FR2957086A1 - Box useful in a system for dynamic cell culture or for seeding cells, comprises a well, a microstructured chamber for dynamic cell culture, a microsystem, and connectors attached to an interface of the microsystem - Google Patents

Abstract

The cell culture box (1) comprises a well (10), a microstructured chamber (15) for dynamic cell culture, a microsystem, and connectors attached to an interface of the microsystem. Each chamber is directly connected to an input well via a hole to a bottom of the well and to an output well, where the bottom of the well has a height above any point of the chamber. The microsystem consists of a bottom plate including an imprint of the microstructured chamber and a top plate. The connector is a deformable tube forming a part of the top plate. The cell culture box (1) comprises a well (10), a microstructured chamber (15) for dynamic cell culture, a microsystem, and connectors attached to an interface of the microsystem. Each chamber is directly connected to an input well via a hole to a bottom of the well and to an output well, where the bottom of the well has a height above any point of the chamber. The microsystem consists of a bottom plate including an imprint of the microstructured chamber and a top plate. The connector is a deformable tube forming a part of the top plate through which a tip of a pipette is inserted in the chamber. A lower box (100) covered with a tight cover is maintained in the box by a clamping unit (20). The lower box includes a microsystem and a multi-well plate (110). The cover comprises a male cover perfusing the wells, and female inlet connectors. The box further comprises a male connector and a female connector through the input wells, and a male connector and two female connectors through output wells. A membrane insert is disposed in the wells. Independent claims are included for: (1) a system for dynamic cell culture; and (2) a method of seeding cells.

Description

GENERAL TECHNICAL FIELD

The present invention relates to the in vitro culture of cells. More specifically, it relates to a multi-bioreactor box for dynamic cell culture in a nutrient medium.

STATE OF THE ART

Cell cultures are now used as a tool for the toxicological evaluation of substances. There are also bioreactors that reproduce an environment favorable to the development and organization of cells, close to that of an animal or human tissue or organ. Thanks to them, one can predict the reaction of an organ vis-à-vis a substance such as a xenobiotic, a cosmetic, a drug or more generally any active ingredient, and develop relevant in vitro models. These models are increasingly used in pharmaceutical research because they represent a serious alternative to in vivo models, ie animal testing, against which both economic and ethical pressures are emerging at the international level. Cell culture is also the basis for the creation of artificial organs capable of substituting for failed or absent organs, one of the challenges of tissue engineering. The bioreactors comprise at both ends of a microstructured chamber (a culture chamber comprising an upper wall and a lower wall having microstructures) a fluid entry point and a fluid outlet point to allow the passage of a fluid. nutrient needed for cell development. The fluid is circulating, for example mu pump, this is called a dynamic cell culture. Such a device has been described in the patent application FR0954288. 1 This precursor bioreactor offers excellent results and allows optimal cell development, but can not alone be sufficient for industrial use. It would be desirable to be able to increase as much as possible the culture surface of the cells.

One solution for this is to use multi-bioreactors, i.e., parallelized bioreactors. These devices should also have a small footprint. Various integrated cell culture systems have been proposed for this purpose. The Hprel® system, described by patents US5612188 and US7288405, consists of four double bioreactors arranged in parallel and connected to a peristaltic pump. It should be noted that the introduction of the bioreactors into the perfusion circuit requires a long and delicate operation under sterile conditions to avoid the formation of potentially very harmful bubbles for the developing cells.

The addition of a bubble trap in the circuit is also necessary. In addition, these bioreactors are inside a glass block several centimeters thick that does not have connectors. The connection is made via tips placed in holes of the glass block.

This system provides a seal, but is problematic when seeding into cells. Indeed the human cell strains are extremely expensive, and must be implemented carefully. But the only solution for seeding is to inject at the tips, which represent a large dead volume input. Thus a substantial part of the cells is wasted, since they are deposited on the walls of the ducts and not in the bioreactors. This causes an uncertainty on the injected quantity which forces to completely fill the block of glass for safety. It is estimated that 50% of the cells are lost in advance. In addition, the structure of this device requires seeding two consecutive bioreactors with the same cells, hence redundancies.

The BiofluxTM 200 and BiofluxTM 1000 systems, described in US2007 / 0243523, provide a sealed box using a commercially available multiwell plate. A multiwell plate is a standardized plate with many wells acting as small test pieces. This is a tool commonly used in laboratories, available in many models. In this adapted plate, microchannels connecting two wells serve as support for cell growth. However, these systems do not allow dynamic cell culture, because the perfusion of the culture medium is done by applying an overpressure. The problem of bubbles no longer arises, but this system is much less favorable to cell culture. In addition there are no microstructures, but a simple channel between two wells. The adhesion and the effective surface are very weak and the yield drops. A huge quantity of cells must be blindly injected to seed such a device. Knowing that a dose to seed a single microbioreactor, of the order of one million cells, costs several hundred dollars, it is essential to improve cell seeding processes to make it a profitable tool. In addition, these known devices comprise in series in the circuit a reservoir of a few milliliters of nutrient medium. This tank is also used to take samples. In addition to complicating the perfusion network since it requires one of each by parallel bioreactor, the possible reservoirs and bubble traps lengthen the circuits, and increase the internal surface of the pipes. This surface is the seat of adsorption of chemical substances in solution. This adsorption distorts the toxicology tests, because a xenobiotic injected at the beginning of the circuit would see its concentration fall or even drop to zero before even meeting the cells in culture at the end of the circuit. The risk of false negatives is not negligible.

PRESENTATION OF THE INVENTION The present invention aims at solving the above-mentioned difficulties by proposing a device which, by a simple structure, makes it possible to parallelize efficient bioreactors of dynamic cell culture while avoiding reservoirs or external bubble traps. and greatly reducing the necessary circuit length. An object of the invention is to achieve this goal while obtaining a product that is easier to use and no longer presents a risk of destruction of the cellular environment by a bubble.

An ancillary purpose is to allow controlled, rapid, and more economical cell seeding.

The present invention thus relates to a box comprising at least one well and at least one microstructured chamber of dynamic cell culture, characterized in that each microstructured chamber is connected to a well at the inlet, the bottom of the well being at an altitude greater than every point of the room.

Thanks to this structure, the well or wells fulfill a function of bubble trap. The fluid column being located at an altitude greater than the microchamber and each well being infused from above, the bubbles eventually carried off escape upwards by buoyancy. The air can not be drawn into the circuit. The wells also perform a reservoir function. Indeed, the volume of each well is of the order of one milliliter. By ideally connecting each chamber to two wells, one is close to the conventional volume of 2-3mL in the circuit, and an external reservoir becomes useless. Moreover, this automatic emptying facilitates the procedure of filling the circuit at the beginning of culture: it is no longer necessary to purge the entire circuit carefully, since during the first fluid turn the bubbles will escape upstream of the microchamber, it Just check that the level of fluid in the first well is sufficient. More than one hour of work is thus saved. And there is no longer the risk that the crop is destroyed if this work was not done perfectly before seeding. Finally, these wells make it possible to seeding directly into the chamber without any loss, and which requires only a pipette. The operation is fast, accurate, and can be performed on many rooms in a row. There is no need to use a syringe as in the prior art devices. The device is operational in minutes for a minimal cost.

According to other advantageous and non-limiting characteristics: each microstructured chamber is connected directly to a well via a hole at the bottom of the well; the box comprises a microsystem, interface connectors of said microsystem fitting into the hole or holes; - The microsystem consists of a bottom plate comprising the footprint of said at least one microstructured chamber, and an upper plate; the connector is a deformable tube forming part of said upper plate, through which the tip of a seeding pipette can be inserted into the chamber; - The connector is a connector attached to the hole infusing the chamber through holes in the upper plate; each microstructured chamber is connected to an inlet well and an outlet well, each of which is infused with inlet and outlet connections respectively; - The box consists of a lower housing covered with a sealed cover held by clamping means; the lower box comprises the microsystem and a multi-well plate, the wells being wells of said multi-well plate; - The cover includes connectors male perfusing the wells and connectors female input; - the box includes a male connector and a female connector per input well and a male connector and two female connectors per output well; the microsystem consists of polydimethylsiloxane; the multi-well plate is a disposable polystyrene plate; the multi-well plate is an autoclavable polycarbonate plate; - A membrane insert is disposed in at least one of the wells.

The invention also proposes, according to a second aspect of the invention, a dynamic cell culture system characterized in that it comprises: - a box according to the first aspect of the invention, and - at least one fluid circuit comprising a circulation pipe provided with a circulation means and connected to at least one microstructured chamber. According to other advantageous and non-limiting characteristics: the circulation means is a multichannel peristaltic pump.

By removing bubble traps and reservoirs, it is possible to reduce the perfusion circuit length by half. The results of toxicological studies on a cell culture system according to the second aspect of the invention will be all the more reliable with regard to similar tests carried out on known devices.

The invention finally proposes, according to a third aspect, a method of cell seeding a box according to the first aspect of the invention, characterized in that it comprises steps of: a) opening of the cover; b) Introduction of cells to be seeded in a pipette; c) Inserting the tip of the pipette into a connector; d) Injecting cells directly into the microstructured chamber from the pipette; e) Sealing the hood and locking by the clamping means. According to other advantageous and non-limiting characteristics: the pipette is a volumetric micropipette, calibrated to contain a predetermined quantity of cells corresponding to the optimum seeding of a microstructured chamber; steps b), c) and d) are repeated as many times as there are chambers in the microsystem; the pipette is a multichannel pipette with spacing adapted to the multi-well plate.

PRESENTATION OF FIGURES

Other features and advantages of the present invention will appear on reading the following description of a preferred embodiment. This description will be given with reference to the accompanying drawings in which: - Figure 1 is an exploded perspective view of a box according to the invention; - Figures 2a and 2b are two cross sections of the box according to the invention, decapitated in the first and covered in the second. Figure 2a shows the seeding of a microstructured chamber, and Figure 2b shows the fluidic path in the box; - Figure 3 is an exploded perspective view of the lower case of the box according to the invention; FIG. 4 is an exploded perspective view of the microsystem of the box according to the invention; - Figure 5 is a perspective view of another possible embodiment of the lower case of the box according to the invention, wherein the microsystem is detachable; - Figure 6 is an exploded perspective view of the cover of the box according to the invention; FIGS. 7a-7c show three possible types of multiwell plate that can be used in various embodiments of the box according to the invention; FIGS. 8a-8b are examples of microscope observations of cells in culture in various embodiments of the box according to the invention; FIG. 9 is a diagram of a dynamic cell culture system according to the other aspect of the invention.

DETAILED DESCRIPTION

General Architecture

Box 1 according to the invention is preferably aimed at parallelizing the bioreactors. It will thus be designated in some embodiments as a multi-reactor box. However, the invention is not limited to this embodiment and may comprise a well 10, and a microstructured chamber 15. In the preferred embodiment illustrated by the figures, the box 1 consists of a lower housing 100 that comes to cover a sealed cover 200 held by a clamping system 20, for example four screws passing through it, as shown in FIG. 1. This housing 100, which is better seen in detail in FIG. , comprises a multiwell plate 110, among which we will find the wells 10, and a microsystem 150. Threaded spacers 21 may be arranged between the wells 10 in a complementary manner with the clamping means 20. These spacers 21 may be part of some multi-well plates 110, or be tubes inserted into holes drilled in the housing, and glued.

The multi-well plate 110 is an object well known to those skilled in the art which can thus be declined in many forms, and which is sometimes called microtiter plate, with reference to the small calibrated volumes of the wells. By convention, these are arranged in a 2: 3 ratio matrix, and generally 6, 12, 24, 96 or 384 well plates are commercially available. Advantageously, for considerations related to the volume of the wells, the multiwell plate 110 according to the invention is a 24-well plate. Such a box contains six rows of four wells 10 arranged as shown in Figure 2a or 2b. At the bottom of the wells 10 are drilled holes 11 through which fluid communication takes place between the wells 10 and the microstructured chambers 15. Each hole 11 makes it possible to inoculate efficiently, since the cell strains injected into this hole fall through gravity in the microstructured chamber 15 to which it is connected. There is no dead volume in which the cells would be deposited before reaching the chamber, as in the prior art. The microsystem 150 advantageously adapts to a multipurpose plate 110 by being composed of twelve chambers 15 of identical dimensions, the surface of which is microstructured, arranged in such a way that there are two of them per row, each of which is particularly preferred but not limited to a bioreactor according to the patent application FR0954288. By choosing the ratio one chamber for two wells, one obtains a volume of stored liquid of 2-3mL per chamber, which is equivalent to that of the additional tanks required in the previous devices. The invention is however not limited to this architecture, and it is only necessary to have at least one well per chamber, this well being arranged as input. For example, in another embodiment, a box according to the invention may comprise six microstructured chambers and a six-well plate. Two parts visible in FIG. 4 can compose the microsystem 150: a bottom plate 152 bearing the footprint of the chambers 15, and an upper plate 151.

Once these two plates assembled hermetically, the cavities that appear are the chambers 15. These are small volumes in which the cells will develop under very favorable conditions. Indeed, the microstructures form complex channels offering a large contact surface with the fluid. The presence of a microsystem 150 is however optional, other structures that can house a cell culture chamber being known to those skilled in the art.

Embodiments of the top plate of the microsystem

The upper plate 151 may be subject to two advantageous embodiments. It will be appreciated that the invention is not limited to either of these embodiments, the upper plate 151 being an optional element. However, each of these two embodiments combines perfectly with all the features described above.

One material, PolyDiMethylSiloxane (PDMS), is particularly suitable for both of these embodiments, as well as for the rest of the microsystem 150. Indeed, it is transparent, porous to oxygen and gas in general, and very suitable for microstructuring, hence its interest in cell culture. In addition, it is slightly deformable.

In the first embodiment, the upper plate 151 comprises connectors 16 input and output of the microsystem 150, visible in most of the figures and particularly in Figure 4. These small tubes fit into the holes 11 wells 10, and provide fluid communication in normal operation. If the wells 10 are wells of a multi-well plate 110, the seal may be optionally reinforced for example by a silicone material seal cast at the bottom of each well 10. If the material that makes up the connectors 16 is flexible, as is the case with silicone materials, these connectors 16 will be able to accommodate by elasticity the tip of a pipette 2 used as a seeding tool. Indeed a pipette is a conventional instrument for sampling and transferring a precise amount of fluid, generally by suction through a kind of pump called propellant overlying it. The term micropipette is used to designate a precision system preferably used in the invention, in which the pneumatic mechanism is integrated. In such a system, only the tip (which is a disposable cone) is in contact with the fluid, but it will improperly speak pipette generically as being the fluid receptacle in the following description. The pipette 2, or more precisely the tip of the pipette 2, can therefore be filled with cell seeding liquid. Thus inserted into a connector 16, the tip of the pipette 2 has its end at the base of the upper plate 151, that is to say at the ceiling of one of the chambers 15. Direct access to the inside of the room is allowed. Moreover thanks to the tension due to the conical deformation of the connector 16, the seal between the connector 16 and the tip of the pipette 2 inserted is ensured. A piston allows an operator to empty the liquid column of the pipette by compression. The ejected volume can be controlled very precisely, especially if the pipette used is a micropipette. Since injected directly to the ceiling microstructured chambers, the fluid does not touch any other body that this room in which it spreads. The fluid initially present, which may be for example culture medium or a saline solution, is driven by buoyancy and escapes through another connector 16 of the chamber. With these connectors 16, optimal seeding is possible.

In the architecture shown in the drawings, there is a connector 16 at the inlet and the outlet of each chamber 15, the central connectors being fluid inlets, and the side of the fluid outlets. By analogy, the wells 10a at the center of the rows are input wells, and the wells 10b at the row edges are exit wells. Note that even if preferentially there are two connectors 16 per room, it suffices that only one of these connectors is connected as previously described to a well to allow direct cell seeding. The input and output connectors can also be used interchangeably.

In the second embodiment, represented in FIG. 5, the upper plate 151 is provided with holes in place of the connectors 16 of the first embodiment. These are no longer integral with the microsystem 150, but are hard connectors fixed in the holes 11 of the wells 10, for example by screwing, but also by gluing, molding, or any other known technique. These connectors 16 fit removably into the holes to easily separate the microsystem 150 from the rest of the box 1. Their material is preferably polycarbonate or polypropylene. The flexibility of the PDMS is again very significant for the plate 152. By adjusting the diameter of the holes, a hermetic junction between the microsystem and the connectors 16 is possible, similarly to the junction between the connectors 16 and the pipette 2 in the previous embodiment. This allows operation without risk of leakage, even after successive uncoupling and resolidarizations of the microsystem 150.

Hood structure

It is recalled that the sealed cover 200 is an optional element of the invention that can be the subject of many embodiments. It is compatible with any of the embodiments of the lower case 100 mentioned above. An advantageous cover 200, detailed in FIG. 6, is preferably made up of a 10 mm thick polycarbonate plate in which 24 male polypropylene connectors 204 and 36 female polypropylene connectors 201 are screwed (04 mm 10-32 UNF). 24 of these female connectors 201a and 201c are located on the top of the cover 200, and are aligned with the male connectors 201. The other twelve 201b are on the side of the cover 200. Thus, when the box is closed, the sealing being provided by a flat seal PDMS 203 2 mm thick, there is a male connector 204 and one or two female connectors 201 per well 10: - for the input wells 10a, there is a single connector of each type (201 a and 204). The fluid inlet is on top of the hood 200. - For the output wells 10b, there are two female connectors 201 b-c. Only the lateral connector 201b serves for the passage of the fluid. The female connector 201 c serves as an access hatch for taking samples.

Integrated debuller system

Figure 2b shows the fluidic path. The fluid enters through the female connector 201a. It is transmitted into the input well 10a by a male connector 204. The fluid contained in this input well 10a is then perfused by the input connector 16 of the microstructured chamber 15, to be collected in the output well. 10b via the connection 16 output of the chamber 15. The excess fluid contained in the outlet well 10b is then sucked by a male connector 204 and routed to the female connector 201b of the cover. By taking care of the volume used, it is thus possible to continuously infuse the culture chamber. Each well 10 having a volume of the order of one mL, the two wells 10a-b per chamber 15 are equivalent to an external reservoir of 3 mL.

Moreover, as can be seen in FIG. 2b, any point of the well 10a is situated at an altitude greater than a point of the chamber. At higher altitudes, conditions such that any drop of fluid in chamber 15 has less potential gravitational energy than any drop of well 10a are implied. This is the case by placing the box 1 according to the invention in a normal operating position, that is to say with the bottom of the lower housing 100 placed on a surface close to the horizontal.

Due to this difference in altitude, even if an air bubble would come out of the connector 204, Archimedes' pushing would bring it back to the top of the well 10a. It would be contrary to the mechanics that it could reach the bottom of the well, and enter the chamber 15. As has already been said, thanks to this structure it is possible to automatically debulle the circuit provided only to be careful volume of fluid in the well 10a. This is also true during the initial filling of the circuit, during which it was necessary to purge carefully the pipes of any air bubble for the prior art devices. In the box according to the invention, there is a direct communication between the wells 10 and the chamber 15 via the connector 16. Once the latter has been seeded and therefore filled by an operator, it suffices to fill each well 10 with nutritious liquid to ensure that there is no air bubble enclosed in the lower housing 100, by gravity. The cover 200 is closed, and the air bubbles possibly present in the rest of the circuit will be captured by the wells 10a. Tests have been carried out, and it appears that an operator only needs seven minutes to infuse after seeding a complete multi-reactor box, that is, twelve bioreactors. This operation previously took between one hour and one and a half hours, during which the operator had to gently inject fluid into each bioreactor by means of a syringe, slowly filling the perfusion circuit, then degassing it, which no longer takes place being.

Microscopic observation of cells during culture

Thanks to their materials, the cover 200 and the microsystem 150 are autoclavable: according to the needs of the users, it is possible to reuse them in some cases for a new culture after having cleaned and sterilized them. For the multiwell plate 110, many materials exist. It is first possible to choose a polystyrene plate, like that of Figure 7a. A plate 110 of this material is disposable and must be discarded at the end of culture. On the other hand, it is inexpensive and perfectly transparent, making it easy to observe the cells during culture: the overall size of the multi-reactor box according to the invention is not greater than that of a Petri dish, it can be placed whole under a microscope. It is indeed desirable to be able to monitor the cells at any time without disturbing them, both to ensure good growth and to observe the direct toxicological impact of a substance. Alternatively one can choose a plate 110 of polycarbonate, that is to say the same material as the cover 200. The assembly is then autoclavable, but the polycarbonate has the disadvantage of becoming opaque when machined, which is problematic for observation of chamber parts under wells. A first solution, if one chooses a polycarbonate multi-well plate 110 as shown in FIG. 7b, consists in cutting the bottom of the wells 10, and interposed between the box 110 and the microsystem 150 a thin extra plate 111 of polycarbonate and a PDMS seal ensuring a seal identical to that between the housing 100 and the cover 200. A clamping means similar to the means 20 then holds the lower housing uni. Thanks to this additional plate 111 perfectly transparent because not machined, observation under the microscope is perfect and at the same time the whole is autoclavable. A second, more clever, solution consists in choosing a plate 110 with half-moon shafts, represented in FIG. 6c. Indeed, we note for example in Figure 4 that the most interesting area of observation is the central part of the rooms 15. Indeed this is where the heart of the cell culture, the side parts of a room 15 being rather fluid distribution channels. And this central zone is not located under the wells 10, but between a well 10a and a well 10b. In the case of a half-moon box, it is an open rectangular area that corresponds to the "flat" of the half-moon wells. A plate with half-moon wells is thus perfectly adapted to the observation, without there being any need to modify the bottom of the wells.

The images obtained by observing the cells throughout the entire multi-bioreactor box are thus of remarkable precision, despite the thickness of plastic traversed. Figure 8a is an example, a precision of ten microns is reached, largely sufficient to count the cells. However, it is possible to achieve an even higher optical accuracy in the case of a micro-system box 150 detachable (Figure 5) described above. During the cell culture, it is thus sufficient to stop the flow of fluid, and to separate the bioreactors from the housing 100 by gently pulling on the microsystem 150 PDMS. The latter can be introduced into a sterile transparent enclosure and observed with a level of detail and unequaled contrast, since the vision is direct, through only a few millimeters of plastic. It can then be observed to the inside of the cells, as shown in FIG. 8b, for example to analyze in a very thorough manner certain effects of a test substance. During the uncoupling phase, the upper housing 100 remains filled with liquid: the circulation being stopped, the fluid is maintained in the thin channels of the screwed connectors 16 by simple capillarity provided that the cover 200 is not open. The microsystem 150 is then reconnected to the rest of the box 1 via the connectors 16, to continue and resume the infusion. It has been tested experimentally that it is sufficient to place a drop of culture medium in each inlet and outlet hole of the chambers before reconnecting the assembly to avoid the risk of introduction of an air bubble into the chamber. a room 15.

Dynamic cell culture system

According to another aspect, the invention relates to a dynamic cell culture system comprising a box 1 according to the first aspect of the invention, at least one fluid circuit 300 and a pump 320, preferably a peristaltic pump, or any other means of fluid circulation known to those skilled in the art. The various members are connected on the circuit 300 by a pipe 310 consisting of tubes, preferably the shortest possible tubes, and the least adsorbent internal surface possible to limit any change in the concentration of a substance injected into the circuit 300 The fact that the culture is dynamic means that there is closed circuit recirculation of the fluid. In such a configuration, the cell development conditions are closer to those of the body than in the prior art systems in which the fluid is static, or never performs a complete circuit rotation (velocity movement). and / or fluid in a chamber, which in particular does not allow comparison between the fluid entering the chamber and that exiting the chamber, or single passage of the chamber with a fluid that is not reused).

A peristaltic pump is a pump in which the fluid is entrained by compression and deformation of a tube, in the manner of muscular contractions around the esophagus, for example. This system is particularly suitable for application to cell culture for many reasons. Firstly, it applies mainly to small flows, for which it is of great precision and flexibility. The stoppage of the pump coincides exactly with the immobilization of the fluid in the circuit. Then, there are no elements other than the flexible tube in contact with the fluid. There is no risk of contamination or leakage. Finally, only one pump can handle several fluid circuits at a time, especially 24 for a particularly preferred Ismatec® IPC-N pump. This corresponds to the totality of the chambers of two multi-reactor boxes previously described. By varying the internal diameter of the pipes 310, it is also possible to work at the same time with different flow rates depending on the circuits, which can for example be useful for comparing different kinetics of exposure to a substance.

An example of a circuit with a twelve channel pump 320 and a box 1 according to the invention is shown in FIG. 9. There is a circuit 300 of fluid per chamber 15. It is again noted that no additional element such as a tank or a trap Bubble is not required. The length of each circuit 300 and therefore the variations in concentrations of the injected substances are minimal. In this example, all cultures are parallelized. Thanks to the connectors 201, it is however possible to realize very varied architectures, with different reactors in series or in parallel. This modular aspect possible thanks to the dynamic aspect of the cell culture of the system according to the invention makes it possible to simulate complex metabolisms, for example a sequence of several bioartificial organs and / or different types of characteristic cells of the same organ. This is called co-culture. Indeed the metabolic response to a substance could be on several levels: not to affect a first organ but to affect a second, to cause the secretion of a substance by a first organ that would affect a second, etc. In a particularly preferred embodiment, each chamber 15 of the multi-reactor box simulates a different organ (liver, kidney, pancreas, etc.), these bioartificial organs being wired in multiple circuits representing the closest human architecture ( kidney downstream of the liver for example).

Membrane inserts Even more advantageously, the box according to the invention makes it possible to simulate certain "filters" present in the body. Thus, the blood-brain barrier is an anatomical barrier that filters and controls the passage of blood substances and prevents them from freely passing blood to the extracellular liquid of the central nervous system. It isolates the gray matter from the rest of the body and allows it to have a specific environment. Only 2% of the molecules pass through it freely. Substances that would be neurotoxic if injected directly without the brain could be completely safe if injected normally. To take this into consideration, it is possible to place membrane inserts in the wells. These commercially available carriers are in the form of a small disk having the physicochemical properties of the anatomical membrane to be simulated, and are installed in the inlet and outlet wells of the organ to be isolated. The dynamic culture offered by the system according to the invention is again necessary to use this possibility. It is thus possible, for example, to complete the structure described above by simulating the passage into the blood of a molecule ingested by means of an intestinal mucosa of synthesis.

In a last aspect, the invention relates to a method of cell seeding of the multi-bioreactor can. As it has been explained, the connectors 16 can receive the tip of a pipette 2. Before being able to perform seeding as such, it is necessary to access the connectors 16 by opening the cover 200, usually after having unlocked the clamping means 20. Therefore, the bottom of the wells 10, and therefore the connectors 16, are accessible. The operator then uses a pipette 2 as a seeding instrument. The seeding configuration is visible in Figure 2a. Once each room is seeded, it is only necessary to close the hood, connect the circuit 300 or circuits, and start the perfusion with the nutrient fluid, to initiate the development of cells.

Preferably, the pipette 2 is a volumetric micropipette.

Unlike pipette graduates, these pipettes are designed to contain a particular volume, for which they are extremely accurate. Since the volume of a chamber 15 is known, as well as the quantity of cells to be injected therein, it is possible to determine the optimal volume corresponding to the seeding of a chamber, and to choose a perfectly adapted volumetric micropipette. With such an instrument, the operator only has to repeat for each of the chambers of the steps of: filling the pipette 2, introduction of its tip into a connector 16 of the chamber, injection of the content, and removal of the point. Each seeding takes only a few seconds, uses a rigorously known and minimal amount of cell liquid, and guarantees optimal crop conditions. Alternatively, a graduated pipette filled with a large volume of liquid may be used and the chambers 15 may be seeded afterwards, visualizing the volume to be injected each time by means of the graduations. Particularly preferably, a multichannel pipette adapted to the dimensions of the multi-well plate 110 will be used. It is a pipette comprising a single handle, but multiple tips (in this case six in the case of a 24-well box as shown in the figures). This instrument makes it possible to seed simultaneously a chamber of each row, and thus to seed the whole box in only two manipulations.

A multichannel pipette can even be equipped on an automated dispensing system. Such a system includes a robotic arm and a supply or a fluid reservoir. It moves itself from one position to another in order to automatically seed all the chambers of a multi-bioreactor box, or even several. This kind of device opens the way to a mass culture of bioartificial organs.

Claims (21)

REVENDICATIONS1. Box (1) comprising at least one well (10) and at least one microstructured dynamic cell culture chamber (15), characterized in that each microstructured chamber (15) is connected to a well (10) at the inlet, the bottom of the well (10) being at an altitude greater than any point in the chamber (15). 2. Box according to the preceding claim, characterized in that each microstructured chamber (15) is connected directly to a well (10) via a hole (11) at the bottom of the well (10). 3. Box according to the preceding claim, characterized in that it comprises a microsystem (150), interface connectors (16) of said microsystem (150) to be inserted into the hole or holes (11). 4. Box according to the preceding claim, characterized in that the microsystem (150) consists of a bottom plate (152) comprising the footprint of said at least one microstructured chamber (15), and a top plate (151). ). 5. Box according to the preceding claim, characterized in that the connector (16) is a deformable tube forming part of said upper plate (151), by which one can insert the tip of a seeding pipette (2) in the bedroom (15). 6. Box according to claim 4, characterized in that the connector (16) is a connector attached to the hole (11) infusing the chamber (15) through holes in the upper plate (151). 7. Box according to one of claims 3 to 6, characterized in that each microstructured chamber (15) is connected to an inlet well (10a) and an outlet well (10b) each infused with a connector (16) respectively input and output. 8. Box according to the preceding claim, characterized in that it consists of a lower housing (100) covered with a sealed cover (200) held by clamping means (20). 9. Box according to the preceding claim, characterized in that the lower housing (100) comprises the microsystem (150) and a multiwell plate (110), the wells (10) being wells of said multiwell plate (110). 10. Box according to one of claims 8 to 9, characterized in that the cap comprises male connectors (204) perfusing wells (10) and female connectors (201) input. 11. Box according to the preceding claim, characterized in that it comprises a male connector (204) and a female connector (201a) by inlet well (10a) and a male connector (204) and two female connectors (201 bc ) per outlet well (10b). 25 12. Box according to one of claims 3 to 11, characterized in that the microsystem (150) consists of polydimethylsiloxane. 13. Box according to one of claims 9 to 12, characterized in that the multi-well plate (110) is a disposable polystyrene plate. 15 20 14. Box according to one of claims 9 to 12, characterized in that the multi-well plate (110) is an autoclavable polycarbonate plate. 15. Box according to one of the preceding claims, characterized in that a membrane insert is disposed in at least one of the wells (10). 16. A dynamic cell culture system characterized in that it comprises - a box (1) according to one of the preceding claims, and - at least one circuit (300) of fluid comprising a circulation pipe (310) provided with circulation means (320) connected to at least one microstructured chamber (15). 17. System according to the preceding claim characterized in that the means (320) for circulation is a multichannel peristaltic pump. 18. Method of seeding a box (1) cell according to one of claims 8 to 15, characterized in that it comprises steps of: a) opening of the cover (200); b) Introduction of cells to be seeded in a pipette (2); c) insertion of the tip of the pipette (2) into a connector (16); d) injecting cells directly into the microstructured chamber (15) from the pipette (2); e) Sealing the hood (200) and locking by the clamping means (20). 25 5 10 19. Method according to the preceding claim, characterized in that the pipette (2) is a volumetric micropipette, calibrated to contain a predetermined amount of cells corresponding to optimal seeding of a microstructured chamber (15). 20. Method according to one of claims 18 to 19, characterized in that steps b), c) and d) are repeated as many times as there are chambers (15) in the microsystem (150). 21. Method according to the preceding claim, characterized in that the pipette (2) is a multichannel pipette spacer adapted to the multiwell plate (110).