] Step 130: Media recovery

[0094] Following drying, the supports 1 can be easily separated from the receptacle 10 or the plate 20 due to their hydrophobic nature. During recovery, the supports 1 can be collected on a recovery medium, preferably a flexible one, such as a cloth. This could be a porous nylon filter. The porosity is optimized to retain the supports 1 while allowing the removal of debris, such as residues of supports that broke during the process, as these fragments pass through the recovery medium. For example, when the diameter (or the largest diagonal) of the supports is 600 µm, the particle size of the recovery medium can be 400 µm. When the diameter of the supports is 200 µm, the particle size can be 150 µm.

[0095] The fact that the receptacle 10 is hydrophobic prevents the formation of OH bonds between the supports, resulting from the implementation of the process, and the receptacle 10. This facilitates the recovery of the supports.

[0096] Step 140: washing

[0097] The supports recovered during step 130, placed on the recovery means, are washed, for example by immersion in a washing solution to remove 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 containing 50% isopropanol by mass. The process may comprise several successive baths, for example, two or three successive baths.

[0098] Step 150: Post-wash drying

[0099] Following step 140, the substrates are dried. Drying can be carried out at ambient temperature or at a higher temperature, for example up to 100°C or above. The drying temperature can be lowered if a partial vacuum is created around the substrates. During drying, the substrates can be placed on the collection device, and the assembly is placed in an oven.

[0100] According to one variant, shown in [Fig. 4], the drops are deposited in clusters, each cluster being formed of drops deposited sufficiently close to one another. Preferably, the drops, or some drops within 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 its width, along a lateral axis Y perpendicular to the longitudinal axis.

[0101] Figure 5 illustrates a variant in which the drops 12 are deposited onto the receptacle 10, as described in connection with step 100 of the first embodiment. In step 110, the flattening of the drops 12 does not result from the application of a plate 20. The flattening of the drops is spontaneous. It occurs, in particular, gradually during drying. This makes it possible to obtain supports 1 with a thickness ranging from 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 with a thin thickness, typically less than 30 µm, or even less than 10 µm. This makes it possible to obtain supports 1 with good buoyancy.

[0102] 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 convex.

[0103] According to this embodiment, step 120 of drying and gelling can be carried out in the open air, without passing through an oven.

[0104] The second embodiment is particularly suitable for obtaining thin supports, for example less than 10 µm. In general, the second embodiment allows for a very high diameter-to-thickness ratio, which is conducive to good buoyancy. The first embodiment allows for better control of the flatness of the supports.

[0105] Steps 130 to 150 of the second embodiment are identical to those described in connection with the first embodiment.

[0106] The second embodiment is preferably implemented by lowering the concentration of precursors in the sol-gel solution compared to the first embodiment of Implementation. Preferably, in the second embodiment, the contact angle θ of each drop is smaller than in the first embodiment, to promote spreading of the drop on the receptacle 10. To lower the contact angle θ, a solvent with a specific tension lower than that of water can be used, for example, an alcohol such as ethanol or isopropanol. A surfactant can also be added to the sol-gel composition.

[0107] Regardless of the embodiment, the receptacle 10 can be structured so that, under the effect of flattening, each drop 12 spreads out on the receptacle 10 according to a predetermined shape, depending on the structure of the receptacle. An example of such a receptacle 10 is shown in [Fig. 6]. The structure of the receptacle 10 allows for the formation of 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.

[0108] Preferably, as shown in [Fig. 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.

[0109] According to one possibility, the receptacle 10 can be microstructured so as to comprise first hollow 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 to the edge delimiting said first part. This yields supports such as those shown in [Fig. 1B], the shape of which corresponds to the shape of each first part 10i.

[0110] According to another possibility, the structure of the receptacle 10 forms first parts 10i that are 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 this angle, the more hydrophobic the material is with respect to the solution forming the droplet. The first parts 10i can be slightly hydrophobic or hydrophilic. Each second part 102 is preferably hydrophobic. By slightly hydrophobic, we mean a part in which the contact angle 0 is at least 5° or 10° smaller than the contact angle on the most hydrophobic part.

[0111] 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 for better control of the support shape. It also allows for the creation of supports whose shape is driven by the geometry of the initial parts. Experimental trials.

[0112] 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 1-300 nozzle to form sol-gel microdroplets. The dispenser was mounted on a Janome 200 mm / 200 mm tri-axis robot, allowing the dispenser to move parallel to a receptacle 10. - Receptacle 10: glass plate 200 mm x 170 mm, thickness 6 mm, previously treated hydrophobically by exposure to dichlorodimethylsilane. - Plate 20: identical to the receptacle. - Spacing e between receptacle 10 and plate: 140 pm. The spacing is obtained by placing 30 adhesive tape type spacers. - Precursor: Tetramethoxysilane 98% (Alfa Aesar). - Solvent: Isopropanol Technical (Alfa Aesar). - Catalyst: 6M HCl (Sigma Aldrich). - Organic functionalization compound: Type 1 bovine collagen: 10mg / ml (Vornia Ltd). - Iron oxide particles with a diameter between 1 pm and 3 pm - mass fraction l / 10th. - Thickening agent: Chitosan diluted in acetic acid.

[0113] 10 mL of tetramethoxysilane was poured into a 50 mL beaker, which was kept under 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 was added 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.

[0114] 2 ml of Chitosan solution was added. The Chitosan solution was prepared by pouring 9 ml of deionized water into a beaker and adding, while stirring, 200 mg of chitosan and 1 ml of glacial acetic acid.

[0115] Next, 1 g of iron oxide particles was added to the solution. The solution was stirred manually for 2 minutes and then allowed to stand for 10 minutes before the formation of the drops described below.

[0116] For drop formation, the sol-gel solution was introduced into a syringe of the dosing 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 a person skilled in the art.

[0117] A hydrophobic glass plate, acting as a receptacle 10, was placed on the previously mentioned robot, which was programmed to move parallel to the first glass plate, describing lines spaced 3 mm apart. The drops were formed at a distance of 2 mm from the hydrophobic plate. The spacing between two adjacent drops, arranged on the same line, was 2 mm. The volume of each drop was 2 pl.

[0118] The number of drops formed was 500. After all the drops had formed, each drop flattened spontaneously during drying. The drops were dried at room temperature for 2 hours and then placed in an oven at 120°C for 20 minutes.

[0119] After drying, the supports were recovered on a porous nylon filter with pores measuring 400 µm in diameter. The filter, retaining the supports, was placed in a crystallizing dish containing 50% (mass fraction) isopropanol diluted in deionized water to perform a wash. The wash lasted two hours and was repeated three times. Following the washes, the filter retaining the supports was dried in a chamber at 100°C for 1.5 hours. (disc-shaped)

[0120] The supports resulting from the implementation of the process had a thickness of 70 µm and a diameter of 1500 µm. They were placed in a chamber 40 filled with water, as shown in [Fig. 7]. The water here represents a culture medium. A magnetic field was applied inside the chamber by placing a magnet 50 outside of it. The magnet was formed: - of a first magnet 50;, positioned opposite an upper part 40s of the enclosure 40, and segmented between a North N polarity and a South S polarity. - of a second magnet 502, positioned opposite a lower part 40; of the enclosure 40, forming a base, and segmented between a North N polarity and a South S polarity.

[0121] Generally, when using permanent magnets, it is preferable for a North pole, facing the upper part of the enclosure, to be positioned opposite a South pole, at the lower part of the enclosure, or vice versa. This allows the formation of magnetic field lines through the enclosure. Other magnetic configurations are possible that also allow the formation of magnetic field lines through the enclosure.

[0122] In [Fig. 7], field lines are shown as dashed lines. Under the effect of the applied magnetic field, the supports aligned themselves along the field lines. The magnetic means 50 are magnets, whose North and South (N or S) polarities are indicated. The magnets can be static or displaced relative to at enclosure 40. In this example, the magnets are rotated around the enclosure. As the magnets rotate, the magnetic field lines rotate. The supports have followed these field lines. This allows the supports to be moved in the water without disturbing the water.

[0123] Preferably, regardless of the embodiment, the field lines extend parallel, or substantially parallel, to each other. Substantially parallel means parallel within an angular tolerance, for example, up to + / - 10° or + / - 20°. The fact that the field lines are parallel prevents collisions between the supports when they are set in motion. The motion can be translational and / or rotational.

[0124] Alignment along the field lines is more pronounced when the carrier has a higher diameter-to-thickness ratio or a high length-to-width ratio. Therefore, it is preferable to use planar supports, and / or, if possible, supports that are longer than they are wide. This is a significant advantage compared to isotropic geometries, for example, spherical geometries. The process's ability to produce thin supports, whose in-plane geometry can be controlled, is taken advantage of.

[0125] Such a configuration is particularly advantageous because it allows the supports to be moved within a culture medium without having to agitate the medium using 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. Moving the supports within the chamber homogenizes the culture medium and ensures homogeneous culture conditions: each cell can be considered as having grown in a culture medium identical to any other cell growing within the chamber.

[0126] An interesting aspect of the invention is that each support acts both as a cell culture support and as a means of agitation, complementary to the magnet, due to the magnetic field-sensitive particles it contains.

[0127] The fact that the supports spontaneously align along the field lines prevents their sedimentation. This eliminates the need for agitation to keep the supports suspended in the culture medium. The alignment of the supports along the field lines allows for the formation of a fixed and stable support network within the chamber. Movement of the magnet 50 causes the network to shift, with the supports remaining stationary relative to each other, as long as the movement speed is low. Collisions between the supports are prevented due to their alignment along the field lines.

[0128] The arrangement of the supports according to a network, distributed in the culture medium, allows alternating phases during which the supports are moved and phases during which the supports are immobile in the enclosure.

[0129] As the speed 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, short movements. This can facilitate cell detachment. In this case, the strong agitation is preceded by the addition, to the culture medium, of a detachment agent, for example, an enzyme, which promotes cell detachment from the supports. This could, for example, be an enzyme such as Trypsin or trypLE (trademark). The combination of such an agent and strong agitation, or, preferably, jerky agitation, facilitates cell detachment from the supports. The cells can then be recovered from the culture medium.The use of a detachment 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.

[0130] A notable advantage of the supports according to the invention is that, after cell detachment, the supports can be held along the field lines while the culture medium is drained and / or renewed to extract the cells. This avoids the need for a filter to separate the supports from the culture medium and the associated problems, in particular filter clogging by the supports and / or the passage of support debris through the filter. In one embodiment, the supports are moved to a predetermined part of the bioreactor by means of a magnet. They can, for example, be placed in a part far from the bioreactor's discharge.

[0131] The magnet 50 may comprise one or more permanent magnets. In this case, the displacement of the magnetic field lines inside the enclosure results from the displacement of the magnet or of a permanent magnet forming the magnet 50. Alternatively, the magnet may comprise an electromagnet. The displacement of the magnetic field lines may result from the displacement of the electromagnet or from a variation in the electric current flowing through the electromagnet.

[0132] Figure 8A shows a modular parallelepiped-shaped enclosure with a volume of 5 liters, a height of 12.5 cm, and sides of 20 cm. Due to the absence of an intrusive stirring means, several identical magnetic means can be juxtaposed: Figure 8B shows a bioreactor 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 increases the volume of culture medium, and to increase culture yield, while exposing cells to the same environment.

[0133] A difficulty associated with large-volume prior art tanks is that increasing the dimensions changes the agitation parameters, 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 scale-up, between a laboratory bioreactor and a production bioreactor is subject to certain uncertainties due to the increase in size. The culture conditions in a large-volume production bioreactor can be significantly different from the culture conditions in a laboratory bioreactor.

[0134] The use of magnetic supports allows for the juxtaposition of magnetic means for stirring and holding the supports (magnet or electromagnet). This makes it possible to maintain cell culture conditions close to or identical to those of a laboratory. In one scenario, the volume of the reservoir is increased, for example, its cross-section, perpendicular to its height, while duplicating the magnetic stirring means parallel to the cross-section, on either side of the reservoir along its height. The configuration of [Fig. 8B] makes it possible to increase the production volume without altering the culture conditions.

[0135] In the example described above, the term "support" was used. However, the invention can be applied to culture supports of micrometer size, but also to millimeter or even centimeter size. Thus, the invention applies to the production and use of culture supports, the largest dimension of which can vary from a few tens of micrometers to a few centimeters.

[0136] In the example described above, the culture supports are intended for cell culture. However, the invention also applies to the manufacture and use of magnetic objects that do not necessarily have a cell culture support function. The magnetic objects may be used solely for agitating a medium, for example, a culture medium, for the purpose of homogenization.

[0137] The invention may, by way of non-limitation, be implemented for applications related to cell culture, preferably excluding human embryonic stem cells. The invention can be implemented for bacterial or fungal culture applications.

[0138] The sol-gel objects described above can be used for the purpose of absorbing 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

Demands

1. A cell culture method, using at least one cell culture medium, excluding human embryonic stem cells, the method comprising: - (i) bringing the culture medium into contact with at least one cell; - (ii) placing the culture medium in a chamber (40) comprising a culture medium; the method being characterized in that the medium comprises particles sensitive to a magnetic field, and that the medium has, along a longitudinal axis, a dimension greater than the dimensions along another axis, the greater dimension forming a length of the medium, the method comprising: - (iii) applying a magnetic field in the chamber, the magnetic field forming field lines extending through the chamber - (iv) following the preceding step, suspending each culture medium in a culture medium contained in a chamber.

2. A method according to claim 1, wherein the culture support extends: - along a lateral axis, perpendicular to the longitudinal axis, along a width; - perpendicular to a plane formed by the lateral axis and the longitudinal axis, along a thickness, the support being such that the thickness and the width are less than the length.

3. A method according to claim 2, wherein the thickness and / or width are at least 30% or 50% less than the length.

4. A method according to any one of claims 2 or 3, wherein the culture medium takes an essentially longitudinal shape, so as to exhibit increased sensitivity to the magnetic field.

5. A method according to any one of the preceding claims, comprising, following step (iv), a modification of the field magnetic so as to induce a vibration of each growing medium inside the enclosure.

6. Bioreactor, comprising a chamber configured to receive a cell culture medium, the bioreactor comprising means for applying a magnetic field through the chamber, the chamber comprising supports, each support comprising magnetic field-sensitive particles, and each support having, along a longitudinal axis, a dimension greater than the dimensions along another axis, the larger dimension forming a length of each support.

7. Bioreactor according to claim 6, wherein the culture media extend: - along a lateral axis, perpendicular to the longitudinal axis, along a width; - perpendicular to a plane formed by the lateral axis and the longitudinal axis, along a thickness, the media being such that the thickness and the width are less than the length.

8. Bioreactor according to claim 7, wherein the thickness and / or width of the supports are at least 30% or 50% less than the length.

9. Bioreactor according to any one of claims 6 to 8, wherein the magnetic field application means are configured to permit movement of field lines within the enclosure.

10. Bioreactor according to any one of claims 6 to 9, wherein the means for applying the magnetic field can be chosen from: permanent magnet and / or electromagnet.

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