EP3221442A1

EP3221442A1 - Process for assembling a secure cell culture system

Info

Publication number: EP3221442A1
Application number: EP15798335.4A
Authority: EP
Inventors: Nicolas TARDIVEL; Audrey LAMS
Original assignee: Maco Pharma SAS
Current assignee: Maco Pharma SAS
Priority date: 2014-11-20
Filing date: 2015-11-19
Publication date: 2017-09-27
Also published as: FR3028863A1; WO2016078777A1; FR3028863B1

Abstract

The invention relates to a process for using a cell culture system in order to carry out a sequence of operations on said cells, said culture system comprising an assortment of sterile devices, said assortment of sterile devices comprising: - a culture device (1) comprising a culture container (4) suitable for culturing cells, - more than one transfer device (2) intended to enable the movement of fluid towards the culture device (1) or from said culture device (1), said transfer device (2) comprising at least one empty pouch (14) in fluidic communication with a primary transfer tubing (15) connected to which is one or more secondary transfer tubings (16), and - a multiple coupling device (3) in order to couple the culture device (1) to more than one transfer device (2), said multiple coupling device (3) comprising a plurality of coupling tubings (7), said process comprising the steps consisting in: (a) sterilely connecting one of the coupling tubings (7) of the multiple coupling device (3) to one of the transfer tubings (16) of the transfer device (2), (b) carrying out, in a closed system, an operation on the cells to be cultured, (c) once the operation has been carried out, disconnecting said transfer device (2) from said multiple coupling device (3), and (d) repeating the steps (a) to (c) with another transfer device (2) in order to carry out another operation on the cells.

Description

Method for assembling a secure system for cell culture

The invention relates to a method for implementing a cell culture system in order to carry out a succession of operations on said cells.

The invention is typically applied to cell culture, in particular in the context of cell therapy, that is to say when the cells are intended to be injected into a patient for the purpose of treating it. Cell culture refers to a set of techniques used to grow cells out of their body ("ex-vivo") or their medium of origin, for therapeutic purposes, scientific experimentation or biotechnological production. Among these techniques, a first operation consists in seeding the cells. Seeding comprises culturing cells in a culture vessel. This seeding is carried out by introducing into the culture vessel, cells suspended in a culture medium.

The resetting operation consists of a change or renewal of the culture medium. In this case, the culture medium is removed from the culture vessel and replaced with a fresh culture medium. This operation is used to provide cells in culture with the elements necessary for their maintenance, proliferation, expansion and / or differentiation.

At the end of the culture, the cells are subcultured, i.e. they are concentrated and removed from the culture vessel for recovery or reseeding into other culture vessels.

Alternatively, at the end of the culture, the cell-free supernatant may contain products of interest is recovered.

These different operations must be carried out under aseptic conditions in order to avoid any bacterial contamination.

SUBSTITUTE SHEET (RULE 26) However, these different operations require the use of various containers such as syringes, flasks, flanges and pockets. In addition, these containers are either empty or filled with various solutions, such as culture media, culture medium supplements or reagents.

Because of the diversity of the operations to be performed on the cells, the diversity of the containers to be used and the diversity of the solutions to be used, it is not possible to work with closed systems, that is to say systems in which the various components would all be pre-connected manufacturing. In practice, these operations are therefore performed in an open system, generally under a laminar flow hood, in order to reduce the risk of contamination of the cells. These operations carried out under a hood, however, always constitute a risk of contamination due to possible handling errors. On the other hand, some culture vessels being relatively bulky, manipulations in a hood are not always easy. Solutions have already been proposed to secure cell culture operations.

For example, the document EP 1 903 101 A1 proposes a system of pipes, designated transfer system, for performing the medium change of the system step closed, once the connection made between on the one hand the medium bag of fresh culture and transfer system and secondly between the culture vessel and the transfer system. However, these two connections are now performed under laminar flow hood. On the other hand, this system allows to secure only one of the culture operations.

US 2014/0335608 discloses a so-called "closed" culture system for seeding cells in a culture vessel and the introduction, sampling and removal of the culture medium. The system of culture comprises firstly a culture vessel pre-connected to a sampling vessel and a cell harvesting vessel and secondly a culture medium container and a container containing the cells to be cultured, these two containers being, at the time of cultivation, connected via aseptic connectors to a tubing connected to the culture vessel. To ensure the sterility of the system, connections should be made in a laminar flow hood. In addition, this system is not easily scalable according to the culture protocols to be applied. US 201 1/0281358 A1 discloses a closed system for isolating and collecting cells, such as bone marrow progenitor cells, and then seeding them on a biocompatible three-dimensional matrix or tissue graft. However, if this system is adapted to perform the seeding operation, other subsequent operations to be performed require the opening of the system.

Also known from WO 87/06952 is a system for carrying out the introduction of a culture medium into a culture vessel in the form of a flexible bag comprising the cells to be cultivated. The various elements of this system can in particular be interconnected by sterile connectors. But, this system requires to adapt all the useful containers to the culture of cells by equipping them with specific sterile connectors. On the other hand, the number of connections to be made for certain operations can be important, making the use of such a system difficult.

The invention proposes to secure all cell culture operations by providing a closed system that can be manipulated outside a laminar flow hood, thus facilitating the manipulations required for said operations. The invention also proposes to simplify said operations by providing a single device that can be used regardless of the operation to be performed and whatever the particular protocol followed by the user. To this end, the invention relates to a method for implementing a cell culture system for performing a succession of operations on said cells, said culture system comprising an assortment of closed and sterile devices, said assortment of devices comprising :

a culture device comprising a culture vessel adapted for culturing cells,

more than one transfer device for allowing the movement of fluid towards the culture device or from said culture device, said transfer device comprising at least one empty bag in fluid communication with a primary transfer tubing on which is connect one or more secondary transfer tubings, and

a multiple coupling device for connecting the culture device to more than one transfer device, said multi-connection device comprising a plurality of connection tubes, more than one of said connection tubes being intended to be connected to the sterilization device; one of the transfer tubings of said transfer devices, said method comprising the steps of:

(a) connecting one of the connection tubes of the multiple coupling device to one of the transfer tubes of one of said transfer devices,

(b) performing a closed system operation on the cells to be cultured,

(c) after the operation is performed, disconnecting said transfer device from said multi-connection device, and

(d) repeating steps (a) through (c) with another transfer device to perform another operation on the cells.

Other objects and advantages will become apparent from the following description.

Figure 1 schematically shows a culture vessel equipped with a closure member provided with a primary connection pipe.

Figure 2 schematically shows the closure element of Figure 1, provided with a primary connection pipe. Figures 3 and 4 show schematically each an embodiment of a set of connection pipes. Figure 5 schematically shows a particular embodiment of a multiple splicing device.

Figures 6 to 8 each show an embodiment of a transfer device.

Figure 9 schematically shows a container necessary for performing a culture operation that can be connected to a sterile transfer device. Figure 10 schematically shows a culture device, a multiple coupling device and a transfer device assembled together.

The invention provides a method for implementing a secure culture system to perform certain closed system cell culture operations such as seeding, medium changing and cell transplanting. Another operation is the sampling of the culture medium or of the cell suspension contained in a culture vessel.

The cells are, for example, animal cells, in particular human cells intended to be injected to a patient for a therapeutic purpose. The cells are in particular adherent type cells such as mesenchymal stem cells. The human cells are especially non-embryonic cells. The cultivation operations generally require the use of a culture medium which is a biological medium, in particular adapted to the expansion or maintenance of the cells. The culture medium is for example a nutrient medium. In particular, the nutrient medium is a synthetic base medium available in the trade, such as Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), CMRL, EMEM, RPMI1640, DMEM / F-10, DMEM / F12.

This base medium can be supplemented with various additives commonly used by those skilled in the art.

Among the additives, mention may be made of serum or a serum substitute such as platelet lysate, non-essential amino acids, L-glutamine, vitamins, antibiotics, beta-mercaptoethanol, growth factors, cytokines, etc. .

Alternatively, the biological medium is a differentiation medium allowing, for example, the differentiation of stem cells into specific cells.

In connection with the figures, the secure culture system comprises a culture device 1 intended for culturing cells, plus a transfer device 2 intended to allow the movement of fluid towards the culture device or from the device for culture and a multiple coupling device 3 for connecting said culture device 1 and one of said transfer devices 2 so as to put in fluid communication said culture device 1 and said transfer device 2.

According to Figure 1, the culture device comprises a culture vessel 4 adapted to the culture of cells.

The culture vessel 4 is a flexible container such as a pocket or a rigid container such as a bottle, a multi-stage bottle, for example CellStack ™ from Corning or the Cell Factory ™ by Thermo Fisher Scientific, or even rotating bottles.

In connection with FIG. 1, the culture vessel 4 comprises at least one primary orifice to allow the introduction of fluid into said culture vessel 4 or the withdrawal of fluid from said vessel 4. In a particular example, the culture vessel 4 is a rigid and transparent container, made for example of a biocompatible polymer material such as polystyrene. The polymeric material can be chemically and / or physically treated to allow cell adhesion.

Other biocompatible materials for making the culture vessel 4 are polypropylene, ethylene vinyl acetate, Teflon®, polycarbonate, polyethylene terephthalate, glycolized polyethylene terephthalate (PETg) or high density polyethylene (HDPE) .

In Fig. 1, the culture vessel 4 is provided with at least one other vent-forming secondary port 5 comprising a liquid impermeable and gas permeable membrane. The membrane advantageously has a porosity of 0.22 μηι so as to form a sterile vent and to keep the sterility of the culture device. This vent makes it possible in particular to provide the cultured cells with the oxygen necessary for their survival.

At least a part of a multiple coupling device 3 is arranged at the primary orifice of said culture vessel 4.

In connection with FIG. 5, the multiple coupling device 3 makes it possible to connect the culture device 4 to more than one transfer device 2 and comprises a plurality of connecting tubes 7. These connection tubes are closed and able to be connected. connected sterile to other tubings.

According to a first embodiment (not shown), the multiple coupling device comprises a closing element of the culture vessel, such as a stopper, from which extends a plurality of connecting tubes capable of being connected in a sterile manner to other tubings. An example of a stopper incorporating a plurality of tubings is described in the document US 2014/0103077. According to a second embodiment and in connection with FIGS. 1 to 5, the multiple coupling device 3 comprises, on the one hand, a primary connection pipe 6 in fluid communication with the culture vessel 4 and, on the other hand, a set of pipes comprising a plurality of secondary connecting pipes 7, at least one being connected (FIG. 5) or intended to be connected (FIGS. 1 to 4) to said primary connection pipe 6. In particular, and as shown in FIG. 5, the multiple coupling device 3 comprises a closing element 8 of the culture vessel 4, such as a plug, from which the primary connection pipe 6 extends.

Advantageously, the culture device 1 and the primary connection tubing 6 of the multiple connector are pre-connected for manufacturing, the user then having to connect the primary connection tubing 6 to one of the secondary connection pipes 7 of the assembly of tubings to form a closed subsystem consisting of the culture device 1 and the multiple coupling device 3.

Even more advantageously, the culture device 1 and the multiple coupling device 3 are pre-connected to manufacture so as to form said closed subsystem constituted by the culture device and the multiple coupling device.

In FIG. 1, the culture vessel 4 comprises a neck defining said primary orifice arranged to receive the closure element 8. For example, the neck of the container 4 comprises a thread on its outer surface and the closure element 8 is a plug that can be screwed onto said neck of the container 4.

For greater safety and to ensure the sterility of the system, the closure element 8 is permanently placed on the primary orifice of the culture vessel 4, so that a user can not remove it under normal conditions. use. According to an alternative embodiment, a vent orifice comprising a liquid impermeable and gas permeable membrane is provided on the closure member of said container. Advantageously, the membrane has a porosity of 0.22 μηι so as to obtain a sterile vent.

In another variant shown in Figures 1 and 2, an aeration pipe 9 extends from said closure member 8, said aeration pipe 9 comprising at its end a vent 10 comprising a liquid impermeable membrane and permeable to gases. Advantageously, the membrane has a porosity of 0.22 μηι to preserve the sterility of the culture device.

This vent 10 makes it possible not only to introduce the oxygen necessary for the survival of the cells cultured in the culture vessel 4, but also, and in particular in the case of a rigid culture vessel, to facilitate the fluidic movements towards the container. 4 or the culture vessel 4. The ventilation tube 9 is provided with a selective opening and closing element 1 1 of the fluid communication in said tubing such as a clamp. This clamp, in the closed position, is particularly useful when adding or removing fluid from the culture vessel 4 in order to avoid wetting said membrane of the vent 10, which would lead to subsequently preventing the entry of oxygen in said culture vessel 4.

In connection with FIGS. 3 to 5, the tubing assembly comprises a plurality of connecting tubings 7, more than one of said connecting tubings being intended to be connected in a sterile manner to a tubing of one of the transfer devices as described below.

In particular, the set of pipes comprises a first pipe on which is connected at least one multi-channel connector 12, others tubing being connected to each of the other branches of the multi-channel connector 12, so as to obtain an assembly comprising a plurality of connection pipes 7 intended to be connected to other tubules sterilely.

In particular, the set of pipes comprises at least three, in particular between three and eight pipes 7 can be connected sterilely.

For example, in Figures 3 and 4, the multi-channel connectors 12 are four-way connectors. In a variant, each connector may comprise between five and eight branches.

Each of the pipes 6, 7 of the multiple coupling device 3 is equipped with a selective opening and closing element 13 of the fluid communication in said tubing, such as a clamp.

Each of the pipes 6, 7 of the multiple coupling device 3 intended to be connected to another tubular is closed at its free end, that is to say the end which is not connected to any other element. For example, each of the tubes 6, 7 is closed by a stopper or any other small conical or cylindrical part which fits by pressure in the said end of the tubing and which guarantees the closure and therefore the sterility (after sterilization) of the device. multiple connection. Another sterile device of the culture system is the transfer device 2 intended to allow the movement of fluid towards the culture device 1 or from said culture device 1.

The fluid is in particular in the form of a liquid such as a culture medium, a cell suspension, supplements or additives for culture medium, reagents necessary for culturing cells, rinsing solutions, etc. In particular, said transfer device 2 is intended, via the multiple coupling device 3, to introduce solutions such as fresh culture medium into the culture vessel 4, to supplement fresh culture medium with additives or additives for medium of culture. culturing, introducing reagents such as dissociation reagent, into said culture vessel 4, or collecting the spent culture medium from the culture vessel 4.

In connection with FIGS. 6 to 8, the transfer device 2 comprises at least one empty bag 14 in fluid communication with a primary transfer tube 15 to which is connected more than one secondary tubing 1 6 transfer.

According to a particular embodiment, the primary transfer pipe 15 comprises at least one multi-channel connector 17 to which is connected more than one secondary transfer pipe 16, in particular between two and eight secondary transfer pipes.

Thus, the transfer device 2 comprises a plurality of tubings 15, 1 6 able to be connected to other tubules sterilely.

Each of the transfer tubes 15, 16 intended to be connected sterilely to other tubings is closed at its free end, that is to say the end which is not connected to any other element. For example, each of said tubes is closed by a plug or other small conical or cylindrical piece which fits by pressure in said end of the tubing, thus ensuring the sterility of the device (after sterilization).

In a variant shown in FIG. 7, one of the transfer tubes 18 is provided at its end with a filter 19 comprising a sterilizing membrane. The membrane has a porosity of 0.22 μηι. Said filter 19 comprises a connector 20, in particular of Luer type, able to connect a container, such as a syringe, equipped with a suitable compatible connector. This tubing 18 equipped with a sterilizing filter 19 may be used for example to connect a syringe filled with a dissociation solution such as trypsin or any other additive solution such as heparin. In particular, each of the tubes 15, 16, 18 of the transfer device is equipped with a selective opening and closing element 21 of the fluid communication in said tubing such as a clamp.

The transfer device 2 has the advantage of being able to be used to carry out in a secure manner, that is to say by reducing the risk of pathogenic contamination, several culture operations and also to be adapted to the different culture protocols. the end user.

Indeed, thanks to the plurality of tubings available for making sterile connections, the user will be able to sterilely connect the different containers necessary for the execution of a cell culture operation, such as an empty container to collect the cells. cultivated, a container containing fresh culture medium or any other solution. Such a container is shown in FIG. 9. The container is in the form of a bag 22 from an orifice of which a tubing 23 extends. This tubing is closed at its other end, in particular by a stopper or any other small conical or cylindrical piece which fits by pressure in said end of the tubing, thus ensuring the sterility of the device (after sterilization).

In particular, the container 22 comprises fresh culture medium, supplements of culture medium such as platelet lysate, trypsin, etc.

The tubing 23 is intended to be connected in a sterile manner to one of the tubings 15, 16 of the transfer device. Alternatively, such containers 22 are pre-connected or integrated manufacturing the transfer device.

In the case where the solutions necessary for a cell culture operation are not provided in receptacles provided with a tubing capable of being connected to a tubular of the transfer device, they must be reconditioned in receptacles equipped with tubing.

The sterile connection between the various tubes of the secure culture system is notably performed using a sterile tubular tube connection device such as the TSCD ™ device (Terumo Medical Corp.), the TCD ™ (Genesis BPL). or the Biowelder ™ (Sartorius Stedim).

The tubings intended to be connected sterilely are made of a weldable and sterilizable material. In particular, these pipes are made of polyvinyl chloride or other heat-sealable thermoplastic elastomer such as C-Flex ™ Saint-Gobain.

The culture system devices or parts thereof are closed and sterile, i.e. they have been sterilized by a conventional sterilization method such as steam sterilization or gamma irradiation or gamma irradiation sterilization. 'ethylene.

A method according to the invention is now described for implementing a cell culture system as just described in order to carry out a succession of operations on said cells, said culture system comprising an assortment of closed devices. and sterile, said assortment of devices comprising:

a culture device 1 comprising a culture vessel 4 suitable for culturing cells,

- more than one transfer device 2 intended to allow the movement of fluid to the culture device 1 or from said culture device, said transfer device 2 comprising at least one empty bag 14 in communication fluidic with a primary transfer line 15 to which is connected one or more secondary tubing 16 transfer, and

a multiple connection device 3 for connecting the culture device 1 to more than one transfer device 2, said multiple connection device 2 comprising a plurality of connection tubes 7, more than one of said connection tubes being intended for be connected sterilely to one of the tubing 1 6 transfer of said transfer devices 2.

According to the invention, the method comprises the steps of:

(a) connecting one of the connection pipes 7 of the multiple coupling device 3 to one of the transfer pipes 1 6 of one of said transfer devices 2,

(b) performing a closed system operation on the cells to be cultured,

(c) after the operation is performed, disconnecting said transfer device 2 from said multi-connector device 3, and

(d) repeating steps (a) through (c) with another transfer device 2 to perform another operation on the cells.

Each operation to be performed on the cells is chosen in particular from the group consisting of seeding, changing the medium or subculture.

After the step of connecting (a) one of the connecting pipes 7 of the multiple coupling device 3 to one of the transfer pipes 16 of one of said transfer devices 2, a closed system is obtained as shown in Figure 10.

In step (d), the other operation to be performed on the cells may be of the same type or not. For example, if the first operation is a medium change operation, the next operation is another medium change operation or a dunnage operation. In particular, the method comprises, prior to step (b), the step consisting in connecting at least one of the transfer tubes 1 6 of transfer of said transfer device 2, at least one tubing 23 equipping a container 22 necessary for perform the operation to be performed on the cells, for example a container comprising a liquid such as a culture medium, a supplement or additive for culture medium, a buffer solution or a dissociation reagent.

After carrying out the operation, the disconnection of the transfer device 2 from said multiple coupling device 3 is achieved by welding and cutting the connection pipe 7 of the multiple coupling device 3 which has been connected to the tubing 1 6 of the device. transfer 2 to step (a).

Advantageously and in order to reduce the risk of contamination in the event of a faulty connection, the disconnection is performed by welding cutting the connection pipe 7 as close as possible to the multi-lane connector 12, thus preventing the re-use of the remaining portion of tubing.

Specific examples of implementation of the secure culture system as shown in the figures comprising a culture device 1, a multiple coupling device 3 and a transfer device 2 are now described.

The method comprises the preliminary step of:

if necessary, connect the primary connection pipe 6 of the multiple connection device 3 to one of the secondary pipes 7 for connecting the pipe assembly to form the multiple coupling device 3.

- Sterile connect one of the tubings 7 connecting the set of pipes to one of the pipes 1 6 of a transfer device 2.

These preliminary steps can be performed outside the laminar flow hood since the connections are sterile connections. A method of implementing the system for performing a seeding operation is now described.

In order to carry out the seeding, in addition to the culture devices 1, the multiple connector 3 and the transfer device 3, a fresh culture medium bag, a container containing a cell suspension and optionally a container containing supplements of culture medium. The bag and each of the containers are equipped with a tubular in fluid communication with the interior volume of said container or said bag.

The method comprises the following preliminary steps:

- Sterile connect the bag of fresh culture medium to one of the transfer tubes 1 6 of the transfer device,

- Sterile connect the container containing the cell suspension to be seeded to one of the tubes 1 6 of the transfer device,

where appropriate, sterilely connecting one or more other containers containing culture medium supplements to one or more other tubings 16 of the transfer device. The seeding operation comprises the steps of:

transfer the cell suspension and, where appropriate, culture medium supplements to the fresh culture medium bag,

transfer the fresh culture medium containing the cells and optionally complemented in the culture vessel 4 of the culture device 1,

- Disconnect the transfer device 2 by welding and cutting advantageously the connecting pipe 7 connected to the transfer pipe 1 6 as close to the multi-lane connector 12 to which the connecting pipe is connected, to prevent reuse.

After this seeding operation, the culture system comprising the culture device 1 and the multiple coupling device 3 is ready to be used to perform another operation. The method of implementing the system for performing the medium change operation is described below. In order to carry out the change of medium, in addition to the culture devices 1, the multiple connector 3 and the transfer device 2, a fresh culture medium bag and one or more containers containing a complement of medium of culture are required. culture. The bag and each of the containers are provided with a tubular in fluid communication with the interior volume of said container or said bag.

The following steps can be performed outside the hood.

The method comprises the preliminary steps of:

- Sterile connect the bag of fresh culture medium to one of the transfer tubes 1 6 of the transfer device 2,

- if necessary, connect one or more other containers containing culture medium supplements to one or more other tubings 1 6 of the transfer device 2 in a sterile manner.

The medium change operation comprises the steps of:

transfer the used medium into the empty bag 14 of the transfer device 2,

- if necessary, transfer the culture supplements to the bag of fresh culture medium,

- Transfer the fresh culture medium optionally complemented in the culture vessel 4,

- Disconnect the transfer device 2 by welding and cutting the connecting pipe 7 connected to the transfer pipe 1 6.

After this medium change operation, the culture system comprising the culture device 1 and the multiple connector device 3 is ready to be used to perform another operation. Finally, a method of implementing the system for carrying out a subcapping operation is described. For transplanting, that is to say the recovery and concentration of cells, it is necessary, in addition to the culture devices 1, multiple connector 3 and transfer 2, a dissociation solution of cells of trypsin type, a PBS buffer solution, an empty syringe with tubing and an empty cell collection bag. The bags and containers are equipped with tubing in fluid communication with the interior volume of said containers or pockets and a balance

The method comprises the following preliminary steps:

- Sterile connect the buffer solution bag, the cell collection bag, the syringe and trypsin to one of the transfer tubing 16 of the transfer device 2, so as to form a closed system.

The transplanting operation consists of:

transfer the used culture medium into the empty bag 14 of the transfer device 2,

- take a certain volume of culture medium in the empty syringe,

rinse the culture vessel 4 with a buffer solution,

add a dissociation solution in the culture vessel 4 in order to detach the cells from the container,

transfer a determined volume of culture medium to neutralize the dissociation solution by placing the culture vessel on the scale,

transfer the detached cells to the cell collection bag, and

- Disconnect the transfer device 2 by welding and cutting the connecting pipe 7 connected to the transfer pipe 1 6. For each of these steps, it is advantageous to use a connection pipe 7 which has not already been used to avoid any risk of contamination. If the syringe is not equipped with a tubing, then it is possible to use a transfer system as shown in FIG. 7 which comprises a tubing 18 provided at its end with a filter 19 comprising a sterilizing membrane. The syringe is then connected to the sterilizing filter to securely perform the transplanting operation.

During each of these operations, the clamp 1 1 on the ventilation tube is in the closed position. Between each operation, the culture device assembled with the multiple coupling device is placed in an incubator at 37 ° C, the clamp 1 1 on the ventilation duct 9 being in the open position.

Claims

1. A method for implementing a cell culture system for performing a succession of operations on said cells, said culture system comprising an assortment of closed and sterile devices, said assortment of devices comprising:

- a culture device (1) comprising a culture vessel (4) adapted for culturing cells,

- more than one transfer device (2) intended to allow the movement of fluid towards the culture device (1) or from said culture device (1), said transfer device (2) comprising at least one empty bag (14) in fluid communication with a primary transfer line (15) to which one or more secondary transfer tubings (16) are connected, and

a multi-connection device (3) for connecting the culture device (1) to more than one transfer device (2), said multi-connector device (3) comprising a plurality of connection tubes (7), plus one of said connection tubes being intended to be connected to one of the transfer tubes (1 6) of said transfer devices (2), said method comprising the steps of:

(a) connecting one of the connecting tubes (7) of the multiple coupling device (3) to one of the transfer tubes (16) of one of said transfer devices (2),

(b) performing a closed system operation on the cells to be cultured,

(c) after the operation is performed, disconnecting said transfer device (2) from said multi-connector device (3), and

(d) repeating steps (a) through (c) with another transfer device (2) to perform a new operation on the cells.

2. Method according to claim 1 comprising, prior to step (b), the step of connecting sterilely to at least one of the transfer tubes (1 6) of said transfer device, at least one tubing (23) equipping a container (22) necessary to perform the operation to be performed on the cells.

3. Method according to claim 2 wherein the container necessary for carrying out the operation to be performed on the cells comprises a liquid such as a culture medium, a complement or additive for culture medium, a buffer solution or a dissociation reagent. .

The method of any one of claims 1 to 3 wherein the step (b) operation is selected from the group consisting of seeding, changing of medium or subculture.

5. Method according to any one of claims 1 to 4 wherein the disconnection of the transfer device (2) of said culture device (4) is performed by welding and cutting the connecting pipe (7) of the multiple coupling device ( 3) which has been connected to the tubing (1 6) of the transfer device (2) in step (a).

6. Method according to any one of claims 1 to 5 wherein the culture vessel (4) is rigid.

7. Method according to any one of claims 1 to 6 wherein the multiple coupling device (3) comprises a closure member (8) of said culture vessel (4) from which extends a plurality of connecting pipes (7).

8. Method according to any one of claims 1 to 6 wherein the multiple coupling device (3) comprises on the one hand a primary connection pipe (6) in fluid communication with the culture vessel (4) and on the other hand, a set of pipes comprising a plurality of secondary connecting pipes (7), at least one being connected or intended to be connected to said primary connection pipe (6).

9. The method of claim 8 including if necessary the preliminary step of connecting the primary connection tubing (6) sterilely from the multiple coupling device to one of the secondary tubings (7) for connecting the tubing assembly.

10. The method of claim 8, wherein the multiple coupling device (3) comprises a closure member (8) of said container (4) from which extends said primary connection pipe (6).

1 1. Method according to one of claims 8 or 10, in which a ventilation duct (9) extends from said closure element (8), said ventilation duct (9) comprising at its end a vent (10). ) comprising a liquid impermeable membrane permeable to gases.

12. A method according to any one of claims 1 to 1 1 wherein each of the tubings (7, 1 6) of said multiple coupling devices (3) and transfer device (2) to be connected sterilely is closed at its end. free.

13. Method according to any one of claims 1 to 12 wherein at least one of the transfer tubes (18) is provided at its end with a filter (19) comprising a sterilizing membrane.

14. A method according to any one of claims 1 to 13 wherein each of the tubings (7, 1 6) of said multiple coupling devices (3) and transfer device (2) are equipped with a selective opening element and closing (13,21) the fluid communication in said tubing.