Automated process for the production of cell cultures

Description

[0001] The present invention relates to an automated cell culture system, in particular for the culture of stem cells (of the CD34+ type for example), as well as to a cell culture method using this automated system.

[0002] Among the fields that utilize cell culture, cell therapy is the least advanced in terms of industrialization. Therefore, there is a significant need to find a technology capable of producing cells in sufficient quantities and under optimal conditions for therapeutic applications.

[0003] Some cell therapy procedures require stem cell culture or amplification before re-injection into a patient, as the quantities harvested are sometimes insufficient to have a therapeutic effect. It is essential to guarantee the integrity of the cells' therapeutic properties during culture. In current techniques, the proposed solutions for culturing stem cells are... vivo They are artisanal, very empirical and not very effective.

[0004] Furthermore, current techniques do not allow for the production of stem cells in sufficient quantities for therapeutic applications. Therefore, there is a real need to develop a bioreactor-type technology with a compact geometry that allows for the large-scale cultivation of cells.

[0005] The use of bioreactors for stem cell culture has already been proposed. However, the amplification phase remains an essentially manual step, and the environmental conditions for cell culture (temperature, CO2, etc.) are not controlled with great precision.

[0006] Examples include membrane perfusion bioreactors, hollow fibre bioreactors, fluidized bed bioreactors, and micro-bioreactors with continuous perfusion of O2, nutrient medium and growth factors (see application WO 00 / 73411).

[0007] US-A1-2008 / 0118977 describes a therapeutic protocol for reconstructing a patient's heart after a myocardial infarction. Reconstruction is achieved by injecting specific stem cells (CD34+), isolated from a blood sample, amplified ex vivo, and purified after culture, into the patient's heart.

[0008] The document EP 1 978 089 A1 is known.

[0009] The present invention aims in particular to provide a technology capable of ensuring the culture, amplification or expansion of this type of cell, and also to bring about significant improvements in terms of standardization, traceability and control for operations which are mostly carried out manually in the prior art.

[0010] To this end, it proposes an automated cell culture system, comprising reservoirs of culture medium, growth factors and cells to be cultured, a thermostatically controlled incubator in which a cell culture or cell expansion container is housed, and a computer control system including means for data entry and recording and intended to regulate the culture conditions in the enclosure and to control fluid distribution valves according to a predefined sequence, characterized in that it comprises a support and agitation device for the cell culture or cell expansion container which is controlled by the computer system and which is housed in the enclosure, and in that the container is formed by a cell expansion pouch having at least one inlet port connected to the aforementioned reservoirs and an outlet port connected to means for retrieving and storing the cells after culture,These storage means and reservoirs are located outside the enclosure and are connected to the openings of the cell expansion pouch by conduits which, together with the cell expansion pouch, form a pre-assembled module placed within the enclosure and which pass through a wall element of the enclosure, so as to allow the cell expansion pouch to be supplied with culture medium, growth factors, and cells to be cultured, and to retrieve the contents of the cell expansion pouch into the storage means, while keeping the enclosure closed. The support and agitation device includes a support tray for the cell expansion pouch, which is rotatable about a first horizontal axis and which is movable about this axis between a substantially horizontal position for cell culture and a substantially vertical position for cell retrieval after culture.

[0011] The automated system according to the invention essentially automates the steps of the biological culture protocol and the management of environmental conditions (temperature control, CO2 level, etc.) for cell culture in the incubator, in order to produce an optimal cell amplification yield. It also ensures the distribution of the culture medium, growth factors, and cells to be cultured to a cell expansion pouch (which can have a relatively large volume) located in the incubator, by controlling means such as valves and pumps. The automated system further ensures the agitation of the cell expansion pouch and the transfer of the cultured cells from the expansion pouch to the storage facilities.

[0012] The automated system according to the invention can generate large quantities of cells, such as stem cells, from cells taken from a patient. The cell culture bag can have a volume greater than 100 ml, 200 ml, 300 ml, or 500 ml, and is, for example, approximately 650 ml or even more (1 L, 2 L, 3 L, etc.). Culturing stem cells in such a bag makes it possible to generate a sufficient quantity of cells to perform cell therapy in a patient, such as a patient who has suffered a myocardial infarction, according to the biological protocol described in US patent application US-A1-2008 / 0118977.

[0013] The cell expansion bag preferably comprises flexible walls that are liquid-tight and gas-permeable. It preferably exhibits good permeability to oxygen and carbon dioxide, allowing for good aeration of the bag's contents without opening it and thus without risk of contamination. In a specific example of the bag's design, it has the following permeability characteristics (in cc per day, at 37°C): O₂ (gas) ≈ 418, CO₂ (gas) ≈ 966, N₂ (gas) ≈ 157, and H₂O (liquid) ≈ 0.05.

[0014] The cell expansion pouch preferably has little affinity for chemical and biological products, particularly for the cells to be cultured, and does not absorb such products. The pouch is, for example, made of a thin film of FEP (fluoroethylene-propylene) copolymer. The pouch can be made up of different types of ports (modifiable interfaces), including, for example, FEP fir tree fittings. These are assembled to the pouch to limit the risk of contamination.

[0015] The automated system is designed to perform cell culture and includes all the necessary means and resources for this purpose, eliminating the need for an operator to manipulate valves, replace bags or reservoirs, etc. Cell culture is carried out according to a precise biological protocol that is entirely managed by the computer system. This system allows, for example, the control of valves, the stirring device, and the regulation of environmental conditions within the incubator. The operator can input data into the computer system, including patient identification information, details of the cells collected, and the nature and origin of the various reservoirs or bags, so that all this data is recorded in the system. The invention thus enables the execution of biological protocols with excellent reproducibility and ensures precise traceability and control of the protocols and resources used.

[0016] The control and traceability of the steps in the biological protocol can be ensured by the computer system and an appropriate human-machine interface (HMI), which allow, for example: define an automated culture process in which the parameters specific to the biological culture protocol are not modifiable, ensure good security by limiting access to the computer system data, by user identification and the need for a password (in accordance with FDA 21 CFR Part 11 regulations), allow a recording of events and the different stages of the process, and establish a report generation (including results of sampling analysis and a posteriori analysis of graft characteristics, for example).

[0017] The cell expansion bag is housed within the enclosure and includes at least two openings connected to conduits that pass through a wall element of the incubator and are linked to reservoirs and storage devices located outside the enclosure. The cell expansion bag and conduits form a pre-assembled, single-use module that is easily installed and replaced by the operator.At least part of the contents of the tanks located outside the enclosure is intended to be distributed into the cell expansion bag located inside the enclosure, the contents of which, after culture, are intended to be transferred to the storage means located outside the enclosure. All these fluid distribution operations are carried out while keeping the incubator enclosure closed by means of conduits that pass through a wall element of the incubator, which ensures optimal environmental conditions within the enclosure throughout the biological protocol and limits the risks of contamination of the cell culture medium.

[0018] Advantageously, the cell expansion bag includes a sampling port connected by a conduit to sampling means located outside the enclosure. This conduit passes through the aforementioned wall element of the enclosure and is part of the pre-assembled module. The cell expansion bag thus comprises three ports with different functions (feeding, retrieval, and sampling) connected to different conduits.

[0019] In one embodiment of the invention, the incubator comprises a cabinet having an opening and a door for sealing this opening. Passage means for the aforementioned conduits are mounted on the peripheral edge of this opening and have substantially parallel grooves for housing the conduits. These grooves are intended to be covered by the door when it is in the closed position. The conduits can be easily inserted into (and removed from) these means by an operator when the door is open, by translating the conduits in a direction perpendicular to the longitudinal axes of the grooves, which facilitates the installation of consumables.

[0020] The reservoirs for growth factors and cells to be cultured are preferably formed by pockets located higher than the inlet of the cell expansion pocket, so that the contents of each pocket of culture medium and cells to be cultured can flow by gravity into the cell expansion pocket. This ensures the integrity of the cells and growth factors during their transfer to the cell expansion pocket. Using a pump or any other mechanical means to circulate the cells and growth factors through the channels would risk damaging them.

[0021] The storage means may include one or two pouches that are at least partially located lower than the outlet of the cell expansion pouch so that, after culture, the contents of the cell expansion pouch can flow by gravity into the pouch(es) of the storage means. This also helps ensure the integrity of the cells after culture, upon retrieval.

[0022] The automated system may include a peristaltic pump to control the supply of culture medium to the cell expansion pouch and the growth factor and cell culture reservoirs, for the purpose of rinsing these reservoirs. The peristaltic pump has the advantage of not coming into direct contact with the culture medium, thus avoiding any risk of contamination.

[0023] The automaton may also include two air-trapping bags, one of which is connected to the growth factor and cell culture reservoirs, and the other of which is connected to the cell expansion bag, and which are intended to collect and store the air contained in the ducts, the cell expansion bag and / or the reservoirs.

[0024] Advantageously, the conduits are formed by flexible tubes, at least some of which pass through valves designed, in the closed position, to pinch these tubes. Each tube is, for example, designed to be easily inserted into a groove of a valve by the operator, for example by translation in a direction substantially perpendicular to the longitudinal axis of the tube.

[0025] As described previously, the support and agitation device comprises a cell expansion pouch support tray, which is rotatably mounted around a first horizontal axis and is movable around this axis between a substantially horizontal cell culture position and a substantially vertical cell retrieval position. This latter position facilitates cell retrieval after culture, with the cells flowing directly by gravity into the aforementioned storage means.

[0026] The tray can be mounted to rotate around a second horizontal axis, around which the tray is intended to oscillate for agitation and homogenization of the contents of the cell expansion bag. The first and second axes of rotation of the tray are preferably parallel.

[0027] Preferably, the tray carries valves for controlling the supply of the cell expansion bag, for retrieving the contents of this bag, and for taking samples from this bag.

[0028] The support and agitation device may further include a vertical arm having at its upper end means for attaching an air-trapping bag connected to the cell expansion bag.

[0029] The storage means are advantageously mounted to rotate about a horizontal axis and are movable about this axis between a substantially vertical position and a substantially horizontal position in which these means are entirely located below the cell expansion pocket. This ensures that all the cells contained in the cell expansion pocket will be transferred by gravity into the storage means.

[0030] As an example, in the specific case of CD34+ stem cell culture, the sterility required at the level of the complete CD34+ cell conditioning chain necessitates the use and installation of consumables in the form of a single-use cell culture kit.

[0031] The present invention therefore also relates to a cell culture kit, preferably sterile and single-use, for an automated cell culture system, characterized in that it comprises at least one cell expansion bag and flexible tubing for connecting the bag to other bags or reservoirs, the tubing and the cell expansion bag forming a pre-assembled module, the bag being intended to be placed in a cell culture chamber and the tubing being intended to pass through a wall element of said chamber, the cell expansion bag comprising an inlet port, an outlet port and optionally a sampling port.

[0032] The kit may also include all the necessary fittings for connecting the tubing to each other and to the bags and / or reservoirs, as well as the means connected to the third port of the cell expansion bag to allow for cell sampling. All these elements may be part of the aforementioned pre-assembled module.

[0033] The inlet of the cell expansion pouch is connected by tubing to the inlet and outlet of the growth factor pouch, and to the inlet and outlet of the cell culture pouch. The inlet of the cell expansion pouch is also designed to be connected to an outlet of the cell expansion pouch.

[0034] The kit also includes two air-trapping bags, one of which is connected to the outlet ports of the growth factor and cell culture bags, and the other of which is connected to the inlet port of the cell expansion bag.

[0035] The kit may also include one or two cell recovery bags after culture, which are connected by tubing to the outlet port of the cell expansion bag.

[0036] In one embodiment of the invention, the growth factor and cell culture pockets and the air-trap pockets have an internal volume of approximately 150 ml, the cell expansion pocket has a theoretical volume of approximately 3000 ml, and the two recovery pockets each have a volume of approximately 600 ml. The culture medium dispensing pocket may have a volume of approximately 1000 ml.

[0037] Advantageously, the kit forms or constitutes a closed circuit which, once installed for cell culture, includes all the resources necessary for that culture without requiring the addition of any product or operator intervention. This limits the risk of contamination of the kit and the culture medium.

[0038] The invention further relates to a support and agitation device for a cell culture automaton, characterized in that it comprises a support tray for a cell expansion bag, this tray having three valves and being mounted to rotate about a first horizontal axis for tilting the tray from a substantially horizontal position to a substantially vertical position, and a second horizontal axis about which the tray is intended to oscillate for agitation and homogenization of the contents of the cell expansion bag, the device also comprising controlled means for tilting the tray about the aforementioned horizontal axes.

[0039] The device may include a vertical arm having at its upper end means for attaching an air-trapping bag.

[0040] The invention also relates to an automated cell culture method, using an automated system as described above, characterized in that it comprises the steps of: a) supply the cell expansion bag with culture medium, growth factors and then cells to be cultured, keeping the incubator closed; b) agitate the cell expansion bag to homogenize its contents; c) maintain the cell expansion bag under incubation conditions for several days; and d) retrieve the contents of the cell expansion bag into the storage means, keeping the enclosure closed.

[0041] The method according to the invention may comprise one or more of the following steps: before step a), a step of installing the pre-assembled module by mounting the cell expansion bag on the stirring device, mounting the conduits in the incubator's passageways and valves, and connecting these conduits to the reservoirs or bags; before step a), a step of removing the air contained in the conduits by passing culture medium from the culture medium reservoir to the air-trapping bag(s); after feeding the cell expansion bag with growth factors in step a), a step of rinsing the growth factor reservoir by passing culture medium through this reservoir and then removing its contents to the cell expansion bag;after feeding the cell expansion bag with cells to be cultured in step a), a step of rinsing the reservoir of cells to be cultured by passing culture medium through this reservoir and then draining its contents into the cell expansion bag; during step c), one or more steps of sampling the contents of the cell expansion bag, each of which is preceded by a step of tilting the support tray from a horizontal culture position to an inclined position in which the sampling port of the bag represents the lowest point of the bag; before step c), a step of removing the reservoirs of culture medium, growth factors and cells to be cultured, by cutting and welding or pinching the connecting conduit or tubing of these reservoirs to the inlet port of the cell expansion bag;before or during step d), tilt the tray into a substantially vertical position so that the outlet of the cell expansion pouch represents the lowest point of the pouch.

[0042] The invention also relates to the use of an automated system, kit, or device as described above for culturing CD34+ stem cells or blood mononuclear cells, such as lymphocytes. The stem cells may originate from one or more sources, particularly umbilical cord blood, bone marrow, and whole blood.

[0043] The invention will be better understood and other details, advantages and features of the invention will become more apparent upon reading the following description, given by way of non-limiting example and with reference to the accompanying drawings in which: THE figures 1 and 2are schematic perspective views of the cell culture automaton according to the invention, this automaton comprising a cabinet defining an enclosure which is closed in figure 1 and open in figure 2 ; there figure 3 is a very schematic view of the automaton of figures 1 and 2 , without the computer system; the figure 4 is a very schematic view of the components carried by an agitation device of the automaton figures 1 and 2 ; there figure 5 is a schematic view of a cell culture kit according to the invention; the figure 6 is a schematic perspective view of the means of fluid conduit passage of the automaton figures 1 and 2 ; there figure 7 is a schematic perspective view of the stirring device according to the invention; the figures 8 and 9 are schematic perspective views of the device of the figure 7 and represent two different tilt positions of the platform of this device; the Figure 10is another schematic perspective view of the stirring device of the figure 7 , with partial tearing of the casing of this device; the figure 11 is a schematic perspective view of the platform and controlled means for tilting the platform of the device figure 7 , seen from below; the Figures 12 and 13 schematic views are shown in perspective of a controlled system for locking the rotation of the platform of the device. figure 7 , this blocking system being active in figure 11 and inactive in figure 12 ; there figure 14 is a flowchart showing the steps of a cell culture process according to the invention; and the figures 15 to 24 are views corresponding to the figure 3 and representing steps of the process according to the invention.

[0044] We first refer to figures 1 and 2which represent an example of an embodiment of the cell culture automaton 10 according to the invention, this automaton being particularly but not exclusively intended for the culture of stem cells, for example according to the biological protocol described in US-A1-2008 / 0118977 application, the content of which is incorporated herein by reference.

[0045] In the example shown, automaton 10 essentially comprises three elements: an incubator 12 with a thermostatically controlled chamber 14 in which is housed a device 16 for supporting and agitating a cell expansion bag (not shown), a frame 18 for supporting bags (not shown) containing the media necessary for cell culture, and carrying means (valves 20, pump 22, etc.) for distributing and regulating the flow of fluids between the bags, and a computer system 24 connected to the incubator 12 and to the means 20, 22 for their control as well as for entering and recording data and managing the biological protocol.

[0046] In the example shown, the incubator 12, the frame 18 and the computer system 24 are arranged side by side on a support 26 which is mounted on casters, the frame 18 being located between the computer system 24 and the incubator 12.

[0047] Typically, the computer system 24 includes means for data entry and recording, means for data processing, means for display, and means for emitting control and piloting signals for the incubator 12 and the means 20, 22 of the frame 18. Preferably, the computer system 24 includes a touch screen for displaying and entering data.

[0048] To limit access to pre-recorded data in the computer system 24, several security levels can be implemented. The PLC manufacturer can have maximum access rights, via a specific password, allowing access to all information recorded in the computer system 24, while an administrator and an operator with lower access levels will only have access, via specific passwords, to certain information.

[0049] The computer system 24 is advantageously connected to a computer network via a connection of the type Ethernet or Wifi for example, so that information contained in the system 24 can be accessed from a computer station on the network, remote from the automaton 10, and possibly that actions can be requested and ordered from the automaton from this station.

[0050] The computer system 24 controls, for example, the opening and closing of the valves 20, which are, for example, of the on / off type, the flow rate of the pump 22, the heating regulation of the chamber 14 of the incubator 12 (so that it has, for example, a temperature of approximately 37°C), and the supply of gases to the chamber, such as CO2 (at a concentration of approximately 5%, for example). The system 24 can also regulate other parameters within the chamber 12, if necessary, to define optimal environmental conditions for cell culture.

[0051] For clarity, the means of connecting the computer system 24 to the means 18, 20 and to the incubator 12, the means of heating and gas supplying the incubator 12, and the means of electrical supply are not shown in the drawings.

[0052] The frame 18 has a parallelepiped shape and includes a vertical front face 28 on which are schematically drawn rectangles 30 representing the positions of reservoirs of biological media, in the form of pockets, as well as lines 32 representing the location of fluid conduit(s) between these pockets.

[0053] The upper part of this front face 28 includes four drawn rectangles 30 which inform an operator about the nature of each of the pockets which must be positioned at the level of these rectangles, these pockets belonging to a consumables kit which will be described in more detail below.

[0054] A large rectangle is drawn in the upper left corner of the front face 28 of the frame 18 and represents the position of a pocket containing a culture medium (pocket referenced as 34 in figure 3Three smaller rectangles 30 are drawn in the upper right of face 28 and represent respectively the positions of a growth factor pocket, a cell culture pocket, and an air trap pocket (which are respectively referenced 36, 38 and 40 in figure 3 ).

[0055] The middle part of the front face 28 of the frame 18 includes mounting holes for the valves 20 and the pump 22 mentioned above, each of these elements (valves and pump) being located on a line 32 representing a fluid conduit, which is formed by a flexible tube from the consumables kit.

[0056] The lower part of the front face 28 has two coplanar plates 42 arranged side by side. These plates 42 are pivotally mounted at their lower ends around a common horizontal axis extending parallel to the front face 28. The plates 42 can be rotated about this axis between a vertical position (shown in figures 1 and 2 ) in which they extend parallel and at a short distance from the front face 28, and a horizontal position in which they can rest on the support 26.

[0057] Rectangles 44 are drawn on the front faces of the plates 42 when they are in a vertical position. These rectangles 44 inform the operator about the type of pockets that must be carried by these plates 42. Pockets for cell recovery and storage after culture (referenced 46 in figure 3 ) are intended to be carried by these plates 42.

[0058] Pockets 34, 36, 38, 40 and 46 of the consumables kit are intended to be fixed or hung on the front face 28 of the frame 18 and on the plates 42 by appropriate means which are not shown.

[0059] The incubator 12 includes a cabinet defining the enclosure 14 and having an opening which can be hermetically closed by a double door, these two doors 48, 50 being mounted pivotally on one side of the opening, for example the right side.

[0060] The internal door 48 is a glass door designed, in the closed position, to bear against a peripheral seal 52 of the cabinet opening, this seal 52 being visible in figure 6 The external door 50 is thermally insulated and has a peripheral seal designed to rest against the peripheral edge of the cabinet opening.

[0061] The computer system 24 can be connected to sensors detecting the position (open or closed) of each door 48, 50, and can control the locking of these doors in particular during the incubation and cell culture phase.

[0062] For example, enclosure 14 of incubator 12 has an internal volume of approximately 200 L.

[0063] In the example shown in figures 3 to 5 The consumables kit is for single use in cell culture and includes the bags 34, 36, 38, 40 and 46 and the aforementioned tubing, as well as a cell expansion bag 54 and a second bag 56 forming an air trap, these bags 54, 56 being carried by the stirring device 16 which will be described in more detail below, with reference to figures 7 to 13 .

[0064] Cellular expansion pocket 54 is more visible in Figures 4 and 5may have an internal volume greater than 500 ml, and which is for example 650 ml, and includes three ports, a sampling port 58 connected by a tube 60 to sampling means 62, an outlet port 64 connected by a tube 66 to the cell recovery bags 46 after culture, and an inlet port 68 connected by tubes to the bags 34, 36, 38 and 56.

[0065] The inlet port 68 of the cell expansion pocket 54 is connected by a tube 70 to an inlet port of the culture medium pocket 34. The growth factor and cell pockets 36 and 38 each comprise an inlet port which is connected to one end of a tube 72, the other end of which is connected to the tube 70, and an outlet port which is connected to one end of a tube 74, the other end of which is connected to the tube 70 (downstream of the connection point(s) of the tubes 72 to the tube 70). The pouch 40 comprises two orifices which are connected by tubes 76 to tubes 74, and the air-trap pouch 56 comprises an orifice connected by a tube 78 to the tube 70, in the vicinity of the inlet orifice 68 of the pouch 54 ( Figures 4 and 5 ).

[0066] The cell expansion bag 54 and the tubing 60, 66, 70, 72, 74, 76, and 78 are preferably pre-assembled and supplied sterile. Bags 34, 36, 38, 40, 46, and 56 are also supplied sterile. Bags 40, 46, and 56 are supplied empty and can be pre-assembled with the cell expansion bag 54 to the aforementioned tubing. The cell culture bag 38 is also supplied empty and can be pre-assembled to the tubing or connected to the tubing during kit installation in the automated system. Bag 38 can be filled with a medium containing the cells to be cultured before or after kit installation in the automated system. Bags 34 and 36 are preferably supplied filled with culture medium and growth factors, respectively.

[0067] All tubing and tubing-to-bag connections, as well as sampling devices, are also preferably part of a pre-assembled module, which is schematically represented in figure 5 pockets 34, 36 and 38, which are not necessarily part of this module, are represented by dotted lines.

[0068] Bags 36, 38, 40 and 56 have a capacity of approximately 150 ml, bag 46 has a capacity of approximately 600 ml, and bag 34 of culture medium has a capacity of approximately 1000 ml.

[0069] In the case where the automaton 10 is used to culture CD34+ stem cells, bag 38 includes cells of this type taken from a patient and possibly isolated and purified, and the growth factors in bag 36 are cytokines.

[0070] The cell expansion pouch 54 and the air-trap pouch 56 are carried by the stirring device 16 and are housed in the enclosure 14 of the incubator 12 ( figure 3 ). The other bags 34, 36, 38, 40 and 46 and the sampling means 62 are located outside enclosure 14.

[0071] The connecting tubes 60, 66 and 70 of the cell expansion pouch 54 to the elements located outside the enclosure 14 pass through a component of the incubator, which allows the enclosure 14 to be sealed airtight, this component being shown in figures 3 And 6 .

[0072] This component is a wall element formed by a block 80 of material (for example, plastic) which is fixed to the peripheral edge of the opening of the incubator cabinet 12 and which includes three parallel grooves 82 for engaging and passing the aforementioned tubing 60, 66, and 70. These grooves 82 are substantially straight and spaced apart. The block 80 has a substantially flat shape and extends in a vertical plane. It comprises a rear face bearing against the peripheral edge of the cabinet opening and a front face on which the grooves 82 are formed. These grooves are substantially horizontal and extend over the entire transverse dimension of the block.

[0073] The grooves 82 have a substantially circular cross-section and an internal diameter slightly greater than that of the tubes 60, 66, 70. These tubes are intended to be fully engaged in these grooves and possibly to pass through cutouts 84 of the peripheral seal 52 of the edge of the cabinet opening.

[0074] In the closed position of the enclosure 14, the peripheral edge of the inner door 48 is intended to bear against the seal 52 and to cover the parts of the tubes 60, 66, 70 extending into the aforementioned cutouts 84 of the seal 52, and the peripheral seal of the outer door 50 is intended to bear against the front face of the block and to cover the grooves 82 and the parts of the tubes 60, 66, 70 extending into these grooves.

[0075] In the example shown, the lower groove of block 80 forms a passage for the tubing 66 connecting the cell expansion bag 54 to the recovery bags 46, the middle groove forms a passage for the tubing 60 connecting the bag 54 to the collection means 62, and the upper groove forms a passage for the tubing 70 connecting the bag 54 to the bags 34, 36, 38 and 40.

[0076] As this is represented in figure 3 , the tubing 70 is engaged in the pump 22 in the vicinity of the bag 34 of culture medium, this pump being a peristaltic pump to avoid the risks of contamination of this medium.

[0077] The aforementioned valves 20 are solenoid valves, twelve in number in the example shown, referenced from 86 to 108 in figure 3 .

[0078] The pipes 66, 60, 70 and 78 are engaged respectively in four valves 86, 88, 90 and 92 which are carried by the stirring device 16 ( figures 3 and 4 ).

[0079] The portion of tubing 70 located outside enclosure 14 is engaged in two valves 94 and 96, positioned at a distance from each other. One valve, 94, is located near the culture medium pouch 34. The other valve, 96, is located downstream of the connection between tubing 70 and tubing 72 and upstream of the connection between tubing 70 and tubing 74.

[0080] The tubing 72 connected to the inlet ports of the bags 36 and 38 are engaged in valves 98 and 100, respectively, and the tubing 74 connected to the outlet ports of the bags 36 and 38 are engaged in valves 102 and 104, respectively.

[0081] The tubes 76 connected to the bag 40 forming an air trap are each engaged in a valve 106, 108.

[0082] The pump 22 and the valves preferably include a transverse groove for mounting a tube by translation in a direction perpendicular to the longitudinal axis of the tube or the groove.

[0083] As schematically represented in figure 3 and visible in figure 2 The bags 34, 36, 38, 40, and 56, and the tubing 40, 72, 74, 76, and 78 connecting these bags to the cell expansion bag 54, are all located above this bag 54 when the latter is positioned horizontally. The bags 46, the sampling devices 62, and the tubing 60 and 66 connecting these devices to the cell expansion bag 54 are all located below this bag 54 when the latter is positioned horizontally.

[0084] Pockets 36, 38 and 56 are substantially located in the same horizontal plane which is located below a horizontal plane in which pockets 34 and 40 are located.

[0085] The consumables kit can be installed in the controller as follows. Doors 48 and 50 of incubator 12 are open. Bags 34, 36, 38, 40, and 46 are attached to the frame, and bag 56 is attached to the arm of the stirring device 16. Bag 54 is placed flat on the stirring device 16. Tubing 70 is engaged in valves 94 and 96 as well as in pump 22; tubing 72 is engaged in valves 98 and 100; tubing 74 is engaged in valves 102 and 104; and tubing 76 is engaged in valves 106 and 108. Tubing 66, 60, 70, and 78 are engaged respectively in valves 86, 88, 90, and 92 carried by the device 16, and then tubing 66, 60, and 70 are engaged respectively in the grooves 82 of the block 80. The tubing is connected to the bags which are not already pre-assembled to the tubing, then the doors 48, 50 of the incubator 12 are closed.

[0086] We now refer to figures 7 to 13 which represents an embodiment of the stirring device 16 according to the invention.

[0087] The stirring device 16 includes a support tray 110 for the cell expansion pouch 54 (not shown in the figures 7 to 13 ), this platform being mounted to rotate about a first horizontal axis A for the movement of the platform from a substantially horizontal position represented in figures 7 And 11 up to a roughly vertical position represented in figures 8 And 10 (the 110 plate can adopt any position between these extreme positions, such as a position shown in figure 9 in which it is inclined at approximately 45° relative to a horizontal plane), and around a second horizontal axis B around which the plate 110 is intended to oscillate (over an angular range of approximately + / - 8°) to agitate and homogenize the contents of the cell expansion pocket.

[0088] The tray 110 has a rectangular shape with dimensions slightly larger than those of the cell expansion bag 54 (approximately 40 cm long and 22 cm wide), which is intended to be placed flat on the tray. The tray 110 includes peripheral rims 112 for retaining the bag and is perforated so that the face of the bag 54 against the tray can be directly exposed, at least partially, to the environmental conditions within the enclosure 14 of the incubator 12.

[0089] The tray 110 includes at one of its ends, corresponding to one of the short sides of the tray, a hook 114 for fixing the cell expansion pouch 54, this hook being intended to represent the highest point of the device 16 when the tray is in a vertical position ( figure 8 ). The plate 110 includes at its end opposite the hook 114 three mounting holes for the aforementioned valves 86, 88, 90.

[0090] The device 16 includes a U-shaped piece 116 whose free ends of the two lateral arms are articulated on pivots 118 fixed on the lateral edges of the end of the plate 110 carrying the valves 86, 88, 90. These pivots 118 are aligned and define the aforementioned first axis A of rotation of the plate 110.

[0091] The arms of the U-shaped part 116 carry, substantially in their middle, pivots 120 which are articulated on a chassis 122 of the device 16, these pivots 120 being aligned and defining the aforementioned second axis B of rotation of the plate 110.

[0092] When the 110 plate is in a substantially horizontal position ( figure 7 ), the U-shaped piece 116 extends along three sides of the tray, namely the longer sides and the shorter side bearing the hook 114.

[0093] The movement of the plate 110 around the axis A is ensured by a cylinder 124 which is mounted between the arms of the U-shaped part 116 and whose cylinder is fixed to the middle part of this part 116 and the piston rod is fixed to the end of the plate carrying the valves 86, 88 and 90.

[0094] As can be seen in Figures 10 and 11 The piston rod of the cylinder 124 is articulated on an axis carried by a yoke 121 fixed to the end of the plate 110 carrying the valves, this axis being substantially horizontal. The cylinder of the cylinder 124 is articulated on a substantially vertical axis carried by a first yoke 123 which is itself articulated on a substantially horizontal axis carried by a second yoke 125, this second yoke 125 being fixed to the middle part of the component 116, substantially at its center.

[0095] When the piston rod of cylinder 124 is in the extended position, the plate 110 is in its substantially horizontal position shown in figures 7 And 11 When the piston rod of cylinder 124 is in the fully retracted position, the plate 110 is in its substantially vertical position shown in figures 8 And 10 In the case of the figure 9 , the piston rod of cylinder 124 is partially retracted or extended.

[0096] The movement of the platform around axis B is ensured by an electric motor 126 whose output shaft drives, via a belt 127, a drive wheel of one of the pivots 120 carried by the U-shaped part 116 ( Figure 10 ). The motor 126 is fixed to the chassis 122 of the device by appropriate means.

[0097] As depicted in Figures 12 and 13 , the chassis 122 of the stirring device 16 carries a system 129 for blocking the rotation of the plate 110 around the axis B, this system 129 comprising a retractable finger 131 which cooperates with an element 133 carried by the U-shaped part 116 for blocking the plate.

[0098] Finger 131 is movable from an extended position shown in figure 12 up to a retracted position represented in figure 13 the movement of this finger being controlled by the computer system 24.

[0099] The element 133 carried by the U-shaped part 116 has an elongated shape and comprises a first end fixed to one of the pivots 120 of the part and a second end having a notch into which the finger 131 is intended to be engaged to block the rotation of the plate 110 around the axis B. When the finger 131 is deployed ( figure 12 ), the lateral faces of the notch of element 133 can come to rest against the finger, thus preventing any rotation of the plate around axis B. When the finger is in the retracted position ( figure 13 ), part 116 and plate 110 can be moved in rotation around axis B.

[0100] The locking of the rotation of the tray 110 around the axis B can be activated by the computer system 24 when the tray is moved around the axis A to an inclined or vertical position, for the purpose of sampling or retrieving cells from the cell expansion bag 54, to prevent the tray from moving around the axis B due to the force exerted on one side of the tray by the weight of the cell expansion bag.

[0101] The stirring device 16 also includes a vertical arm 128 for attaching the aforementioned valve 92 and for hooking the bag 56, which forms an air trap. The valve 92 is located approximately halfway up the arm 128, and the upper end of the arm includes a hook 130 for attaching the bag 56 ( figures 7 to 9 ).

[0102] The device 16 further includes sensors 132 for the position of the platform 110 around the axes A and / or B, which are carried by the chassis 122.

[0103] There figure 14 is a flowchart representing the steps of the process according to the invention.

[0104] The first step 130 of the process consists of entering and recording culture parameters specific to the biological protocol using the computer system 24. This entry is performed by an operator, and the parameters entered include, for example, patient identification, consumables kit identification, the volume of the cell expansion bag 54, etc. To facilitate the entry of these parameters, the computer system 24 can be equipped with a barcode reader. The consumables kit may contain a barcode that directly informs the computer system 24 of the kit number and type, as well as the volume of each bag.

[0105] The process includes a second step 132 of installing the consumables kit in the PLC 10, as described above. This installation can be guided and supervised by the computer system 24. The installation can be carried out in several sub-steps, with the computer system 24 displaying installation instructions to the operator, indicating whether to validate or invalidate the completion of a sub-step and proceed to the next. These sub-steps are, for example: The placement of the various bags on the frame 18 and in the enclosure 14 of the incubator 12; the placement of the tubing 70 of the culture medium bag 34 into valves 94 and 96 (the computer system 24 then commands the opening of valves 94 and 96, which are subsequently closed as soon as the operator has validated this sub-step); the placement of the tubing 72, 74, and 76 of the growth factor bag 36 and the air trap bag 40 into valves 98, 102, and 106 (the computer system 24 commands the opening of these valves, which are subsequently closed as soon as the operator has validated this sub-step); the placement of the tubing 72, 74, and 76 of the cell culture bag 38 and the air trap bag 40 into valves 100, 104, and 108 (the computer system 24 commands the opening of these valves which are then closed as soon as the operator has validated this sub-step),the placement of tubing 78 of the air-trapping bag 56 in valve 92 (computer system 24 commands the opening of this valve, which is then closed as soon as the operator has validated this sub-step), and the placement of tubing 70, 60, 66, one after the other, in valves 90, 88 and 86 (computer system 24 commands the opening of each of these valves, one after the other, which are then closed as soon as the operator has validated each sub-step).

[0106] The process according to the invention includes a third test step 134 called "self-test" during which the computer system 24 checks the proper functioning of the valves as well as the tilting means (cylinder 124 and motor 126) of the tray 110 of the stirring device 16. The operation of the incubator 12 can be implicitly controlled at the initiation of the biological protocol, the latter being able to be initiated only if the temperature and the CO2 level, for example, in the enclosure 14 are stabilized at the incubation setpoint values.

[0107] The process according to the invention includes another fluid distribution step 136, which comprises several substeps schematically represented in figures 15 to 19 .

[0108] The first sub-step of distribution step 136 is represented in figure 15The process involves removing the air contained in tubing 70, 72, and 74. To achieve this, valves 94, 96, 106, and 108 are opened, and pump 22 is activated by computer system 24, causing culture medium to flow from bag 34 through tubing 70, 72, and 74 to bag 40, which acts as an air trap. Tubing 70, 72, and 74 then fill with culture medium, and bag 40 fills at least partially. Pump 22 is set to a predetermined flow rate and operates for a predetermined duration, at the end of which the pump is stopped and valves 94, 96, 106, and 108 are closed.

[0109] The second sub-step of distribution step 136 is represented in figure 16The process involves removing the air from tubing 70 and 78. To achieve this, valves 94, 96, and 92 are opened, and pump 22 is activated by computer system 24, causing culture medium to flow from bag 34 through tubing 70 and 78 to bag 56, which acts as an air trap. Tubing 78 then fills with culture medium, and bag 56 fills at least partially with culture medium. Pump 22 is set to a predetermined flow rate and operates for a predetermined duration, at the end of which the pump is stopped and valves 94, 96, and 92 are closed.

[0110] The third sub-step of distribution step 136 is represented in figure 17The procedure involves supplying the cell expansion bag 54 with culture medium. Valves 94, 96, and 90 are opened, and pump 22 is activated by the computer system 24 to circulate culture medium from bag 34 to bag 54. Bag 54 then fills with culture medium. Pump 22 is set to a predetermined flow rate and operates for a predetermined duration according to the parameters of the biological protocol, which specifies the volume of culture medium to be dispensed into bag 54 and its feed rate. Pump 22 is then stopped, and valves 94, 96, and 90 are closed.

[0111] The fourth sub-step of distribution step 136 is represented in figure 18The procedure involves supplying cell expansion pouch 54 with growth factors, then rinsing growth factor pouch 36 with culture medium and transferring the contents of pouch 36 to cell expansion pouch 54. Initially, valves 104 and 90 are opened, allowing the growth factor-containing medium to flow by gravity from pouch 36 to pouch 54 through tubing 74 and 70 (arrows 138). Pouch 54 fills with growth factors. Valves 104 and 90 remain open for a predetermined period, depending on the volume of growth factor-containing medium to be distributed to pouch 54. After this period, valves 104 and 90 are closed. Valves 94 and 100 are then opened and pump 22 is operated (according to a predetermined flow rate and duration) to supply bag 36 with culture medium for rinsing.Valves 94 and 100 are closed, and valves 104 and 90 are reopened to allow the rinsing solution from bag 36 to flow by gravity into the cell expansion bag 54. Valves 104 and 90 remain open for a predetermined time, depending on the volume of rinsing solution to be dispensed into bag 54. After this time, valves 104 and 90 are closed. These phases of rinsing bag 36 and transferring the rinsing solution to the cell expansion bag 54 can be repeated one or more times, depending on the parameters of the biological protocol, so that, for example, all the growth factors originally contained in bag 36 end up in bag 54.

[0112] The fifth sub-step of distribution step 136 is represented in figure 19The procedure involves supplying cell expansion pouch 54 with cells to be cultured, then rinsing pouch 38 containing these cells with culture medium and transferring the contents of pouch 38 back to cell expansion pouch 54, in a manner similar to that performed in the fourth substep. Initially, valves 102 and 90 are opened, allowing the medium containing the cells to be cultured to flow by gravity from pouch 38 to pouch 54 through tubing 74 and 70 (arrows 140). Pouch 54 fills with cells to be cultured. Valves 102 and 90 are then closed, valves 94 and 98 are opened, and pump 22 is activated (at a predetermined flow rate and duration) to supply pouch 38 with culture medium for rinsing.Valves 94 and 98 are closed, and valves 102 and 90 are reopened to allow the rinsing solution from pocket 38 to flow by gravity into cell expansion pocket 54. Valves 102 and 90 are then closed. These phases of rinsing pocket 38 and draining the rinsing solution into cell expansion pocket 54 can be repeated one or more times, depending on the parameters of the biological protocol, so that, for example, all the cells to be cultured that were originally in pocket 36 end up in pocket 54.

[0113] The distribution step 136 of the process can be followed by a homogenization step of the contents of the cell expansion pocket 54, which is schematically represented in Figure 20During this step, the computer system 24 controls the stirring device 16 so that the platform 110 oscillates around axis B, as explained above (arrows 142). The amplitude, frequency, duration, and periods (rest-stirring, stirring, etc.) of these oscillations are determined according to the parameters of the biological protocol.

[0114] The process according to the invention then includes an incubation step 144 which can last several days, for example, about ten days. Periodically, according to the protocol parameters, the contents of the cell expansion pocket 54 can be homogenized by rotating the tray around axis B as explained above. This homogenization (periods, frequency, amplitude) is determined by the protocol parameters independently of the homogenization step following the distribution step 136.

[0115] During the incubation step 144, the operator may perform one or more samplings 146 in the cell expansion pocket 54 ( Figures 14 And 22 Some of these samples may be required by the computer system. For example, there are three mandatory samples, which can be taken immediately after the distribution stage, three days after the start of incubation stage 144, and seven days after the start of this stage 144. Other samples may be taken at the operator's discretion; the computer system may prompt the operator to perform these optional samples.

[0116] When the operator confirms to the computer system 24 that they are ready to take a sample, the computer system activates the cylinder 124 so that the plate 110 of the stirring device 16 is moved in rotation around the axis A to an inclined position, for example of approximately 45°, relative to a horizontal plane, as schematically represented in figure 9 And 22 The computer system 24 can then detect the correct position of the platform 110 via the sensors of the device 16.

[0117] The computer system 24 controls the opening of the valve 88 so that part of the contents of the cell expansion bag 54 flows by gravity from the bag 54 into the tubing 60 to the sampling means 62 located outside the enclosure 14 of the incubator 12. The operator can take a sample from the cell expansion bag 54 using a syringe 148 equipped with "Luer lock" type connection means which are engaged in the sampling means 62. After sampling, the valve 88 is closed and the tray 110 of the stirring device 16 is returned to a substantially horizontal position.

[0118] The operator can then carry out analyses of the sample taken, the results of these analyses being entered and recorded in the computer system by the operator.

[0119] During incubation step 144, the operator can also remove part of the consumables kit (first phase of consumables removal 150 - Figures 14 And 21 The components of the consumables kit that can be removed are all the bags (34, 36, 38, 40) and tubing (72, 74, 76) connected to tubing 70. To do this, the operator must cut tubing 70 upstream of its passage through the aforementioned block 80 and must simultaneously weld or crimp the cut end of tubing 70 remaining in chamber 14 of the incubator 12, to prevent any contamination of the cell expansion bag. This operation can be performed by the operator using appropriate cutting pliers that create a tight seal on the end of the tubing when it is cut. Valves 94 to 108 are then opened to allow the operator to remove tubing 70, 72, 74, 76 from these valves, as well as pump 22. figure 21). Once the operator has confirmed the removal of these items to computer system 24, the latter commands the closure of valves 94 to 108.

[0120] The process according to the invention further includes a step 152 of cell recovery after culture ( Figures 14 , 23 and 24 ). At the end of the incubation step 144 and at the operator's request, the computer system 24 blocks the rotation of the plate 110 around axis B and actuates the cylinder 124 so that the plate 110 of the stirring device 16 moves around axis A to a substantially vertical position shown in figures 8 , 23 and 24 The computer system 24 can then detect the correct position of the platform 110 via the sensors of the device 16.

[0121] The computer system 24 then controls the opening of the valve 86 so that the contents of the cell expansion bag 54 flow by gravity from the bag 54 into the two recovery bags 46 by circulating in the tubing 66 ( figure 23 ).

[0122] The plates 42 supported by the frame 18 and on which the recovery bags 46 are attached can be moved from their vertical position shown in figures 2 And 23 to their horizontal position represented schematically in figure 24either manually by the operator or via means of movement controlled by the computer system 24. The tilting of the plates 46 allows the bags 46 to be entirely located below the bag 54 and at least part of the tubing 66 so that the contents of the cell expansion bag 54 are, if possible, entirely transferred into the recovery bags 46. When the operator has confirmed to the computer system 24 that the collection is complete, this system commands the closure of the valve 86 and the deployment of the cylinder so that the platform 110 returns to a substantially horizontal position.

[0123] The bags 46 are then removed from the automated unit 10 for possible cell processing and reinjection into a patient's body, for example, for cell therapy. For this purpose, the tubing 66 can be cut and welded using the aforementioned clamp, or the bags 46 can be disconnected from the tubing 66.

[0124] The final step of the process consists of a second phase of removing consumables from the automated unit 10, specifically bags 54, 56, and the remaining tubing 66, 60, 70, and 78. To this end, the computer system 24 commands the opening of valves 86, 88, 90, and 92 to allow the operator to remove tubing 66, 60, 70, and 78. Once the operator has confirmed the removal of these components to the computer system 24, the latter commands the closing of valves 86, 88, 90, and 92.

[0125] When the biological protocol is complete, the computer system can generate a culture report. This report may include the following information to ensure proper protocol traceability: manufacturer-specific information (the instrument's identification number, the version of the computer system's operating software, the version of the biological protocol monitoring software), all protocol parameters unless at least one of these parameters is at its default value, all culture parameters, actions performed by the operator (including the date of each action in YYYYMMDDTHhmiss format, the person who performed the action via a login ID, and the nature of the action via a code to be defined if no ambiguous label exists), and "system" events (including the date of the event in YYYYMMDDTHhmiss format and the nature of the event (alarm, user alert, failure detection, etc.)).etc.) via a coding system to be defined in the absence of an ambiguous wording), the results of sample analysis (including the date of each sample, the person who took the sample, the sample analysis results, etc.), and graft information (from the analysis of cells recovered after culture). This culture report can be accessed via the computer workstation on the aforementioned network.

Claims

1. Automated (10) apparatus of cell culture, comprising tanks (34, 36, 38) of culture medium, growth factors and cells to be cultured, an incubator (12) with a thermostated enclosure (14) in which is housed a container (54) for cell culture or expansion, and a control computer system (24) including means configured for entering and recording data intended to control the culture conditions in the enclosure and to manage valves (20) for dispensing fluids in a predefined sequence, characterized in that it comprises an agitation device (16) for supporting and agitating the cell culture or expansion container which is controlled by the control computer system and which is disposed in said enclosure, and in that said container is formed by a cell expansion bag (54) having at least one inlet port (68) connected to said tanks and one outlet port (64) connected to means (46) configured for harvesting and storage of the cells after culture, these harvesting and storage means and said tanks being located outside the enclosure and being connected to said ports of said cell expansion bag by conduits (66, 70, 72, 74) which form with said cell expansion bag a preassembled module placed in said enclosure and which pass through a wall of said enclosure so as to allow to feed the cell expansion bag with said culture medium, growth factors and cells to be cultured, and to harvest the contents of said cell expansion bag in the harvesting and storage means while maintaining the enclosure closed, the agitation device (16) comprising a tray (110) for supporting the cell expansion bag (54), the tray being rotatably mounted about a first horizontal axis (A), the tray being moveable about this axis between a substantially horizontal position of cell culture to a substantially vertical position of harvesting cells after culture.

2. Automated apparatus according to claim 1, wherein said cell expansion bag (54) further comprises a sampling outlet (58) which is connected by a conduit (60) to a sampling device (62) located outside of said enclosure (14), said conduit passing through said wall of said incubator and being part of said preassembled module.

3. Automated apparatus according to claim 1 or 2, wherein said incubator (12) includes a cabinet having an opening and equipped with a sealed closure door (50), means (80) for the passageway of said conduits (60, 66, 70) being mounted on the peripheral edge of said opening and having grooves (82) which are slightly parallel and into which are engaged said conduits, said grooves being intended to be covered by said sealed closure door when in closed position.

4. Automated apparatus according to one of the preceding claims, wherein said tanks of growth factors and of cells to be cultured are formed by bags (36, 38) which are located above the inlet port (68) of said cell expansion bag (54), so that the content of each of the bags of growth factors and of cells to be cultured can flow by gravity to said cell expansion bag.

5. Automated apparatus according to one of the preceding claims, wherein said harvesting and storage means comprises one or two bags (46) which are at least partially located below the outlet port (64) of said cell expansion bag (54) so that, after culturing, the content of said cell expansion bag can flow by gravity to said one or two bags of the harvesting and storage means.

6. Automated apparatus according to one of the preceding claims, wherein said cell expansion bag (54) comprises flexible liquid-tight and gas permeable walls, in particular to CO2, and preferably having properties that minimize the adhesion of the cells to be cultured to the walls of the bag.

7. Automated apparatus according to one of the preceding claims, which comprises a peristaltic pump (22) for controlling the supply of said culture medium to said cell expansion bag (54) and to said tanks (36, 38) of growth factors and of cells to be cultured, for rinsing said tanks.

8. Automated apparatus according to one of the preceding claims, which comprises two bags (40, 56) forming air trap, one of which being connected to the tanks (36, 38) of growth factors and of cells to be cultured, and the other being connected to said cell expansion bag (54), said two bags being intended to collect and store the air contained in said conduits (70, 72, 74), said cell expansion bag (54) and / or said tanks (36, 38).

9. Automated apparatus according to one of the preceding claims, wherein said conduits are formed by flexible tubes (60, 66, 70, 72, 74, 76, 78), some of which going through valves (86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108) which are intended in closed position, to pinch the tubes.

10. Automated apparatus according to one of the claims 1 to 9, wherein said tray (110) is mounted in rotation around a second horizontal axis (B) around which the tray is intended to oscillate for agitation and homogenization of the content of said cell expansion bag.

11. Automated apparatus according to one of the claims 1 to 10, wherein the tray (110) bears valves (86, 88, 90) for controlling the supply of said cell expansion bag (54), the harvesting of the content of said cell expansion bag, and the sampling of said cell expansion bag.

12. Automated apparatus according to one of the claims 1 to 11, wherein the supporting and agitation device (16) comprises a vertical arm (128) having at its upper end means (130) for attaching a bag (56) forming an air trap connected to said cell expansion bag (54).

13. Automated apparatus according to one of the preceding claims, wherein said means for harvesting and storage (46) are mounted in rotation around a horizontal axis and are movable around said axis between a substantially vertical position and a substantially horizontal position in which said means for harvesting and storage are located completely below said cell expansion bag.

14. Sterile and disposable cell culture kit for a cell culture automated apparatus (10), which comprises at least one cell expansion bag (54) and flexible tubes (60, 66, 70, 72, 74) connecting said at least one cell expansion bag to other bags or tanks, said tubes and said cell expansion bag being pre-assembled, the bag being intended to be placed within a cell culture enclosure and the tubes being intended to pass through a wall element of said enclosure, the cell expansion bag comprising an inlet port (68), an outlet port (64), and optionally a sampling port (58), the inlet port (68) of the cell expansion bag (54) being connected by tubes (70, 72, 74) to the inlet and outlet ports of a growth factor bag (36), and to the inlet and outlet ports of a bag (38) for cell culture, the kit further comprising two bags (40, 56) forming air trap, one of which is connected to the outlet ports of the growth factor and cell culture bags (36, 38), and the other of which is connected to the inlet port (68) of the cell expansion bag (54).

15. Kit according to claim 14, wherein it comprises one or two bags (46) for harvesting the cells after culture, which are connected by tubes (66) to said outlet port (64) of said cell expansion bag (54).

16. Automated apparatus according to one of the claims 1 to 13, comprising a supporting and agitation device (16) for a cell culture automated apparatus (10), characterized in that it comprises a tray (110) for supporting a cell expansion bag (54), this tray bearing three valves (86, 88, 90) and being mounted in rotation about a first horizontal axis (A) for the tilting of said tray from a substantially horizontal position to a substantially vertical position, and about a second horizontal axis (B) around which the tray is intended to oscillate for agitation and homogenization of the contents of said cell expansion bag, wherein the device further comprises controlled means (124, 126) for tilting the tray around the aforesaid horizontal axes.

17. Automated apparatus according to claim 16, which comprises a vertical arm (128) having at its upper end means (130) for attaching a bag (56) forming an air trap.

18. An automated method of cell culture by means of an automated apparatus (10) according to one of claims 1 to 13, 16 and 17, which comprises the steps of: a) feeding said cell expansion bag (54) with said culture medium, with said growth factors then with said cells to be cultured, while maintaining said enclosure (14) of said incubator (12) closed; b) agitating said cell expansion bag in order to homogenize its content; c) maintaining said cell expansion bag in incubation conditions for a period of several days, and d) harvesting the content of said cell expansion bag in said harvesting and storage means (46) while maintaining the enclosure closed.

19. Method of claim 18, which comprises: - prior to step a), a step of installing said preassembled module by fitting said cell expansion bag (54) on said supporting and agitation device (16), by mounting said conduits (60, 66, 70, 72, 74) in the passageway means (80) of said incubator (12) and into said valves (20), and by connecting said conduits to tanks or bags (34, 36, 38), and / or - prior to step a), a step of evacuating air contained in said conduits (70, 72, 74) by passage of culture medium from said culture medium tank (34) to said bags (40, 56) forming air trap, and / or - after supplying said cell expansion bag with growth factors in step a), a step of rinsing said growth factors tank (36) by flowing culture medium in said growth factors tank and then by draining its content to said cell expansion bag (54), and / or - after supplying said cell expansion bag with cells to be cultured in step a), a step of rinsing the tank (38) containing said cells to be cultured by flowing culture medium in said tank of cells to be cultured and then by draining its content to said cell expansion bag (54), and / or - during step c), one or more steps of sampling the contents of said cell expansion bag (54), which are each preceded by a step of tilting said tray from a horizontal position of cultivation to an inclined position in which the sampling outlet (58) of said cell expansion bag is the lowest point of this bag; and / or - prior to step c), a step of removing the tanks (34, 36, 38) of culture medium, of growth factors and of cells to be cultured by cutting and welding or pinching the conduit or tube (70) connecting these tanks to the inlet port (68) of said cell expansion bag (54), and / or - before or during step d), a step of tilting said tray (110) in a substantially vertical position so that the outlet port (64) of said cell expansion bag (54) represents the lowest point of this bag.

20. Use of an automated apparatus according to claim 1 to 13, 16, 17 or of a cell culture kit according to claims 14 or 15, for the culture of stem cells of type CD34 + or blood mononuclear cells, such as for example lymphocytes.

21. Use according to claim 20, herein the stem cells are issued from one or more sources, more particularly such as umbilical cord blood, bone marrow and and / or whole blood.