Cell culture device and method

Abstract

The invention relates to a cell culture device (10, 10a, 10b) comprising - a culture chamber (15, 15a; 15b) comprising a cell culture medium (25), - a plurality of cell culture supports (30, 30a; 30b) present in the cell culture medium (25) contained in the culture chamber (15, 15a; 15b), the cell culture supports (30, 30a; 30b) having a density different from that of the cell culture medium (25) such that these cell culture supports (30, 30a; 30b) are located at an interface (27) at least constituted on one side by the cell culture medium (25), at least one of the cell culture supports (30, 30a; 30b) carrying adherent cells (C), and - a system for moving the cell culture supports (30, 30a; 30b) along the interface (27).

Description

[0001] Description

[0002] Title: Cell culture device and method

[0003] The present invention relates to a cell culture device. It also relates to a cell culture method, in particular using the cell culture device.

[0004] Technical field

[0005] Current cell culture techniques require a large number of manipulations to multiply adherent cells and collect bioproducts, including the following steps: changing the cell culture medium, enzymatic treatment, subculturing, centrifugation, filtration, adding reagents, contamination control, various treatments, visualization by microscopy, cell counting. In particular, cell culture techniques aimed at substantially increasing the number of cells involve steps of seeding cells onto one or more first supports on which these cells multiply, then collecting multiplied cells from one or more of the first supports, then subculturing the cells thus collected into a plurality of second supports, etc., until the desired number of cells is obtained for the intended application.Many of these steps are aimed at increasing the accessible surface area for cell growth and controlling the confluence of cells in culture. These operations must also be adapted to each cell type or line.

[0006] It is known, notably from the article Haeusner Sebastian, et al "From Single Batch to Mass Production-Automated Platform Design Concept for a Phase II Clinical Trial Tissue Engineered Cartilage Product" Frontiers in Medicine, 8-2021n and Shariatzadeh, M., et al. "Distributed automated manufacturing of pluripotent stem cell products." Int J Adv Manuf Technol 106, 1085-1103 (2020), to replace human intervention with a robot, without the culture process itself being modified. However, the accessible culture surface is predefined and cannot be changed. The multiplication of the culture surface is usually done by subculturing on a new support in a new culture medium.

[0007] It is known, in particular from international application WO2017223269, to carry out variable volume cultures to vary the accessible surface area. Such volume variations are complex to implement and control. To avoid the use of proteolytic enzymes such as trypsin, which is harmful to cell integrity, Ohlson et al in Cytotechnology (1994), vol 14, pages 67-80 describes a technology for transferring adherent cells from microcarrier to microcarrier (bead to bead transfer) in the absence of any enzymatic treatment. The close contact between microcarriers covered with adherent cells and bare microcarriers promotes the transfer of cells onto the bare microcarriers from which the cells can amplify.To increase cell growth, bare microcarriers are added to a culture medium containing microcarriers covered with adherent cells and the culture medium is agitated intermittently to promote contact between microcarriers.

[0008] Also known from international patent application WO2011077035 is a culture in a fixed volume container on microcarriers kept suspended in a culture medium containing adherent cells and increasing the number of microcarriers in the culture medium to increase the culture surface accessible to the adherent cells in the culture medium and then harvesting by enzymatic treatment of the cell population produced. Cell culture is carried out in the volume of the culture medium from the cells present in the latter. The supports are only used for cell growth and remain in the container and the cells are recovered after proliferation for further treatment, subculturing or cell passage.

[0009] There is a need to provide a cell culture device capable of carrying out a cell culture process preserving the integrity of the cultured cells, for example free from recurring steps of detaching adherent cells from the surface of the culture supports, and which allows minimal exposure of the cells to the external environment, in particular a source of contamination.

[0010] There is therefore a need to enable all or part of the above-mentioned steps to be carried out in a controlled, automatable and adaptable manner, in particular for different uses and different cell types, in a simple manner. There is also a need to simply control the confluence of cells in culture and to facilitate the handling of cells in a controlled sterile environment.

[0011] Statement of the invention

[0012] The invention meets these needs using a cell culture device comprising a culture chamber comprising a cell culture medium, a plurality of cell culture supports present in the cell culture medium contained in the culture chamber, the cell culture supports having a density different from that of the cell culture medium so that these cell culture supports are located at an interface at least constituted on one side by the cell culture medium, at least one of the cell culture supports carrying adherent cells, and a system for moving the cell culture supports along the interface.

[0013] The term "cell culture medium" means a medium suitable for the cell culture of adherent cells of at least one cell type on a support. It contains in particular the nutrients essential for cell growth, in particular salts and / or sugars and / or buffers. The culture medium may or may not be optimized. It may be an impoverished culture medium containing only the nutrients essential for maintaining the cell population. Such an impoverished medium may in particular make it possible to promote the adhesion of cells to the supports, particularly in the case of prokaryotic cells.

[0014] By "cell culture support" is meant an object whose surface is configured to allow the adhesion of adherent cells of at least one cell type to a support and the growth of said cells on its surface.

[0015] By "along the interface" we mean that the cell culture media displacement system generates a displacement force at least in a direction parallel to the interface.

[0016] By "density" of cell culture media, we mean the average density of the media, i.e. its average density, corresponding to the weight of the media over its volume, compared to the density of water.

[0017] The fact that the device presents cell culture supports at an interface allows the formation of a two-dimensional cultured cell expanse for the culture of the adherent cells on the supports by contact between the supports. The adherent cells propagate by multiplication on all the supports in direct or indirect contact with the support(s) initially seeded with the adherent cells. It is thus possible to control the surface available for cell growth by varying the number of supports present in the culture chamber of the device. Furthermore, the fact that the supports are at the interface makes it possible to simulate a culture surface while having the possibility of modulating the extent of the culture surface by simply adding or removing culture supports.Furthermore, the fact that the adherent cells are on supports, the surface of which colonized by the cells is in contact with the culture medium, and not in suspension in the culture medium allows their easy manipulation by simple manipulation of the supports and a simple control of their propagation by control of the contacts in the culture medium. It is then possible to control cell growth and to extract all or part of the supports carrying adherent cells after growth for subsequent operations. The supports then constitute cell culture modules that are easily manipulated individually.

[0018] The media movement system allows the media to be manipulated in the growing chamber.

[0019] Such a device therefore makes it possible to facilitate cell culture and the manipulation of cultured cells.

[0020] Furthermore, the use of supports and a movement system along the interface allows easy manipulation of these supports along the interface, i.e. in 2D and, consequently, control of the number of adherent cells present in the culture chamber at a given time and whether or not they are in contact. This control can be automated in the absence of external intervention, which allows easy maintenance of the sterility of the culture medium by reducing the need for manual manipulation or using external tools. The movement system allows 2D control of the supports.

[0021] Preferably, the interface is flat but it could be otherwise, particularly in the case of an interface between the culture medium and a wall of the culture chamber.

[0022] Preferably, the device is devoid of a system for suspending the cell culture supports in the cell culture medium.

[0023] Cell culture media movement system

[0024] Preferably, the cell culture media displacement system is configured to generate on the cell culture media a displacement force in a predetermined sense and direction parallel to the interface.

[0025] Preferably, the cell culture media movement system generates a force on the cell culture media perpendicular to the interface and less than a force holding the cell culture media at the interface. This prevents the media movement system from causing the cell culture media to become suspended in the culture medium. It is important in the context of this invention that the cell culture media remain at the interface.

[0026] The cell culture device preferably comprises at least two openings of the culture chamber between which the support movement system can generate a flow of a fluid, in particular cell culture medium or a second fluid, which may be a liquid or a gas, in particular air, extending on the other side of the interface as we will see later, circulating in one direction or the other at least partially along the interface.

[0027] Preferably, the openings are configured to allow flow therebetween parallel to the interface. The openings may extend perpendicular to the interface, particularly on either side of the culture chamber. This allows flow along the interface, which generates a displacement force along the interface on the cell culture supports. The displacement force generated on the cell culture supports therefore does not suspend them but allows control of their movement at the interface.

[0028] The system for moving the cell culture supports may comprise a member for circulating the fluid, in particular the cell culture medium or a second fluid, which may be a liquid or a gas, in particular air, from one of the two openings forming a fluid inlet to the other of the two openings forming a fluid outlet, the circulating member being configured to allow the circulation of the fluid between the two openings in one direction or the other. The fluid circulating member may be a pump, a syringe or any other means for circulating a fluid in a fluid system between two openings.The fluid circulation member may be configured to receive commands for controlling the circulation of the fluid between the two openings, in particular the direction of circulation of the fluid between the two openings, the commands being able to be generated by a control member, in particular a processor, external or from the device, in particular from the system for moving the cell culture supports.

[0029] The fluid circulation member may be configured to change the direction of circulation of the cell culture medium, the second fluid and / or the supports in the fluid chamber. The fluid circulation member is configured to generate a flow between the two openings in a direction substantially parallel to the interface. Preferably, the fluid circulation member is configured to circulate the cell culture medium in the fluid chamber.

[0030] Alternatively, the system for moving the cell culture supports comprises a member for moving the supports globally or individually, independent of whether or not a fluid is circulating, in particular based on the intrinsic properties of the cell culture supports or of the culture chamber. For example, at least a portion of the cell culture supports may have a magnetic property and the member for moving the supports may be a magnetic member arranged outside the chamber and controllable automatically or manually. The magnetic member may allow movement of the cell culture supports at the interface between the two openings, in particular by moving the magnetic member along a wall parallel to the interface to generate a movement force by tracking the magnetic member on the cell culture supports opposite the magnetic member through the wall.Alternatively, the magnetic member is arranged at one of the openings or near one of the openings to generate on the cell culture supports having a magnetic property a displacement force towards one of the openings. Alternatively, the displacement system may be mechanical, in particular comprising a movable barrier at the interface mechanically exerting a mechanical force on the supports by its displacement at the interface.

[0031] Grow room

[0032] The device may comprise a channel for supplying the culture chamber with cell culture medium and / or cell culture supports, in particular opening into the culture chamber through one of the openings.

[0033] The device may include a media storage chamber. The storage chamber may open directly into the cell culture chamber through one of the openings or be connected to the cell culture chamber through the culture chamber feed channel.

[0034] The device may comprise a channel for extracting the cell culture supports from the culture chamber, in particular opening into the chamber through one of the openings, in particular the opening into which the supply channel or the storage chamber does not open.

[0035] The device may comprise a cell processing chamber downstream of the cell culture chamber. The cell processing chamber may open directly into the cell culture chamber through one of the openings, in particular the opening into which the feed channel or the storage chamber does not open, or be connected to the cell culture chamber by the media extraction channel.

[0036] The device may comprise a channel for renewing the cell culture medium of the culture chamber which may or may not be identical to the channel for extracting the cell culture supports. The device may further comprise a filtering member for the culture medium at the channel for renewing the cell culture medium of the culture chamber. Such a filtering member makes it possible in particular to eliminate planktonic cells, i.e. free cells, and cellular debris from the culture medium.

[0037] The culture chamber may have a width greater than that of the supply channel. Alternatively, the device comprises a fluidic channel of substantially constant section, a part of which forms the culture chamber.

[0038] The culture chamber can have a variable width, in particular a continuously variable width, in particular a width decreasing towards one of the two openings, in particular towards the extraction channel. This allows the supports to be brought closer together and therefore promotes their contact.

[0039] The chamber is preferably delimited by surfaces made of a material limiting cell adhesion, in particular surfaces modified to limit cell adhesion, for example by PEGylation, microstructuring, or any other method, or surfaces made of hydrophobic polymers or having a hydrophobic coating, in particular Teflon or any other suitable material. This makes it possible in particular to limit the colonization of the walls of the culture chamber by living or dead cells which detach from the cell culture supports.

[0040] The device may comprise a thermal regulation member. The thermal regulation member may be configured to maintain the temperature of the culture chamber within a predetermined temperature range depending on the type of cells to be cultured, in particular an optimal temperature range for the multiplication of the cells in the culture medium. The thermal regulation member may be configured to control the temperature of the storage chamber within a predetermined temperature range depending on the type of cells to be cultured, in particular a temperature range limiting the multiplication of the cells in the storage chamber. The thermal regulation member may be configured to control the temperature of the cell treatment chamber within a predetermined temperature range depending on the type of cells to be cultured, in particular a temperature range facilitating the treatment of the cells in the cell treatment chamber.Such temperature ranges are known to those skilled in the art depending on the type of cells.

[0041] Cell culture supports

[0042] Preferably, the supports are of millimetric or micrometric sizes. The size of the supports is chosen according to the type of cells to be cultured. They have in particular a largest dimension greater than or equal to 1 μm, better greater than or equal to 10 μm, better greater than or equal to 50 μm and / or less than or equal to 1 cm, better less than or equal to 5 mm, even better less than or equal to 3 mm,

[0043] The supports can be monolithic, multi-layered or aggregates of particles.

[0044] Preferably, the supports are made of plastic material, preferably hydrophobic, in particular polyethylene (PE), polypropylene (PP), polystyrene (PS), polylactic acid (PLA) or polydimethylsiloxane (PDMS) or mixtures thereof.

[0045] Preferably, the supports are elastic on the surface. This makes it possible in particular to limit the crushing of the cells when the supports come into contact with each other and to facilitate contact between supports by limiting the rebound effects between them.

[0046] The supports may all be of substantially the same shape, in particular spherical or polyhedral. The supports may have a shape allowing them to fit together and / or be assembled with each other at the interface. This may facilitate cell culture between adjacent supports, facilitate the formation of a “sheet” of supports for cell culture and densify the “sheet” of supports for cell culture, which limits the movements of supports between them and therefore the effects of rebound.

[0047] The supports may be anisotropic in shape, in particular elongated in a direction of elongation, for example ovoid or rice grain in shape. In this case, the cell culture supports may be oriented in the cell culture medium so that the direction of elongation of each support extends parallel to the interface. The directions of elongation of the cell culture supports may all extend in the same direction, under the action of a flow of the cell culture medium between the two openings or a flow of a second fluid constituting the other side of the interface as we will see below.

[0048] The supports may all be the same size larger in the direction transverse to the interface. The supports may all be substantially identical.

[0049] Alternatively, at least some of the supports may be different in at least one characteristic, notably color and / or shape. This can make it easy to discriminate them, particularly for their study.

[0050] The supports may have a density strictly lower than the density of the culture medium, in particular a density strictly lower than 1 g / cm3, better still lower than or equal to 0.97 g / cm3, better still between 0.9 and 0.97 g / cm3, even better still between 0.95 and 0.97 g / cm3, in particular for polyethylene supports. This allows the supports to extend at an upper interface of the cell culture medium as we will see later, in particular an interface between the cell culture medium and water. Alternatively, the supports have a density strictly higher than the density of the culture medium. This allows the supports to extend at a lower interface of the cell culture medium as we will see later.

[0051] The supports can be solid or expanded.

[0052] In some embodiments, the supports are porous, so as to promote the circulation of nutrients, oxygen and metabolic waste between the cells and the culture medium. In this case, the pore size is preferably less than or equal to 0.4 μm. Such a size limit makes it possible to limit the colonization of the pores by the cells. In other embodiments, the supports are non-porous.

[0053] The supports may have an external surface configured to allow and / or promote cell adhesion. The supports may have a surface treatment that promotes cell adhesion.

[0054] Preferably, the cell culture supports have a surface coating of a biocompatible material, for example a biocompatible material intended to promote cell adhesion to the supports. Examples include extracellular matrix (ECM) coatings such as collagen, coatings obtained by bringing the surface into contact with serum proteins, for example with fetal calf serum, fibronectin or Matrigel. Examples include coating materials such as poly-L-lysine or gelatin. Chemical coating materials, possibly ionized, may also be used, such as polyethyleneimine (PEI) which is cationic, 3-aminopropyltriethoxysilane acid (APTES), or even a surface treatment by silanization.

[0055] Preferably, the device is configured so that the supports extend over at least 50% of their surface area in the cell culture medium. This allows the supports to be, at least for a portion of them, in contact with each other within the cell culture medium and thus to facilitate the growth of the cells from one support to another by contact of the supports in the cell culture medium.

[0056] The device may be configured so that the supports are held in the cell culture medium at the interface by pressing against a wall of the fluidic chamber or by equilibrium at the interface between the cell culture medium and a second fluid constituting the other side of the interface as explained below.

[0057] At least two supports can be in contact with each other. This allows the colonization of cells from one support to the other.

[0058] A plurality of the supports may be in contact with each other at the interface so as to form a two-dimensional web of supports having therebetween a continuity of contact with the support carrying the adherent cells. The web of supports may form, by the surface of the supports extending into the culture medium, a continuous cell culture surface in the culture medium on which the adherent cells may be cultured from the support carrying the adherent cells.

[0059] Adherent cells

[0060] Adherent cells can be eukaryotic cells, including yeast, and / or prokaryotic cells. In the case of prokaryotic or yeast cells, the support can support the cells, possibly forming a corresponding biofilm.

[0061] Preferably, the device comprises only one type of adherent cells. Alternatively, the device comprises several types of adherent cells. The cells of the different cell types may be introduced into the culture chamber on different cell culture supports or may be introduced onto one or more of the same supports on which they are in coculture.

[0062] The device may include different first and second media configured to culture different cell types, the first media being a good adhesion media for a first cell type and a poor adhesion media for a second cell type and the second media being a good adhesion media for the second cell type and a poor adhesion media for the first cell type.

[0063] Interface

[0064] Preferably, the interface consists on one side of the cell culture medium and on the other of a second fluid immiscible with the cell culture medium.

[0065] The second fluid may have a density greater than that of the cell culture medium, the interface being at the lower interface of the cell culture medium in the culture chamber and the cell culture supports having a density strictly greater than that of the cell culture medium and a density strictly less than that of the second fluid to extend at the interface. In this case, the second fluid is preferably a liquid.

[0066] The second fluid may have a lower density than the cell culture medium, the interface being at the upper interface of the cell culture medium in the culture chamber and the cell culture supports having a density strictly lower than that of the cell culture medium and a density strictly higher than that of the second fluid to extend at the interface. In this case, the second fluid may be a liquid or a gas, in particular air, a mixture of gases suitable for maintaining the cells, for example air having a carbon dioxide content of approximately 5% (v / v).

[0067] The second fluid may not be a fluid suitable for cell culture, that is, it may not contain all the nutrients essential for cell culture.

[0068] Alternatively, the interface consists of one side of the cell culture medium and the other of a solid wall of the culture chamber.

[0069] Barrier

[0070] Preferably, the device comprises at least one barrier for the cell culture supports extending into the culture chamber at least partially in the cell culture medium and configured to assume a closing configuration preventing the displacement of the cell culture supports by the displacement system at the interface in at least one direction and an opening configuration allowing the displacement of the cell culture supports by the displacement system in said direction. The barrier may be perpendicular or inclined to the interface and to the direction of the displacement force that the displacement system exerts on the supports.

[0071] Preferably, the barrier extends from one side wall to the opposite side wall of the growth chamber, spaced therefrom by a distance preventing the passage of media between the side wall and the barrier.

[0072] Preferably, the barrier is at least on the surface made of a material limiting the adhesion of cells, in particular metal, or hydrophobic polymer, or polymer whose surface is functionalized or microstructured. This makes it possible to limit the colonization of the walls of the barrier by living or dead cells which detach from the cell culture supports.

[0073] The barrier is preferably configured to allow the culture medium to pass through, in particular between the two openings, in the opening configuration and the closing configuration. The barrier may extend over only a portion of the height of the culture chamber, the height being the dimension perpendicular to the interface or the barrier may be perforated. Preferably, the height of the barrier in the culture medium is less than the height of the culture medium in the culture chamber, more preferably less than or equal to 50% of the height of the culture medium in the culture chamber.

[0074] The barrier may be immersed in the cell culture medium to at least 50%, better still at least 70%, better still at least 100% of the average height of the cell culture supports.

[0075] The barrier may be a physical barrier, in particular a plate or a grid, or a fluidic barrier, in particular a bubble curtain or a liquid flow, in particular of the vertical culture medium, in particular perpendicular to the interface.

[0076] The barrier can be movable in translation between the two openings in the cell culture chamber.

[0077] The barrier can be at a fixed position in the grow room.

[0078] The barrier may be a physical barrier and move from one configuration to another by rotation around an axis, notably perpendicular or parallel to the interface, or by translation in a direction perpendicular or inclined relative to the interface.

[0079] The device may comprise a motor for moving the barrier, in particular for translational movement between the two openings and / or for movement between the two open and closed configurations. Method

[0080] The invention also meets this need using a method of cell culture and / or cell culture control and / or cell culture monitoring, in particular using the cell culture device described above, comprising, in a culture chamber, bringing into contact with each other at least a portion of cell culture supports extending at an interface, the interface being constituted at least on one side by a cell culture medium, at least one of the cell culture supports brought into contact carrying adherent cells and the culture of the cells on the supports at the interface with the cell culture medium.

[0081] Bringing supports extending in a cell culture medium into contact at an interface with at least one of them carrying adherent cells makes it possible to form a sheet of supports forming a continuity of contact between them so that the cells adherent on one of the supports spread by growth in the culture medium onto the other supports in a substantially continuous surface by simple contact.

[0082] The method may further comprise the release of cells from the supports by carrying them in the culture medium and the adhesion of free cells in the culture medium to the cell culture supports, in particular those lacking them. This allows, in particular in the case of yeast or bacteria, the passage of cells from the support(s) into the culture medium and the transfer of the latter onto a support.

[0083] If all the supports carry adherent cells and the culture is at confluence, the method can allow the confluence of the cell culture to be maintained by adding new supports.

[0084] The surface area accessible to cells can easily be controlled by the number of supports in contact with each other.

[0085] The characteristics described above in connection with the device apply alone or in combination to the process and this independently of the device.

[0086] Preferably, the contacting comprises applying a displacement force to the supports in a direction at least partially parallel to the interface and blocking at least a portion of the supports subjected to this force. The blocking may be implemented by a barrier forming an obstacle to the supports at least in the direction of the displacement force in at least one closing configuration. The displacement force may be formed by a force applied only to the supports, for example a magnetic force, or a flow of fluid formed in the culture chamber between two openings, in particular a flow of the cell culture medium or a second fluid, in particular air, extending on the other side of the interface. The method may comprise circulating a fluid, in particular the culture medium, between the two openings substantially parallel to the interface.This allows the supports to be held against each other at the barrier to form a support span against the barrier. Preferably, the displacement force is configured to have no component perpendicular to the interface or to have a component perpendicular to the interface less than the holding force at the interface.

[0087] The barrier is configured to allow the circulation of cell culture medium in the culture chamber in the closed configuration. This allows in particular the renewal of the culture medium during contact between the supports, which facilitates cell growth and / or the elimination of free or dead cells present in the culture medium.

[0088] In the case of elongated supports, the fluid circulation can be configured to orient the elongation directions of the supports in the direction of the fluid flow.

[0089] Preferably, when contacting the supports, the contact area of ​​the adjacent supports extends into the culture medium.

[0090] The method may include providing additional cell culture media into the culture medium, the media being arranged at the interface, and bringing the additional media into contact with the culture media already present. The additional culture media may or may not include adherent cells. Preferably, they do not. This increases the range of media available for cell growth in the culture chamber.

[0091] The method may include spacing and / or mixing the cell culture supports together, including by applying a reverse or oscillating force parallel to the interface configured to detach the supports from each other. The reverse or oscillating force may be generated by a flow of the cell culture medium or a second reverse or oscillating fluid between the two openings. This may include mixing the supports and / or separating the supports for extraction at a subsequent step.

[0092] The method may include transferring, storing, or extracting one or more supports, optionally after spacing them from the culture chamber. The extraction may include passing the barrier into an opening configuration allowing circulation at the interface of the supports under the action of the displacement force. This allows, in particular, the recovery of the supports with the adherent cells for a subsequent step, in particular a subsequent passage or an analysis.

[0093] The method may comprise introducing the extracted supports into a second cell culture chamber and bringing the supports into contact with each other at an interface in this second culture chamber, the two culture chambers being connected in series with each other with continuity of fluid flow between them.

[0094] The method may comprise, after culturing the adherent cells, the extraction of a portion of the cell culture supports on which the adherent cells have been cultured by opening the barrier, then providing additional culture supports to enable the culture of the adherent cells on new supports from the supports still present in the culture chamber and carrying adherent cells. In this case, the method may comprise mixing the supports before culturing the adherent cells on the supports and the supports on which the cells have multiplied, if necessary until confluence, in particular by applying a reverse or oscillating force parallel to the interface. This makes it possible in particular to distribute the supports lacking them between the supports carrying adherent cells to enable cell multiplication on the supports which are initially lacking them.

[0095] The method may comprise placing in series and / or in parallel several culture chambers each containing cell culture supports in contact with each other with at least one support carrying adherent cells, the adherent cells being identical or not. The cells may differ in their cell types and / or their cell species and / or their cell lines. The method may comprise the coculture, in particular for eukaryotic cells, of different adherent cells, the formation of biofilm in the presence of different bacterial species or populations or the formation of interactions between a pathogen and its host, in particular in the case of a mixture of eukaryotic cells and bacteria.The method may comprise one or more additional steps of handling the cell culture supports, in particular a step of filtering the bioproducts secreted by these cells into the culture medium, the extraction of the adherent cells from the supports, in particular by enzymatic treatment, the sorting of the supports according to the types of cells or the cell culture parameters, in particular number of adherent cells, confluence, morphology of the supports, markers or biomarkers of cell culture and / or study of cell biology and / or in vitro toxicology and / or microbiology, the imaging of the supports and / or adherent cells on the supports, the counting of the supports to determine the quantity of adherent cells cultured.

[0096] The method may include identifying the supports by identifying culture markers present on the supports, in particular by changing the color of probes, size or shape.

[0097] The invention also meets this need by a cell culture and manipulation system comprising, fluidically mounted in series, a cell culture device as described above and an ancillary device for cell culture or for manipulating cell culture supports or, in particular a channel for circulating the culture medium, a member for filtering bioproducts, for extracting adherent cells from the supports, in particular by enzymatic treatment, for sorting the supports according to the types of cells or cell culture parameters, in particular the number of adherent cells, the confluence, the morphology of the supports, another device as described above and / or a device for studying cell biology and / or in vitro toxicology and / or microbiology.

[0098] The cell culture and manipulation system can allow the implementation of the cell culture method and / or cell culture control and / or cell culture monitoring as described above.

[0099] Brief description of the drawings

[0100] [Fig 1] schematically represents an example of a cell culture device viewed from below,

[0101] [Fig 2] is a side view of the device of Figure 1, [Fig 3] is a top view of the device of Figure 1 after growth of the adherent cells on the supports,

[0102] [Fig 4] schematically represents in top view a variant of the device after growth of the adherent cells on the supports,

[0103] [Fig 5] is a side view of the device of Figure 4, the

[0104] [Fig 6 A] represents figure 5, the barrier being in the opening configuration

[0105] [Fig 6B] represents a variant of the device of figure 4, the barrier being in the opening configuration,

[0106] [Fig 6C] represents a variant of the device of figure 4, the barrier being in the opening configuration.

[0107] [Fig 7A] schematically represents a variant of cell culture device in profile view,

[0108] [Fig 7B] schematically represents a variant of cell culture device in profile view,

[0109] [Fig 8A] schematically represents a variant of cell culture device in profile view,

[0110] [Fig 8B] schematically represents a variant of cell culture device in profile view,

[0111] [Fig 9] schematically represents a variant of cell culture device in profile view,

[0112] [Fig 10] schematically represents a variant of cell culture device in profile view,

[0113] [Fig 11] schematically represents a variant of cell culture device in profile view,

[0114] [Fig 12] schematically represents a variant of cell culture device in bottom view,

[0115] [Fig 13] schematically represents a variant of cell culture device in bottom view,

[0116] [Fig 14] schematically represents a variant of cell culture device in bottom view,

[0117] [Fig 15] schematically represents the steps of a cell culture method according to the invention, [Fig 16] schematically represents the steps of a variant of the cell culture method according to the invention,

[0118] [Fig 17] schematically represents the steps of a variant of the cell culture method according to the invention,

[0119] [Fig 18] schematically represents a variant of cell culture device in bottom view,

[0120] [Fig 19] schematically represents a variant of cell culture device in bottom view,

[0121] [Fig 20] schematically represents a variant of cell culture device in bottom view, and

[0122] [Fig 21] schematically represents a variant of cell culture device in bottom view.

[0123] Detailed description

[0124] Figures 1 and 2 illustrate a device 10 according to the invention. It comprises a culture chamber 15 having two side walls 17 and 18 and a bottom 20. The culture chamber 15 contains a cell culture medium 25 in which cell culture supports 30 are present.

[0125] In the illustrated example, the culture chamber 15 is in the form of a fluidic channel.

[0126] The supports 30 have a density different from that of the cell culture medium, here a lower density than the cell culture medium, so that they float on the surface. Thus, they extend to an interface 27 defined at least on one side by the cell culture medium 25. In the example illustrated, the supports 30 have a lower density than the culture medium 25. They therefore float in the culture medium 25. The supports 30 may have a density strictly lower than the density of the culture medium 25, in particular a density strictly lower than 1 g / cm3.

[0127] At least one of the supports 30 has on the surface, preferably on a surface of the support in contact with the culture medium, adherent cells C. As a variant, there may be several supports 30 having adherent cells C on the surface.

[0128] The adherent cells C may be eukaryotic cells, including yeasts, or prokaryotic cells. In the case of prokaryotic cells or yeasts, the support may carry the cells, if necessary, in the form of a corresponding biofilm. In the example illustrated in FIGS. 1 and 2, the culture chamber 15 is open at the top and the interface 27 is an interface between the culture medium 25 and the outside air 45.

[0129] The device 10 comprises a system for moving the supports 30 along the interface 27. This movement system is not illustrated, but an arrow indicates in the figures the direction of the movement force F exerted on the supports 30.

[0130] The displacement system can be of different natures, it can act on the supports 30 directly or indirectly. It can be a system for moving the culture medium 25 in the fluid chamber 15 between two openings, or ends, 32 and 34 of the culture chamber 15, in particular a pump or any other flow circulation member. Alternatively, in the case of an interface between two fluids, it can be a system for moving the fluid forming the other side of the interface 27 in the fluid chamber 15 between two openings, or ends, 32 and 34 of the culture chamber 15. As a further variant, it can be a system for moving the supports 30 directly, that is to say by direct application of a force on the supports 30. This force can be a magnetic or mechanical force.In the case of a magnetic force, the supports may have magnetic properties and the displacement system may include a magnetic member, not shown, configured to magnetically move the supports at the interface, in particular a magnet located at one of the openings 34 or 35 depending on the direction of the force to be applied or a magnet moving on or under the culture chamber 15 and causing in its movement the supports subjected to its magnetic field. In the case of a mechanical force, the system may be a mobile barrier as we will see later in relation to figure 12.

[0131] In the example illustrated, the supports 30 are all substantially identical. They may be millimeter or micrometer in size. They are monolithic. But it could be otherwise. They could be formed of several layers or an aggregate of particles. They are preferably made of a plastic material, preferably hydrophobic, in particular polyethylene (PE), polypropylene (PP), polystyrene (PS), polylactic acid (PLA) or polydimethylsiloxane (PDMS) or mixtures thereof.

[0132] Preferably, their external surface is configured to allow and / or promote cell adhesion. The supports 30 may comprise a surface intended for cell culture, for example a biocompatible material intended to promote cell adhesion to the supports. Mention may be made of extracellular matrix (ECM) coatings such as collagen, coatings obtained by bringing the surface into contact with serum proteins, for example with fetal calf serum, fibronectin or Matrigel. Mention may also be made of coating materials such as poly-L-lysine or gelatin. It is also possible to use chemical coating materials, where appropriate ionized, such as polyethyleneimine (PEI) which is cationic, or even a treatment of the surface by silanization.

[0133] In the illustrated examples, the supports 30 are spherical. However, the invention is not limited to such a shape of the supports. Alternatively, they may be polyhedral or anisotropic in shape, in particular elongated in a direction of elongation, for example ovoid in shape or in the shape of a grain of rice. In this case, the supports 30 may be oriented in the cell culture medium 25 so that the direction of elongation of each support extends parallel to the interface 27 in the direction of flow. The directions of elongation of the supports 30 may all extend in the same direction.

[0134] The supports 30, the culture medium 25 and the culture chamber 15 are configured so that the contacts between supports 30 at the interface 27 are made in the culture medium 25. To do this, they extend into the cell culture medium 25 so that more than half of their surface is in the culture medium 25. In the example of Figures 1 and 2, the proportion of each support 30 extending into the culture medium is governed by the Archimedes pressure and depends in particular on the difference in density between the supports 30 and the culture medium 25. Those skilled in the art will easily know how to adapt the density of the supports to obtain contacts between supports 30 in the culture medium 25 and not in the air.

[0135] The supports 30 may have surface elasticity to limit the crushing of the cells C when the supports 30 come into contact with each other and facilitate contact between supports 30 by limiting rebound effects.

[0136] The supports 30 may be solid, hollow, or expanded. This may allow the density of the supports 30 to be adjusted.

[0137] Such a device allows the culture of adherent cells C on the part of the supports 30 immersed in the culture medium 25 by growth and propagation of the adherent cells C of the support 30 carrying them towards the supports 30 not carrying them in contact as can be seen in Figure 3. A sheet N of adherent cells C is then formed in the culture medium 25 on the “raft” of supports 30 formed by the supports 30 in contact with each other. The supports 30 which are not in direct or indirect contact with the supports 30 carrying adherent cells C do not have a cell culture on their surface as can be seen in Figure 3.

[0138] Preferably, illustrated in figures 4 and 5, the device may comprise at least one barrier 40. The barrier 40 may take a closing configuration, illustrated in figures 4 and 5, and an opening configuration, illustrated according to two different variants in figures 6A to 6C.

[0139] In the closed configuration, the barrier 40 is perpendicular or inclined relative to the interface 27 and to the direction of the displacement force F applied to the supports 30. In the closed configuration, it extends into the culture medium 25 from the interface 27 over a height H in the cell culture medium 25 greater than or equal to 70% of the height s of the supports 30, here greater than the height of the supports 30. It also extends over substantially its entire width of the culture chamber 15, i.e. from the side wall 17 to the side wall 18. It may be spaced from the side walls 17 and 18 by a distance that does not allow the passage of the supports 30 or be in contact with the latter. Thus, in the closed configuration, the barrier 40 blocks the supports 30 extending at the interface upstream of the barrier relative to the direction of the displacement force F.The latter come into contact with the barrier 40 and come into contact with each other against the barrier 40 under the action of the displacement force F. In the example illustrated, the barrier 40 is a solid physical barrier. It may not extend into the culture medium 25 over the entire height M of the latter so that the culture medium 25 can circulate in the culture chamber 15, in particular between the two openings 34 and 35. This allows the application of the displacement force F by circulation of the culture medium 25 as indicated previously. The invention is however not limited to such a barrier 40 as we will see later in relation to FIGS. 9 to 11.

[0140] In the opening configuration, the barrier 40 releases the supports 30 which can then circulate at the interface 27 under the effect of the displacement force F.

[0141] The transition from the closing configuration to the opening configuration can be done by rotation of the barrier along an axis perpendicular to the direction of the displacement force F, as illustrated in FIGS. 6B and 6C, or by translation in a direction perpendicular or inclined relative to the interface 27, as illustrated in FIG. 6A, so as to release at the interface 27 a height allowing the displacement of the supports 30 along the interface 27 by the action of the displacement force F.

[0142] The embodiments of Figures 7A to 8C differ from those of Figures 4 and 5 in the structure of the interface 27 and / or the density of the supports 30 relative to the culture medium 25.

[0143] In Figures 7A and 7B, the interface 27 is formed on one side by the culture medium 25 and on the other by an upper wall 22, as illustrated in Figure 7A, or the bottom 20, as illustrated in Figure 7B, of the culture chamber 15, depending on the density of the supports 30. If the supports are denser than the culture medium, they are positioned at the interface with the lower wall 20, and conversely with the upper wall 22 if they are less dense. The supports 30 are held in the culture medium by pressing against the wall 20 or 22 of the culture chamber 15. The barrier 40, when it is, can extend from the upper wall 22 or from the lower wall 20 depending on the density of the supports 30. In this case, the displacement force can be applied by a flow of the culture medium 25 in the culture chamber 15 as described previously or by a mechanical action.

[0144] In Figures 8A and 8B, the interface 27 is formed on one side by the culture medium 25 and on the other by another fluid 45, in particular another liquid. This fluid 45 may be less dense than the culture medium 25, as illustrated in Figure 8A, and therefore be located above the culture medium 25 so that the interface is in the upper part of the culture medium 25. In a variant illustrated in Figure 8B, this fluid 45 may be denser than the culture medium 25 and therefore be located below the culture medium 25 so that the interface is in the lower part of the culture medium 25.

[0145] The density of the supports is preferably closer to that of the culture medium 25 than to that of the fluid 45. This allows the supports to extend further into the culture medium 25 than into the fluid 45.

[0146] In Figures 8 A and 8B, the displacement force F can be generated by a flow of the culture medium 25 as described previously. It can also be generated by a flow of the fluid 45 on the other side of the interface 27 or by any other means. In the case of a flow of the fluid 45, the barrier 40 can extend over the entire height of the culture chamber 25 in the culture chamber 15 or not, the height being taken perpendicular to the interface 27. In the case of a flow of the culture medium 25, the barrier 40 can extend over the entire height of the fluid 45 in the culture chamber 15.

[0147] The embodiments of Figures 9 to 11 differ from those of Figures 4 and 5 by the shape of the barrier 40.

[0148] As illustrated in Figure 9, the barrier 40 may be an openwork barrier, in particular a grid, allowing the passage of the culture medium 25 and preventing the passage of the supports 30. Such a grid may extend over the entire height of the culture chamber 15 to the extent that it allows the flow to pass. The transition to the open configuration may be done as in the case described previously, by rotation around an axis perpendicular to the direction of the displacement force or translation along an axis perpendicular or inclined relative to the interface 27.

[0149] In the embodiment of Figure 10, the barrier 40 is formed of a bubble curtain. The device may comprise at the bottom of the culture chamber a bubbler 42 configured to generate a vertical bubble curtain 40 in the culture chamber 15. This bubble curtain acts as an openwork barrier by allowing the flow of the culture medium 25 to pass through if necessary and by constituting an obstacle to the circulation of the supports 30. The opening and closing of the barrier 40 in this case can be done simply by controlling the bubbler 42 automatically or manually to stop or put in place the bubble curtain. This embodiment is mainly possible in the case of a device open on the surface or comprising an air evacuation member to evacuate the air emitted by the bubbler 42.

[0150] In the embodiment of Figure 11, the barrier 40 is a flow of fluid or culture medium perpendicular to the interface 27 or inclined and oriented in the direction opposite to the direction of the displacement force F. Such a flow can be established by an inlet 44 of culture medium 25 or liquid fluid 45 in the culture chamber 15 extending perpendicularly or inclined and oriented in the direction opposite to the direction of the displacement force F at the interface 27 in the culture medium 25 or liquid fluid 45. The start of culture medium 25 or liquid fluid 45 forming the barrier 40 is preferably greater than the displacement flow of the supports 30, when the displacement force F is generated by a flow between the two openings 34 and 35.

[0151] As illustrated in Figure 12, the device 10 may comprise two barriers 40, one upstream and one downstream of the or part of the supports 30. This allows upstream and downstream control of the supports 30 in the culture chamber 15 and / or the introduction or evacuation of supports in the culture chamber 15 and to have a displacement force on the supports 30 in one direction or the other indifferently. The barriers 40 described previously may be fixed longitudinally to the direction of the displacement force F. Alternatively, as illustrated, they are movable along the direction of the displacement force F. This makes it possible in particular to vary the dimension of the culture chamber 15 and / or to exert a mechanical displacement force on the supports 30.

[0152] In the variants illustrated in Figures 13 and 14, the culture chamber 15 is not a fluid circulation channel but an enlarged chamber of a fluidic device comprising on either side channels 50 and 55 opening into the enlarged chamber 15 through the openings 34 and 35. The enlarged chamber can have different shapes in top view, in particular a rectangular shape, as illustrated in Figure 13 or a trapezoid shape, as illustrated in Figure 14. The fact that the side walls 17 and 18 are funnel-shaped towards an opening 34 allows the supports 30 to be brought together when the displacement force is in the direction of bringing the walls 17 and 18 together. The device is however not limited to these shapes of the culture chamber.

[0153] The device is not limited to the embodiments described above. The characteristics of the different embodiments can be combined with each other independently or not when they are technically compatible. In particular, the shape of the culture chamber 15 can be combined with the different barriers 40 described or the different interfaces 27 described.

[0154] Furthermore, modifications of the possible embodiments not described will appear obvious to those skilled in the art, in particular modifications of the shape of the culture chamber 15 or the supports 30.

[0155] Examples of methods for culturing adherent cells using the device described above are now described in connection with Figures 3, 15 to 21.

[0156] As illustrated in Figure 3 and described previously, the device allows the contact at an interface 27 of supports 30 carrying adherent cells C with supports lacking them to form a sheet N of adherent cells C in the culture medium 25 on the “raft” of supports 30 formed by the supports 30 in contact with each other. The presence of a barrier 40 downstream of the supports 30 relative to the displacement force E applied to the supports 30 makes it possible to promote contacts between supports 30 by blocking them in their downstream movement. This then promotes the culture of the adherent cells on the supports.

[0157] In this context, as illustrated in Figure 15, the cell culture method may include increasing the size of the “raft” accessible to the adherent cells C present on the supports 30, in particular when the surface accessible to the adherent cells C on the supports 30 is covered entirely by the adherent cells. For this, it is easy with the device to introduce into the culture chamber 15 new supports 30b, in particular identical to those already present in the culture chamber 15, and devoid of adherent cells. As illustrated in step b of Figure 15, the latter come against the layer of adherent cells on the supports 30 already present due to the displacement force F and the blocking by the barrier 40. The contact with the supports 30 carrying the layer N of adherent cells allows the culture of the adherent cells on these new supports 30b to increase the accessible surface.The propagation on the new supports 30b is done step by step until the entire supports 30 and 30b are covered (provided that the culture medium is suitable for such a culture) and an enlarged sheet Nb is formed. Precise control of the surface accessible to the adherent cells over time is therefore easily possible by adapting over time the number of supports 30 accessible to the adherent cells C upstream of the barrier 40.

[0158] It is possible to add a step c illustrated in figure 16 of mixing the supports 30 and 30b in the culture chamber 15 by applying an oscillating displacement force on the supports. Such an oscillating force Fosc causes a separation of the supports 30 from each other and a chaotic movement of the supports 30 and 30b by the congestion in the area, which mixes the supports 30 with the supports 30b. Such an oscillating force Fosc can be generated by applying a flow of the culture medium or the oscillating fluid between the openings 34 and 35 or by applying an oscillating magnetic or mechanical force, for example by alternately moving two barriers 40 and 42 upstream and downstream of the supports 30 in the direction of the displacement force Fosc. The supports 30 and 30b can then be brought back into contact with each other by resuming a unidirectional displacement force, in particular towards the barrier 40.The supports 30b are then mixed with the supports 30 carrying the adherent cells C, which increases the propagation speed of the adherent cells C on the supports 30b for the formation of the new sheet of adherent cells Nb. Furthermore, the device described above can be integrated into a larger cell culture and manipulation system 5 for carrying out a more complex cell culture method. We will describe below examples of integration of the device into a larger cell culture and manipulation system 5. The invention is not limited to the examples described.

[0159] For example, as illustrated in Figure 17, the device 10 can be integrated in series with upstream relative to the direction of the displacement force F a storage chamber 60 comprising supports 30 devoid of adherent cells identical to those of the device 10 at the interface. The storage chamber 60 comprises at the outlet a barrier 65 similar to those described 40 in relation to the device. This storage chamber 60 is fluidically connected in series to the culture chamber 25 by a channel 55. Preferably, the displacement force F is exerted by a circulation of the culture medium 25 from an inlet 67 in the storage chamber 60 to the opening 34 of the culture chamber 15. Thus,the movements of the supports 30 between the storage chamber 60 and the culture chamber 15 are controlled by the barriers 40 and 65. Such an integration makes it possible, as illustrated in FIG. 17, to: cultivate the adherent cells C on the supports 30 from the supports 30 by carrying them in the culture chamber 15, as illustrated in step a) of FIG. 17, then extract from the culture chamber 15, by opening the barrier 40, only a portion of the supports 30 carrying adherent cells C. It is important at this step not to extract all the supports 30 from the culture chamber 15 in order to keep in the latter supports 30 carrying adherent cells, as illustrated in step b) of FIG. 17, then open the barrier 65 to add to the culture chamber 15 supports 30 devoid of adherent cells C from the storage chamber 60 in a controlled manner, as illustrated in step c) of Figure 17,then mixing the supports 30 in the culture chamber 15 by applying an oscillating displacement force Fosc as illustrated in step d) of Figure 17 and described in relation to Figure 16, culturing the adherent cells C on the supports 30 from the storage chamber 60 from the supporting supports 30 which remained in the culture chamber 15 in the previous extraction step, as illustrated in step a) of Figure 17.,

[0160] These steps can be repeated repeatedly to automatically and continuously culture adherent C cells on supports 30.

[0161] In the embodiment of Figure 18, all of the supports 30 in the culture medium carry adherent cells C and the culture medium 25 is circulated according to a loop 70 to be renewed regularly in the culture chamber 15. The supports 30 are maintained in the culture chamber 15 by a barrier 40 as described previously. This system makes it possible to maintain the viable adherent cells C on the supports 30 for future use of the supports 30 carrying the adherent cells C.

[0162] The embodiment of figure 19 differs from that of figure 18 by the addition in the circulation loop 70 of the culture medium 25 of a bioproduct filtration member 75. This filtration member makes it possible to recover the biomolecules and bioproducts secreted by the adherent cells in the culture medium 25, in particular the proteins, lipids, cytokines, chemokines and / or extracellular vesicles.

[0163] In the embodiment of Figure 20, the system comprises two devices 10a and 10b of culture chambers 15a and 15b substantially identical mounted in parallel with each other and connected by their opening 34 to a coculture chamber 80 substantially identical to the culture chambers 15 of the devices 10a and 10b. The culture chambers 15a and 15b respectively contain supports 30a and 30b carrying different adherent cells Ca and Cb. The assembly as described makes it possible, by controlling the opening of the barriers 40a and 40b, to control the quantity of supports 30a and 30b introduced into the coculture chamber 80 in which the supports 30a and 30b are mixed upstream of a barrier 85.This makes it possible to carry out coculture of two cell types by easily controlling the proportions of each cell type and also making it possible to maintain in the coculture chamber a differentiated location of the cell types on each of its supports 30a or 30b. The supports 30a and 30b may be identical or different, in particular different in shape, size and / or adhesion properties with respect to the two cell types. The invention is not limited to two culture chambers in series. A mixture of more than two cell types by the use of several culture chambers in parallel each containing supports carrying a cell type is possible.In some embodiments in which coculture is performed, some media are initially seeded with a first cell type and some other media are initially seeded with another cell type, such that both types of media coexist in the culture chamber. Of course, coculture can be performed with more than two distinct cell types.

[0164] In certain other embodiments in which coculture is performed, two or more distinct cell types may coexist on a single medium.

[0165] Examples include cell cocultures of fibroblasts and endothelial cells, neurons and glial cells, muscle cells and nerve cells, and more generally cell types originating from distinct organs or tissues. Cocultures can also be achieved when, for example, induced stem cells, which can be co-cultured with feeder cells such as fibroblasts, are involved.

[0166] It is specified that the method described here expressly excludes the use of human and animal embryonic stem cells. On the other hand, the method described here can be used for the culture of cells not involving the destruction of an embryo, such as for example the culture of induced stem cells, which are the result of a dedifferentiation of adult cells, which are then subject to re-differentiation towards a chosen cell type.

[0167] The embodiment of Figure 21 is substantially the opposite of that of Figure 20. It comprises a coculture chamber 80 comprising a mixture of supports 30a and 30b respectively carrying different types of adherent cells, in particular the coculture chamber of Figure 20, and two downstream culture chambers 15a and 15b mounted in series with the coculture chamber 80 and in parallel with each other. The system comprises between the culture chambers 15a and 15b and the coculture chamber 80 a detection member 90 making it possible to differentiate the supports 30a and 30b and to control, depending on the support 30a or 30b detected, a sorting member 95, in particular a mobile orientation barrier, to direct the support 30a or 30b towards one or other of the culture chambers 15a or 15b. This system makes it possible, in particular, to easily sort the supports 30a and 30b carrying different cells in different culture chambers.

[0168] It is understood from the figures that the manipulation of the adherent cells C is made easy by the simple manipulation of supports 30 carrying them, whether for culture, separation, cell mixtures or other steps requiring circulation, manipulation or cell culture. Furthermore, the culture at the two-dimensional interface further facilitates the manipulation by allowing easy control of the circulation of the supports 30 in two dimensions in a fluid circuit. The invention is obviously not limited to what has just been described and the cell culture and manipulation device as described can be easily integrated as a modular unit in a complex cell manipulation system, in particular a closed one. It is then possible by simply manipulating the supports 30 to carry out complex processes in a sterile environment.

Claims

Claims 1. Cell culture device (10, 10a, 10b) comprising a culture chamber (15, 15a; 15b) comprising a cell culture medium (25), a plurality of cell culture supports (30, 30a; 30b) present in the cell culture medium (25) contained in the culture chamber (15, 15a; 15b), the cell culture supports (30, 30a; 30b) having a density different from that of the cell culture medium (25) so that these cell culture supports (30, 30a; 30b) are located at an interface (27) at least constituted on one side by the cell culture medium (25), at least one of the cell culture supports (30, 30a; 30b) carrying adherent cells (C), a system for moving the cell culture supports (30, 30a; 30b) along the interface (27), and at least one barrier (40, 40a; 40b) for the cell culture supports (30, 30a; 30b) extending into the culture chamber (15, 15a;15b) at least partially in the cell culture medium (25) and configured to take a closing configuration preventing the displacement of the cell culture supports (30, 30a; 30b) by the displacement system at the interface (27) in at least one direction and an opening configuration allowing the displacement of the cell culture supports (30, 30a; 30b) by the displacement system in said direction.; 2. Device according to claim 1, comprising at least two openings (34, 35) of the culture chamber (15, 15a; 15b) between which the support movement system can generate a flow, in particular parallel to the interface (27), of a fluid, in particular of the cell culture medium (25) or of a second fluid (45) extending on the other side of the interface (27), circulating in one direction or the other at least partially along the interface (27) and the support movement system comprising a member for circulating the fluid from one of the two openings (34, 35) forming a fluid inlet to the other of the two openings (34, 35) forming a fluid outlet, the circulation member being configured to allow the circulation of the fluid between the two openings (34, 35) in one direction or the other.

3. Device according to any one of the preceding claims, in which the cell culture supports (30, 30a; 30b) have a density strictly lower than the density of the culture medium (25), in particular a density strictly lower than 1 g / cm3, better still lower than or equal to 0.97 g / cm3.

4. Device according to any one of the preceding claims, configured so that the cell culture supports (30, 30a; 30b) extend over at least 50% of their surface in the cell culture medium (25).

5. Device according to any one of the preceding claims, in which the adherent cells are eukaryotic cells, including yeasts, and / or prokaryotic cells.

6. Device according to any one of the preceding claims, in which the interface (27) consists on one side of the cell culture medium (25) and on the other of a second fluid (45) immiscible with the cell culture medium, in particular a liquid or a gas, or of a solid wall (20, 22) of the culture chamber (15, 15a; 15b).

7. Device according to any one of the preceding claims, wherein the barrier (40, 40a; 40b) is configured to allow the cell culture medium (25) to pass, in particular between the two openings, in the opening configuration and the closing configuration.

8. Device according to claim 7, wherein the barrier (40, 40a; 40b) extends from one side wall (17, 18) to the opposite side wall (17, 18) of the culture chamber (15, 15a; 15b) being spaced from the latter by a distance preventing the passage of the cell culture supports (30, 30a; 30b) between the side wall (17, 18) and the barrier (40, 40a; 40b).

9. A method of cell culture, in particular using the cell culture device (10, 10a; 10b) according to any one of the preceding claims, comprising, in a culture chamber (15, 15a; 15b), bringing into contact with each other at least a portion of cell culture supports (30, 30a; 30b) extending at an interface (27) at least by applying a flow of fluid circulating parallel to the interface between two openings and blocking at least a portion of the cell culture supports (30, 30a; 30b) subjected to this flow, the interface (27) being constituted at least on one side by a culture medium cellular (25), at least one of the cell culture supports (30, 30a; 30b) brought into contact with each other carrying adherent cells (C) and the culture of the cells (C) in the cell culture medium (25) on the cell culture supports (30, 30a; 30b).

10. Method according to claim 9, comprising the provision of additional cell culture supports (30, 30a; 30b) into the culture medium (25), the supports being arranged at the interface (27), and bringing the additional cell culture supports (30, 30a; 30b) into contact with the cell culture supports (30, 30a; 30b) already present.

11. Method according to any one of claims 9 or 10, comprising spacing and / or mixing the cell culture supports (30, 30a; 30b) from each other, in particular by applying a reverse or oscillating force (Fosc) parallel to the interface (27) configured to detach the cell culture supports (30, 30a; 30b) from each other.

12. Method according to any one of claims 9 to 11, comprising, after the culture of the adherent cells (C), the extraction of one or more cell culture supports (30, 30a; 30b), optionally after spacing the latter from the culture chamber (15, 15a; 15b), in particular by passing the barrier (40, 40a; 40b) in an opening configuration allowing the circulation at the interface (27) of the cell culture supports (30, 30a; 30b) under the action of the displacement force (F).

13. Method according to any one of claims 8 to 12, comprising one or more additional steps: - handling of cell culture supports (30, 30a; 30b), in particular a step of filtration of bioproducts, - extraction of adherent cells from supports, in particular by enzymatic treatment, - sorting media based on cell types or cell culture parameters, including number of adherent cells, confluence, media morphology or a cell culture marker, and / or - study of cell biology and / or in vitro toxicology and / or microbiology.

14. Cell culture and manipulation system (5) comprising, fluidically mounted in series, a cell culture device (10, 10a, 10b) according to any one of claims 1 to 8 and - an ancillary device for cell culture or for handling cell culture supports, - another device according to any one of claims 1 to 8 and / or - a device for studying cell biology and / or in vitro toxicology and / or microbiology.

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