automated device and automated cell culture process

专利摘要:
AUTOMATED DEVICE AND AUTOMATED CELL CULTURE PROCESS. Automated cell culture device (10), comprising culture medium reservoirs, growth factors and cells to be cultured, an incubator (12) with thermostatic chamber (14) in which a cell culture vessel is housed, and a system control computer (24), characterized by the fact that it comprises a device (16) for supporting and stirring the culture vessel housed in the chamber, as well as the fact that the culture vessel is formed by a pouch containing at least one entrance connected to the aforementioned reservoirs and an exit orifice connected to means (42) for the recovery and storage of cells after culture, with such storage means as well as the reservoirs being located outside the chamber, being connected to the orifices of the cell. cell expansion bag by conduits that form a pre-assembled module with the cell expansion bag, and that pass through a chamber wall element, in order to allow feeding the cell expansion bag in culture medium, growth factors and cells to be cultured, and the recovery of the cell expansion bag contents in the storage media, keeping the (...) closed.
公开号:BR112014022647B1
申请号:R112014022647-4
申请日:2013-03-14
公开日:2020-10-20
发明作者:Philippe Henon；Claire Saucourt；Patrick Gasse；Alain Sundas；Pierre Sugranes；Amandine Verdier；Frédéric Demonchy
申请人:Cellprothera；
IPC主号:

专利说明:

[0001] The present invention relates to an automated cell culture device, intended, in particular, for the culture of stem cells (of the CD34 + type, for example), as well as a cell culture process that uses such an automated device.
[0002] Among the domains related to cell culture, cell therapy is the least advanced in terms of industrialization. There is, therefore, a relevant need to find a technology capable of producing cells in sufficient quantity and under optimal conditions, with a view to the use of such cells in therapeutic applications.
[0003] Certain cell therapy processes require a culture or an amplification of stem cells before reinjection into a patient, as the quantities extracted are sometimes insufficient to produce a therapeutic effect. It is essential to guarantee the integrity of the therapeutic properties of the cells during their culture. In the current state of the art, the solutions proposed for the cultivation of stem cells ex vivo are handcrafted, very empirical and ineffective.
[0004] Furthermore, the current technique does not allow the production of stem cells in sufficient quantity for therapeutic applications. There is, therefore, a real need to develop a technology of the type bioreactor with a compact geometry, which allows the cultivation of cells in large quantities.
[0005] The use of bio-reactors 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] For example, bio-reactors with membrane perfusion, bio-reactors with hollow fibers, bio-reactors on a fluidized bed and micro bio-reactors with continuous perfusion of O2, nutrient medium and growth factors ( according to application WO 00/73411).
[0007] Application US-A1 -2008/0118977 describes a therapeutic protocol that allows the reconstruction of a patient's heart after a heart attack. Reconstruction is achieved by injecting, at the patient's heart level, specific stem cells (CD34 +) isolated from a blood extraction, amplified ex vivo and purified after culture.
[0008] The specific purpose of the present invention is to provide a technology capable of ensuring the culture, amplification or expansion of this type of cells, and to bring still visible improvements in terms of standardization, traceability and control for operations that are, in most, performed manually in the light of the prior art.
[0009] To this end, it proposes an automated cell culture device, comprising reservoirs of culture medium, growth factors and cells to be cultivated, an incubator with a thermostatic chamber where a cell culture container or cellular expansion, and a computer command system that includes means of capturing and recording data, and designed to regulate the culture conditions in the chamber and to pilot fluid distribution valves according to a pre-defined sequence, characterized by the fact that comprise a device for supporting and agitating the culture or cell expansion container, which is controlled by the computer system, and which is housed in the chamber, and also because the container is formed by a pouch that holds at least an entrance orifice connected to the above-mentioned reservoirs and an exit orifice connected to the means of recovery and storage of cells after culture, being such means of and storage as well as the reservoirs located outside the chamber and connected to the holes of the cell expansion bag by conduits that form, with the cell expansion bag, a pre-assembled module placed in the chamber and that cross a wall element of the chamber, in order to allow the feeding of the cell expansion bag in culture medium, growth factors and cells to be cultured, and the recovery of the cell expansion bag contents in the storage media, keeping the chamber closed.
[0010] The automated device according to the invention has the primary purpose of automating the stages of the biological culture protocol and the management of environmental conditions (temperature control, CO2 rate, etc ...) of culture of the cells of the incubator, in order to generate an optimal cell amplification yield. It is also intended to ensure the distribution of the culture medium, growth factors and cells to be cultured into a cell expansion bag (which can be provided with a relatively large volume) located in the incubator, thanks to the control of such media. such as valves and pumps. The automated device also ensures the agitation of the cell expansion bag, as well as the transfer of cells after culture, from the cell expansion bag to the storage media.
[0011] The automated device according to the invention can allow the generation of large quantities of cells, such as stem cells, from cells extracted from a patient. The bag in which the cell culture is carried out may have a volume greater than 100 ml_, 200 ml_, 300 ml_, 500 ml_, or be approximately 650 ml_ or even more (1L, 2L, 3L, etc ...). The culture of stem cells in a pouch of this type allows the generation of cells in sufficient quantity to carry out a cell therapy in a patient, such as, for example, patients who have suffered a heart attack, according to the biological protocol described in the US- A1-2008 / 0118977.
[0012] The cell expansion bag preferably comprises flexible walls, impermeable to liquids and permeable to gases. It preferably presents a good permeability to oxygen and carbon dioxide, which allows a good aeration of the bag's contents without opening it, and therefore without risk of contamination of its contents. In a peculiar example of carrying out the bag, it comprises the following permeability characteristics (in cc per day, at 37 ° C): O2 (gas) = 418, CO2 (gas) = 966, N2 (gas) «157 and H2O (net) «0.05.
[0013] Preferably, the cell expansion bag has little affinity with chemical or biological products, in particular with the cells to be cultured, and does not absorb such products. The pouch is formed, for example, by a thin film of FEP copolymer (fluorine-ethylene-propylene). The exchange may consist of different types of ports (modifiable interfaces), among which, for example, FEP couplings. They are mounted on the bag to reduce the risk of contamination.
[0014] The automated device is intended for carrying out cell culture, and includes all the means and resources for carrying out such culture, without an operator having to manipulate valves, replacing bags or reservoirs, etc ... Cell culture it is carried out according to a precise biological protocol, which is entirely generated by the computer system, and which allows, for example, to control valves and the stirring device, and also to regulate the environmental conditions in the incubator chamber. The operator will be able to provide the computer system with patient identification data, information about the extracted cells and about the nature and origin of the different reservoirs or bags, so that all these data are recorded in the computer system. The invention thus makes it possible to implement biological protocols with excellent reproducibility, and also to ensure traceability and precise control of the protocols and the means used.
[0015] The control and traceability of the stages of the biological protocol can be ensured by the computer system and by an adequate human-machine interface (“HMI”), which allow, for example: - to define an automated culture process, where the parameters specific to the biological protocol of cultures are not changeable, - ensure a good safety margin, restricting access to the data of the computer system, thanks to the identification of the user and the need to use a password (in accordance with the regulation 21 CFR Part 11 of the FDA), - authorize a record of events and the different stages of the process, and - establish an edition of reports (comprising results of the analysis of the extraction and of the posterior analysis of the characteristics of the graft, for example).
[0016] The cell expansion bag is housed in the chamber and comprises at least two holes connected to conduits that pass through a wall element of the incubator and which are connected to the reservoirs and the storage means connected to the outside of the chamber. The cell expansion bag and conduits form a pre-assembled, single-use module that is easily implanted and replaced by the operator. At least a part of the contents of the reservoirs located outside the chamber are intended to be distributed to the cell expansion bag located in the chamber, the contents of which after culture are intended to be transferred to the storage media located outside the chamber, and all these operations must be carried out. distribution of fluids must be carried out keeping the incubator chamber closed thanks to the conduits that pass through an incubator wall element, which allows to ensure, in the chamber, the existence of optimal environmental conditions throughout the implementation of the biological protocol, and, still, limit the risk of contamination of the cell culture medium.
[0017] Advantageously, the cell expansion bag comprises an extraction orifice that is connected, via a conduit, to extraction means located outside the chamber, such conduit having to pass through the aforementioned wall element of the chamber and integrate the pre module - mounted. The cell expansion bag thus comprises three orifices with different functions (feeding, recovery and extraction), and which are connected to different conduits.
[0018] In an embodiment of the invention, the incubator comprises a cabinet that includes an opening and an airtight closing door for such an opening, with the aforementioned means of passage of the conduits having to be mounted on the peripheral edge of this opening and having sensible necks parallel to the conduit housing, such throats being intended to be covered by the door whenever it is in a closed position. The conduits can be easily attached (and removed) to these conduits by an operator, whenever the door is open, by translating the conduits in a direction perpendicular to the longitudinal axes of the throats, which facilitates the assembly of consumables.
[0019] The reservoirs of growth factors and cells to be cultivated are preferably formed by bags located above the entrance hole of the cell expansion bag, so that the content of each of these bags of culture medium and cells to be cultured can drain, by force of gravity, into the cell expansion bag. This allows to ensure the integrity of cells and growth factors when they are transferred to the cell expansion bag. The use of a pump or any mechanical means to promote the circulation of cells and growth factors in the conduits would generate an effective risk of damage to them.
[0020] The storage means may comprise one or two bags that are, at least in part, located below the exit hole of the cell expansion bag, so that, after culture, the contents of the cell expansion bag it can drain, by gravity, to the storage bags (s). This also allows to ensure the integrity of the cells after culture, when recovering.
[0021] The automated device may comprise a peristaltic pump to control the feeding in the culture medium of the cell expansion bag, as well as reservoirs of growth factors and cells to be cultured, with a view to washing such reservoirs. The peristaltic pump has the advantage of not being in direct contact with the culture medium, which avoids any risk of contamination of that medium.
[0022] The automated device can also comprise two bags that form an air collector, one of which is connected to the reservoirs of growth factors and cells to be cultivated and the other is connected to the cell expansion bag, both of which are intended for collect and store the air contained in the conduits, in the cell expansion bag and / or in the reservoirs.
[0023] Advantageously, the conduits are formed by flexible pipes, of which at least some pass through valves that are intended, in a closed position, to clamp such pipes. Each pipe is intended, for example, to be coupled to a valve throat in a simple way by the operator, thanks, for example, to translation in a direction substantially perpendicular to the longitudinal axis of the pipe.
[0024] The support and agitation device can comprise a support plate of the cell expansion bag, which is mounted in rotation around a first horizontal axis, being able to move around that axis between a substantially horizontal culture position. cells and a substantially vertical position of cell recovery after culture. This last position facilitates the recovery of cells after culture, and these cells must flow directly, due to gravity, in the aforementioned storage media.
[0025] The plate can be mounted in rotation around a second horizontal axis, around which the plate is intended to oscillate in order to agitate and homogenize the contents of the cell expansion bag. The first and second axes of rotation of the plate are preferably parallel.
[0026] Preferably, the plate includes control valves for feeding the cell expansion bag, retrieving the contents of this bag and extracting samples from this bag.
[0027] The support and stirring device may also comprise a vertical arm that comprises, at its upper end, means for coupling a bag that forms an air trap, and is connected to the cell expansion bag.
[0028] The storage means are advantageously mounted in rotation around a horizontal axis, being able to be moved around that axis between a substantially vertical position and a substantially horizontal position where the means are entirely located below the bag. cell expansion. This ensures that all cells contained in the cell expansion bag are transferred, by gravity, to the storage media.
[0029] As an example, and in the specific case of CD34 + stem cell culture, the sterility required within the entire conditioning chain of CD34 + cells requires the use and installation of consumables in the form of a cell culture kit single use.
[0030] Therefore, the present invention also concerns a cell culture kit, preferably sterile and single use, for an automated cell culture device, characterized by the fact that it comprises at least one cell expansion bag, as well as tubes flexible connection of the bag to other bags or reservoirs, and the cell expansion tubes and bag must be pre-assembled, and the cell expansion bag must include an inlet hole, an outlet hole and, possibly, an extraction hole .
[0031] The kit can also comprise all the couplings necessary to connect the pipes with each other and with the bags and / or reservoirs, as well as the means connected to the third hole of the cell expansion bag to authorize cell extractions. All of these elements can be part of the aforementioned pre-assembled module.
[0032] Advantageously, the entrance hole of the cell expansion bag is connected by pipes to the entrance and exit holes of the growth factors bag, and to the entrance and exit holes of the cell bag to be cultivated. The inlet hole of the cell expansion bag is also intended to be connected to an outlet hole of the cell expansion bag.
[0033] The kit can also comprise two bags that form an air trap, one of which is connected to the exit holes of the growth factor and cells to be cultured and the other is connected to the entrance hole of the cell expansion bag. .
[0034] The kit can also comprise one or two cell recovery bags after culture, which are connected by tubes to the exit hole of the cell expansion bag.
[0035] In an example of an embodiment of the invention, the bags of growth factors and cells to be cultured and those forming an air collector have an internal volume of approximately 150 ml, while the cell expansion bag has a volume theoretical volume of 3000 mL and the two recovery bags an approximate volume of 600 mL. The culture medium distribution bag can have a volume of approximately 1000 mL.
[0036] Advantageously, the kit forms or constitutes a closed circuit that, once installed for a cell culture, comprises all the necessary resources for that culture, without the need for adding any product or the intervention of an operator. This allows to limit the risks of contamination of the kit and the culture medium.
[0037] The invention also refers to a support and stirring device for an automated cell culture device, characterized by the fact that it comprises a support plate for a cell expansion bag, which plate must contain three valves and be mounted in rotation around a first horizontal axis for dragging the plate from a substantially horizontal position to a substantially vertical position, and a second horizontal axis around which the plate is intended to oscillate to agitate and homogenize the contents of the bag. cellular expansion, and the device must also comprise controlled means of dragging the plate around the aforementioned horizontal axes.
[0038] The device may comprise a vertical arm that comprises, at its upper end, means for coupling a bag that forms an air trap.
[0039] The invention also relates to an automated cell culture process by means of an automated device as described above, and characterized by the fact that it comprises the steps that consist of: a - feeding the cell expansion bag in medium culture, growth factors and then cells to be cultured, keeping the incubator chamber closed; b - shake the cell expansion bag in order to homogenize its content; c - keep the cell expansion bag in incubation conditions for several days; and d - recover the contents of the cell expansion bag in the storage means, keeping the chamber closed.
[0040] The process according to the invention can comprise one or more steps among the following: - before step a), a step of installing the pre-assembled module, mounting the cell expansion bag on the stirring device, mounting the conduits in the incubator passageways and valves, and connecting such conduits to the reservoirs or bags, - before step a), a step of evacuation of the air contained in the conduits, thanks to the passage of the culture medium from the reservoir of the culture medium to the vapor trap (s); - after feeding the cell expansion bag into growth factors in step a), a step of washing the growth factor reservoir, promoting the passage of culture medium in that reservoir and then evacuating its contents to the cell expansion; - after feeding the cell expansion bag into cells to be cultured in step a), a step of washing the reservoir of cells to be cultured, promoting the passage of culture medium in that reservoir and then evacuating its contents until the cell expansion bag; - during step c), one or more steps of extracting samples from the contents of the cell expansion bag, each of which is preceded by a step of dragging the support plate from a horizontal culture position to an inclined position, in which the extraction hole 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 cultivated by cutting, soldering or clamping the conduit or the connecting pipe of such reservoirs in the orifice entry of the cell expansion bag; - before or during step d), in dragging the plate in a substantially vertical position, so that the exit hole of the cell expansion bag represents the lowest point of the bag.
[0041] Finally, the invention refers to the use of an automated device, a kit or a device as described above, for the culture of stem cells of the CD34 + type or mononuclear blood cells, such as, for example, lymphocytes. Stem cells can come from one or more sources, such as, more particularly, umbilical cord blood, bone marrow and whole blood.
[0042] The invention will be more clearly understood, and other details, advantages and characteristics of the invention will be more clearly understood by reading the description below, performed by way of example and not restrictive, and making reference to the attached drawings, in which: - Figures 1 and 2 are schematic views in perspective of the automated cell culture device according to the invention, such device having a cabinet that defines a chamber shown closed in Figure 1, and open in Figure 2; Figure 3 is a very schematic view of the automated device in Figures 1 and 2, without the computer system; Figure 4 is a very schematic view of the components of the stirring device of the automated device of Figures 1 and 2; Figure 5 is a schematic view of a cell culture kit according to the invention; Figure 6 is a schematic perspective view of the fluid conduit passage means of the automated device of Figures 1 and 2; Figure 7 is a schematic perspective view of the stirring device according to the invention; Figures 8 and 9 are schematic views in perspective of the device of Figure 7, and represent two different positions of inclination of the plate of that device; Figure 10 is another schematic view in perspective of the stirring device of Figure 7, with the partial removal of the hood of that device; - Figure 11 is a schematic view in perspective of the plate and means controlled for dragging the plate of the device of Figure 7, seen from below; Figures 12 and 13 are schematic views in perspective of a commanded system for locking the plate rotation of the device of Figure 7, said locking system being shown active in Figure 11 and inactive in Figure 12; Figure 14 is an organization chart showing the steps of a cell culture process according to the invention; and - Figures 15 to 24 are views that correspond to Figure 3, and represent steps of the process according to the invention.
[0043] We refer initially to Figures 1 and 2, which represent an example of realization of the automated cell culture device 10 according to the invention, the said device being specifically but not exclusively intended for stem cell culture following , for example, the biological protocol described in application US-A1 -2008/0118977, the content of which is incorporated into this report by reference.
[0044] In the example shown, the automated device 10 essentially comprises three elements: - a thermostatic chamber incubator 12, in which a device 16 supporting and shaking a cell expansion bag (not shown) is housed, - a construction 18 bag support (not shown), containing the necessary means for cell culture and bringing the means (valves 20, pump 22, etc ...) for distribution and regulation of the fluid flow between the bags, and - a system computer 24 connected to the incubator 12 and the means 20, 22 for the command of these, as well as for the capture and recording of data and for the management of the biological protocol.
[0045] In the example shown, the incubator 12, the construction 18 and the computer system 24 are arranged side by side on a support 26 mounted on wheels, the construction 18 being located between the computer system 24 and the incubator 12.
[0046] Typically, the computer system 24 comprises means for capturing and recording data, as well as means for data processing, means of displaying and emitting signals for command and piloting of the incubator 12, and also means 20, 22 of the construction. Preferably, the computer system 24 comprises a touch screen for displaying and capturing data.
[0047] To restrict access to data previously registered in the computer system 24, several levels of security can be implemented. The manufacturer of the automated device can have a maximum level of access right, by means of a specific password, to have access to all the information registered in the computer system 24, while an administrator and an operator with lower levels of access will only have access, through specific passwords, to certain information.
[0048] The computer system 24 is advantageously connected to a computer network by means of an Ethernet or Wifi connection, for example, so that the information contained in system 24 can be accessed from a station computer network, away from the automated device 10, and so that, eventually, certain measures can be requested and commanded to the automated device from that station.
[0049] The computer system 24 controls, for example, the opening and closing of the valves 20, which are, for example, of the all or nothing type, the flow regulation of the pump 22, the heating regulation of the incubator chamber 14 12 (so that it reaches, for example, a temperature of approximately 37 ° C), and the chamber is fed with gases such as CO2 (for example, at a rate of approximately 5%). The system 24 can ensure the regulation of other parameters, if necessary, inside the chamber 12, in order to define optimal environmental conditions for cell culture.
[0050] For the sake of greater clarity, the means of connection of the computer system 24 with means 18, 20 and with incubator 12, with the means of heating and gas supply of incubator 12 and with means of electrical supply not are represented in the drawings.
[0051] Construction 18 has a parallelepiped shape, and comprises a vertical front face 28, on which are schematically drawn rectangles 30 representing the positions of biological media reservoirs in the form of bags, as well as lines 32 that represent the placement of fluid conduits between such bags.
[0052] The upper part of this front face 28 comprises four rectangles 30 drawn, which inform an operator of the nature of each of the bags to be positioned at the level of the mentioned rectangles, and such bags must belong to a kit of consumables that will be described in more details below.
[0053] A first large rectangle is drawn in the upper left part of the front face 28 of construction 18, and represents the position of a bag containing a culture medium (bag referred to as 34 in Figure 3). Three rectangles 30 of lower dimensions are drawn in the upper right part of the face 28, and represent the positions of a bag of growth factors, a bag of cells to be cultivated and a bag that forms an air collector (referred to in Figure 3, such as 36, 38 and 40 respectively).
[0054] The middle part of the front face 28 of construction 18 comprises mounting holes for the valves 20 and the pump 22 mentioned above, each of these elements (valves and pump) located in a line 32, which represents a fluid conduit formed flexible tubing from the consumables kit.
[0055] The lower part of the front face 28 contains two coplanar plates 42 and arranged side by side. Such plates 42 are pivoted from their lower ends, around the same horizontal axis that extends parallel to the front face 28. The plates 42 can be moved in rotation around that axis, between a vertical position (represented in the Figures 1 and 2), in which they extend parallel and a short distance from the front face 28, and a horizontal position, in which they can rest on the support 26.
[0056] Some rectangles 44 are drawn on the front faces of the plates 42, when they are in a vertical position. Such rectangles 44 inform the operator of the nature of the bags to be brought by such plates 42. Some bags for recovery and storage of cells after culture (referred to in Figure 3 as 46) are intended to be brought by such plates 42.
[0057] The bags 34, 36, 38, 40 and 46 of the consumables kit are intended to be fixed or attached to the front face 28 of the construction, as well as to the plates 42, by the appropriate means that are not represented.
[0058] The incubator 12 comprises a cabinet that defines the chamber 14 and which comprises an opening that can be closed hermetically by a double door, these two doors 48, 50 being pivotable on one side of the opening, by example, on the right side.
[0059] The internal door 48 is a glass door designed, in a closed position, to rest on a peripheral joint 52 of the cabinet opening, said joint 52 being visible in Figure 6. The external door 50 is heat-treated, and comprises a peripheral joint designed to rest on the peripheral edge of the cabinet opening.
[0060] The computer system 24 can be connected to sensors for detecting the position (open or closed) of each door 48, 50, and can command the locking of these doors, more specifically during the incubation and cell culture phase.
[0061] The chamber 14 of the incubator 12 has, for example, an internal volume of approximately 200 L.
[0062] In the example shown in Figures 3 to 5, the consumables kit is for single use for a cell culture, and comprises bags 34, 36, 38, 40 and 46 and the tubes mentioned above, as well as a bag of cell expansion 54 and a second bag 56 forming an air trap, such bags 54, 56 being carried by the stirring device 16, which will be described in greater detail below, with reference to Figures 7 to 13.
[0063] The cell expansion bag 54, most visible in Figures 4 and 5, can have an internal volume greater than 500 ml_, such as, for example, 650 ml_, and comprises three holes, an extraction hole 58, connected by a tubing 60 to extraction means 62, an outlet port 64, connected by a tubing 66 to the cell recovery pouches 46 after culture, and an inlet port 68, connected by tubing to the bags 34, 36, 38 and 56.
[0064] The inlet port 68 of the cell expansion bag 54 is connected, via a tubing 70, to an inlet port of the bag 34 of culture medium. Each of the growth factor and cell pockets 36 and 38 to be cultured comprises an inlet port, connected to one end of a pipe 72 whose other end is joined to tubing 70, and an outlet port, attached to one end a pipe 74 whose other end is joined to pipe 70 (below the connection points between pipes 72 and pipe 70). The bag 40 comprises two holes connected by pipes 76 to the pipes 74, and the bag 56 forming an air collector comprises a hole connected by a pipe 78 to the pipe 70, in the vicinity of the entrance hole 68 of the bag 54 (Figures 4 and 5).
[0065] The cell expansion bag 54 and the pipes 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 in the above-mentioned pipes. The bag 38 of cells to be cultured is also supplied empty, and can be pre-assembled in the pipes or, still, connected to the pipes when installing the kit in the automated device. Bag 38 can be filled with a medium containing cells to be cultured, before or after installing the kit in the automated device. Bags 34 and 36 are preferably supplied filled with culture medium and growth factors respectively.
[0066] All couplings of pipes and tubes to the bags, as well as the extraction means are also preferably part of a pre-assembled module, which is represented schematically in Figure 5, being the bags 34, 36 and 38, that are not necessarily part of this module, represented in dotted lines.
[0067] Bags 36, 38, 40 and 56 have a content of approximately 150 ml, bags 46 have a content of approximately 600 ml, and bag 34 of culture medium has a content of approximately 1000 ml.
[0068] If automated device 10 is used to grow CD34 + stem cells, bag 38 will comprise cells of this type from extraction in a patient and eventually isolated and purified, and growth factors from bag 36 will be cytokines.
[0069] The cell expansion bag 54 and the bag 56 forming the air collector are transported by the shaking device 16, and housed in chamber 14 of incubator 12 (Figure 3). The other bags 34, 36, 38, 40 and 46, as well as the extraction means 62 are located outside the chamber 14.
[0070] The pipes 60, 66 and 70 connecting the cell expansion bag 54 to the elements located outside the chamber 14 pass through an organ of the incubator, which authorizes the hermetic closing of the chamber 14, such organ being represented in Figures 3 r 6.
[0071] This organ is a wall element, formed by a block 80 of material (for example, plastic) that is fixed to the peripheral edge of the opening of the incubator cabinet 12, and which comprises three parallel throats 82 of coupling and passage of the pipes 60, 66 and 70 mentioned above. Such throats 82 are substantially straight and distant from each other. Block 80 has a substantially flat shape, and extends in a vertical plane. It comprises a rear face supported on the peripheral edge of the cabinet opening, and a front face on which the throats 82 are formed, which have a substantially horizontal orientation and extend across the transverse dimension of the block.
[0072] Throats 82 have a substantially circular section and an internal diameter slightly larger than that of pipes 60, 66 and 70. These pipes are intended to be completely coupled to these throats, and, eventually, to pass through cuts 84 of the peripheral joint of the edge of the cabinet opening.
[0073] In the closed position of the chamber 14, the peripheral edge of the inner door 48 is intended to rest on the joint 52 and to cover the parts of the pipes 60, 66 and 70 that extend in the aforementioned cuts 84 of the joint 52 , while the peripheral joint of the external port 50 is intended to rest on the front face of the block and to cover the necks 82 and the parts of the pipes 60, 66 and 70 that extend in these necks.
[0074] In the example shown, the lower throat of the block 80 forms a passage of the connecting pipe 66 of the cell expansion bag 54 to the recovery bags 46, the middle throat forms a passage of the connecting pipe 60 of the pocket 54 to the extraction means 62, and the upper throat forms a passage from the connection pipe 70 of the pocket 54 to the bags 34, 36, 38 and 40.
[0075] As shown in Figure 3, piping 70 is coupled to pump 22, in the vicinity of bag 34 of culture medium, such pump being a peristaltic pump to avoid the risks of contamination of this medium.
[0076] The aforementioned valves 20 are solenoid valves in number of twelve in the example shown, which are listed as references 86 to 108 in Figure 3.
[0077] Pipes 66, 60, 70 and 78 are coupled to four valves 86, 88, 90 and 92 respectively, which are transported by the stirring device 16 (Figures 3 and 4).
[0078] The part of the pipe 70 located outside the chamber 14 is coupled in two valves 94 and 96, distant from each other, one of which 94 is located in the vicinity of the bag 34 of culture medium. The other valve 96 is located below the connection of pipe 70 to pipes 72, and above the connection of pipe 70 to pipes 74.
[0079] Pipes 72 connected to the inlet holes of bags 36 and 38 are coupled to valves 98 and 100 respectively, while pipes 74 connected to the outlet holes of bags 36 and 38 are coupled to valves 102 and 104 respectively.
[0080] Each of the pipes 76 connected to the bag 40 forming an air collector is coupled to a valve 106, 108.
[0081] The pump 22 and the valves preferably comprise a transverse pipe assembly throat by translation, in a direction perpendicular to the longitudinal axis of the pipe or throat.
[0082] As shown schematically in Figure 3, and visible in Figure 2, bags 34, 36, 38, 40 and 56 and pipes 40, 72, 74, 76, 78 connecting these bags to the cell expansion bag are found all located above that pocket 54, whenever the latter is arranged horizontally. The bags 46, the extraction means 62 and the pipes 60, 66 connecting these elements to the cell expansion bag 54 are all located below that bag 54, whenever the latter is arranged horizontally.
[0083] Pockets 36, 38 and 56 are substantially located on the same horizontal plane, which is located below a horizontal plane where pockets 34 and 40 are located.
[0084] The consumable kit can be installed on the automated device as follows. Doors 48 and 50 of incubator 12 are opened. Doors 34, 36, 38, 40 and 46 are coupled to the construction, and bag 56 is attached to the arm of the stirring device 16. Bag 54 is placed flat on the stirring device 16. Piping 70 is attached to valves 94, 96, as well as pump 22, pipes 72 are coupled to valves 98, 100, pipes 74 are coupled to valves 102, 104 and pipes 76 are coupled to valves 106, 108. Pipes 66, 60 , 70 and 78 are coupled to valves 86, 88, 90 and 82 respectively, which are loaded by device 16, and then pipes 66, 60 and 70 are coupled to necks 82 of block 80 respectively. The pipes are connected to bags that are not yet pre-assembled in the pipes, and then doors 48, 50 of incubator 12 are closed again.
[0085] We refer now to Figures 7 to 13, which represent an embodiment of the stirring device 16 according to the invention.
[0086] The stirring device 16 comprises a plate 110 supporting the cell expansion bag 54 (not shown in Figures 7 to 13), which plate must be mounted as a piece of furniture rotating around a first horizontal axis A for the displacement of the plate from a substantially horizontal position, shown in Figures 7 and 11, to a substantially vertical position, shown in Figures 8 and 10 (plate 110 can take any position between such extreme positions, such as, for example, a position shown in Figure 9, where it is inclined at an approximate angle of 45 ° to a horizontal plane), and around a second horizontal axis B, around which plate 110 is intended to oscillate (in an angular plane of approximately +/- 8o) to stir and homogenize the contents of the cell expansion bag.
[0087] The plate 110 has a rectangular shape, the dimensions of which are slightly larger than those of the cell expansion bag 54 (approximately 40 cm long and 22 cm wide), which is intended to be placed on the plate. The plate 110 comprises peripheral edges 112 for retaining the pouch, and is perforated so that the face of the pouch 54 against the plate can be directly exposed, at least in part, to the prevailing environmental conditions in the chamber 14 of the incubator 12.
[0088] The plate 110 comprises, at one of its ends, which corresponds to one of the smaller sides of the plate, a hook 114 for fixing the cell expansion bag 54, said hook being intended to represent the highest point of the device 16, when the plate is in an upright position (Figure 8). Plate 110 comprises, at its end opposite hook 114, three mounting holes for the aforementioned valves 86, 88 and 90.
[0089] The device 16 comprises a U-shaped piece 116, whose free ends of the two lateral branches are articulated on pivots 118 fixed on the lateral edges of the end of the plate 110 that carries the valves 86, 88 and 90. These pivots 118 are aligned and define the aforementioned first A axis of rotation of the plate 110.
[0090] The branches of the U-piece 116 carry, particularly in their midst, pivots 120 which are articulated on a chassis 122 of the device 16, such pivots 120 being aligned and defining the aforementioned second axis B of rotation of the plate 110.
[0091] When the plate 110 is in a substantially horizontal position (Figure 7), the U-shaped piece 116 extends along three sides of the plate, namely, the longest sides and the smallest side that carries the hook 114 .
[0092] The displacement of the plate 110 around the A axis is ensured by a pedestal 124, which is mounted between the branches of the U-shaped part 116, whose cylinder is fixed in the middle part of that part 116 and the piston rod is fixed at the end of the plate that carries valves 86, 88 and 90.
[0093] As seen in Figures 10 and 11, the piston rod of the pedestal 124 is articulated on an axis carried by a plate 121 fixed on the end of the plate 110 that carries the valves, such axis being substantially horizontal. The pedestal cylinder 124 is articulated on a substantially vertical axis, carried by a first plate 123 which is articulated on a substantially horizontal axis carried by a second plate 125, such second plate 125 being fixed on the median part of part 116, more particularly in your midst.
[0094] When the piston rod of the pedestal 124 is in the exit position, the plate 110 is in its substantially horizontal position, shown in Figures 7 and 11. When the piston rod of the pedestal 124 is fully in position return, the plate 110 is in its substantially vertical position, shown in Figures 8 and 10. In the case of Figure 9, the piston rod of the pedestal 124 is partially in the return or in the exit.
[0095] The displacement of the plate around the B axis is ensured by an electric motor 126, whose output shaft drives, through a belt 127, a driving wheel of one of the pivots 120 transported by the U-piece 116 (Figure 10). Motor 126 is attached to the chassis 122 of the device by suitable means.
[0096] As shown in Figures 12 and 13, the chassis 122 of the stirring device 16 comprises a system 129 for blocking the rotation of the plate 110 around the B axis, such system 129 comprising a retractable finger 131 that cooperates with an element 133, transported by the U-piece 116 for the plate lock.
[0097] Finger 131 is movable, starting from an exit position shown in Figure 12 to a return position shown in Figure 13, the displacement of that finger being controlled by the computer system 24.
[0098] The element 133 carried by the U-shaped part 116 has an elongated shape, and comprises a first end solidary of one of the pivots 120 of the part, and a second end comprising a notch, where the finger 131 must be coupled to block the rotation of the plate 110 around the B axis. When the finger 131 unfolds (Figure 12), the side faces of the notch of the element 133 may come to rest on the finger, thus preventing any rotation of the plate around the axis B. When the finger is in the position of return (Figure 13), the piece 116 and the plate 110 can be rotated around the axis B.
[0099] The rotation blocking of the plate 110 around the B axis can be activated by the computer system 24, when the plate is moved around the A axis to an inclined or vertical position, with a view to extracting or recovering the cells cell expansion bag 54, so as to prevent the plate from moving around the B axis due to the force exerted on one side of the plate by the weight of the cell expansion bag.
[0100] The stirring device 16 further comprises a vertical arm 128 for fixing the aforementioned valve 92, and for coupling the bag 56 forming an air collector. The valve 92 is located substantially at half height of the arm 128, and the upper end of the arm comprises a hook 130 for securing the bag 56 (Figures 7 to 9).
[0101] The device 16 also comprises pickups 132 of position of the plate 110 around axes A and / or B, which are transported by the chassis 122.
[0102] Figure 14 is an organization chart that represents the steps of the process according to the invention.
[0103] A first stage 130 of the process consists of capturing and recording, using the computer system 24, culture parameters specific to the biological protocol. The capture is carried out by an operator, consisting of the parameters captured in data such as, for example, the identification of the patient, the identification of the consumables kit, the volume of the cell expansion bag 54, etc ... To facilitate the capture of such parameters, the computer system 24 can be equipped with a barcode reader, the consumables kit being able to comprise a bar code that directly reports to the computer system 24 the number and nature of the kit, as well as the volume of each bag.
[0104] The process comprises a second step 132 of installing the consumables kit in the automated device 10, as described in the previous section. This installation can be guided and supervised by the computer system 24. The installation can be implemented in several substeps, and the computer system 24 must display installation records to the operator, indicating whether to validate or invalidate the implementation of a substep and the passage to the subsequent step. These substeps are, for example, the following: - the placement of the different bags on the construction 18 and in the chamber 14 of the incubator 12; - placing the pipes 70 of the pouch 34 of the culture medium in the valves 94, 96 (the computer system 24 then controls the opening of the valves 94, 96 which are then closed as soon as the operator has validated that substep) , - the placement of the pipes 72, 74, 76 of the bag 36 of growth factors and the bag 40 forming the air collector in the valves 98, 102, and 106 (the computer system 24 controls the opening of these valves which are then , closed as soon as the operator has validated this substep), - the placement of the pipes 72, 74, 76 of the bag 38 of cells to be cultured and of the bag 40 forming an air collector in valves 100, 104 and 108 (the system computer 24 controls the opening of these valves, which are then closed as soon as the operator has validated that substep), - the placement of tubing 78 of the bag forming an air collector in valve 92 (computer system 24 commands the opening of this valve that is then closed like this the operator has validated that substep), and - the placement of pipes 70, 60, 66 in sequence in valves 90, 88 and 86 (computer system 24 commands the opening of each of these valves, in sequence, which are, in then closed once the operator has validated each substep).
[0105] The process according to the invention comprises a third stage 134 of tests, called "self-test", during which the computer system 24 controls the proper functioning of the valves, as well as the means of drag (pedestal 124 and engine 126 ) of the plate 110 of the shaking device 16. The operation of the incubator 12 can be implicitly controlled when the implementation of the biological protocol starts, being certain that it can only be started if, for example, the temperature and the CO2 rate in the chamber 14 are stabilized at the record values for the incubation.
[0106] The process according to the invention comprises another step 136 of fluid distribution, which comprises several substeps represented schematically in Figures 15 to 19.
[0107] The first substep of the distribution stage 136 is shown in Figure 15, and consists of evacuating the air contained in the pipes 70, 72, 74. To do so, valves 94, 96, 106 and 108 are opened, and the pump 22 is activated by the computer system 24 to promote the circulation of culture medium from bag 34 in pipes 70, 72, 74 to bag 40 forming an air collector. Pipes 70, 72, 74 are then filled with culture medium, and pouch 40 is filled, at least in part, with culture medium. Pump 22 is regulated at a predetermined rate, and runs for a predetermined duration, at the end of which the pump is turned off and valves 94, 96, 106 and 108 are closed.
[0108] The second substep of the distribution stage 136 is shown in Figure 16, and consists of evacuating the air contained in the pipes 70 and 78. For this purpose, valves 94, 96 and 92 are opened, and the pump 22 is driven by computer system 24 to promote the circulation of culture medium from bag 34 in pipes 70, 78 to bag 56 forming an air collector. Tubing 78 is then filled with culture medium, and pouch 56 is filled, at least in part with culture medium. Pump 22 is regulated at a predetermined rate, and runs for a predetermined duration, at the end of which the pump is turned off and valves 94, 96 and 92 are closed.
[0109] The third substep of the distribution step 136 is shown in Figure 17, and consists of feeding the cell expansion bag 54 in culture medium. Valves 94, 96 and 90 are opened, and pump 22 is driven by the computer system 24 to promote the circulation of culture medium from pouch 34 to pouch 54. Bag 54 is then filled with culture medium. Pump 22 is regulated at a predetermined rate, and works for a predetermined duration, according to parameters of the biological protocol that specify the volume of culture medium to be distributed to bag 54, as well as its feed rate . Then, pump 22 is turned off, and valves 94, 96 and 90 are closed.
[0110] The fourth substep of the distribution step 136 is represented in Figure 18, and consists of feeding the cell expansion bag 54 into growth factors and then washing the growth factor bag 36 with culture medium, and evacuating the contents of that bag 36 towards the cell expansion bag 54. At first, valves 104 and 90 are opened, so that the medium containing the growth factors flows, by gravity, from the bag 36 up to bag 54, through circulation in pipes 74 and 70 (arrows 138). Stock 54 is filled with growth factors. Valves 104 and 90 are opened for a predetermined duration, depending on the volume of medium containing the growth factors to be distributed to bag 54. After this period, valves 104 and 90 are closed. The valves 94 and 100 are then opened and the pump 22 is activated (according to a predetermined flow rate and duration) to feed bag 36 in culture medium, with a view to washing it. Valves 94 and 100 are closed and valves 104 and 90 are opened again, so that the washing product contained in bag 36 flows, by gravity, into the cell expansion bag 54. Valves 104 and 90 are opened for a duration predetermined, depending on the volume of this washing product to be distributed to bag 54. After this period, valves 104 and 90 are closed again. These phases of washing the bag 36 and evacuating the washing product towards the cell expansion bag 54 can be repeated once or several times, according to the parameters of the biological protocol, so that, for example, all factors growth rates originally contained in stock exchange 36 are in stock exchange 54.
[0111] The fifth substep of distribution step 136 is shown in Figure 19, and consists of feeding cell expansion bag 54 into cells to be cultured and then washing bag 38 containing such cells with culture medium, and in evacuating the contents of that bag towards the cell expansion bag 54, in a manner similar to that performed when the fourth substep was implemented. At first, valves 109 and 90 are opened, so that the medium containing the cells to be cultured will drain, by gravity, from bag 38 to bag 54 through circulation in pipes 74 and 70 (arrows 140 ). Bag 54 is filled with cells to be cultured. Valves 102 and 90 are then closed and valves 94 and 98 are opened, and pump 22 is activated (according to a predetermined flow rate and duration) to feed bag 38 in culture medium, with a view to its washing. Valves 94 and 98 are closed and valves 102 and 90 are opened, so that the washing product contained in bag 38 flows, by gravity, to the cell expansion bag 54. Valves 102 and 90 are then , closed. These steps of washing the bag 38 and evacuating the washing product towards the cell expansion bag 54 can be repeated once or several times according to the parameters of the biological protocol, so that, for example, all cells to be cultivated originally contained in bag 36 are found in bag 54.
[0112] The distribution step 136 of the process can be followed by a step of homogenizing the contents of the cell expansion bag 54, which is represented schematically in Figure 20. When implementing this step, the computer system 24 controls the stirring device 16, so that the plate 110 oscillates around the B axis, as explained in the previous section (arrows 142). The amplitude, frequency, duration and periods (agitation, rest, agitation, etc.) of these oscillations are determined according to the parameters of the biological protocol.
[0113] The process according to the invention then comprises an incubation step 144, which can last for several days and, for example, a dozen days. Periodically, according to the protocol parameters, the contents of the cell expansion bag 54 can be homogenized, thanks to the displacement, in rotation, of the plate around the B axis, as explained above. This homogenization (periods, frequency, amplitude) is determined by the protocol parameters independently of the homogenization stage subsequent to the distribution stage 136.
[0114] During incubation step 144, the operator can perform one or more extractions 146 in the cell expansion bag 54 (Figures 14 and 22). Some of these extractions can be imposed by the computer system. These mandatory extractions are, for example, in number of three, and can be performed right after the distribution stage, three days after the start of the incubation stage 144, and seven days after the beginning of this stage 144. Other extractions can be performed according to the operator's wishes, the computer system may invite you to perform such optional extractions.
[0115] When the operator confirms to the computer system 24 his availability to perform an extraction, the computer system activates the pedestal 124, so that the plate 110 of the stirring device 16 is moved in rotation around the A axis to an inclined position , for example, approximately 45 ° in relation to a horizontal plane, as shown schematically in Figures 9 and 22. The computer system 24 can then detect the desirable position of the plate 110, by means of the device 16 pickups.
[0116] The computer system 24 controls the opening of valve 88, so that a part of the contents of the cell expansion bag 54 flows, by gravity, from the bag 54 in the pipeline 60 to the extraction means 62 located outside the chamber 14 from the incubator 12. The operator can extract a sample from the cell expansion bag 54 using a syringe 148, equipped with “Luer lock” connection means, which are coupled to the extraction means 62. After extraction, valve 88 is closed, and plate 110 of the stirring device 16 is returned to a substantially horizontal position.
[0117] The operator can then carry out analyzes of the extracted sample, and the results 148 of such analyzes can be captured and recorded in the computer system 24 by the operator.
[0118] During incubation stage 144, the operator can also remove part of the consumables kit (first phase of removing 150 consumables - Figures 14 and 21). The elements of the consumable kit that can be removed are all the bags (34, 36, 38, 40) and the pipes (72, 74, 76) connected to pipe 70. To do this, the operator must cut pipe 70 above the part that crosses the aforementioned block 80, and must also weld or clamp the cut free end of the tubing 70 that remains in the chamber 14 of the incubator 12, in order to avoid any contamination of the cell expansion bag. This operation can be carried out by the operator by means of a suitable cutting clamp, making the hermetic lock of the end of the pipe, when cutting it. Valves 94 to 108 are then opened to allow the operator to remove pipes 70, 72, 74, 76 from these valves, as well as pump 22 (Figure 21). As soon as the operator has confirmed the removal of these elements to the computer system 24, the latter will command the closing of valves 94 to 108.
[0119] The process according to the invention also comprises a recovery step 152 of the cells after culture (Figures 14, 23 and 24). At the end of the incubation stage 144, and at the request of the operator, the computer system 24 blocks the rotation of the plate 110 around the B axis and activates the pedestal 124, so that the plate 110 of the agitation device 16 moves around the axis A, up to a substantially vertical position shown in Figures 8, 23 and 24. The computer system 24 can then detect the desirable position of the plate 110 by means of the pickups of the device 16.
[0120] Then, the computer system 24 controls the opening of the valve 86, so that the contents of the cell expansion bag 54 drain, by gravity, from the bag 54 in the two recovery bags 46, through the circulation in the pipe 66 (Figure 23).
[0121] The plates 42, carried by construction 18 and on which the recovery pockets 46 are attached, can be moved from their vertical position shown in Figures 2 and 23 to their horizontal position represented schematically in Figure 24, either manually by the operator or thanks to displacement means controlled by the computer system 24. Dragging the plates 46 allows the pockets to be entirely located below the pocket 54 and at least part of the pipe 66, so that the contents of the expansion pocket cell phone 54 is, if possible, transferred in its entirety to the recovery bags 46. When the operator has confirmed to the computer system 24 the closure of the extraction, that system will command the closing of the valve 86 and the opening of the pedestal, so that that plate 110 returns in a substantially horizontal position.
[0122] The pouches 46 are then removed from the automated device 10, with a view to an eventual treatment of the cells and the reinjection of these cells in the body of a patient to carry out a cell therapy, for example. For this purpose, tubing 66 can be cut and welded by the aforementioned clamp, or bags 46 can be disconnected from tubing 66.
[0123] The last stage of the process consists of a second phase of removing 154 of the consumables from the automated device 10, with the remaining bags 54, 56 and pipes 66, 60, 70 and 78 being removed. For this purpose, the computer system 24 commands the opening of valves 86, 88, 90 and 92 to allow the removal, by the operator, of pipes 66, 60, 70 and 78. Once confirmed by the operator to the computer system 24, the removal of these elements, the system controls the closing of valves 86, 88, 90 and 92.
[0124] When the biological protocol is closed, the computer system can edit a culture report, which can include the following information to ensure good protocol traceability: information specific to the manufacturer (the identification number of the automated device, the version of the computer system operating software, the version of the biological protocol supervision software), the set of protocol parameters provided that at least one of these parameters is not in its lowest value, the set of culture parameters, the actions carried out by the operator (comprising the date of each action in the format yyyymmddThhmiss, the author of that action by means of a connection identifier, the nature of the action by means of a coding to be defined, in the absence of a misleading document), events “system ”(Comprising the date of the event in the format yyyymmddThhmiss, the nature of the event (alarm, user alert, fault detector, etc ...) through of a codification to be defined in the absence of an equivocal document), results of the extraction analysis (including the date of each extraction, the author of the extraction, the results of the extraction analysis), and graft information (obtained from the analysis cells recovered after culture). This crop report can be accessed via the computer network of the aforementioned network.

权利要求:
Claims (21)
[0001]
1. Automated cell culture device (10), characterized by the fact that it has reservoirs (34, 36, 38) of culture medium, growth factors and cells to be cultivated, an incubator (12) with chamber (14) thermostatic, in which a cell culture cell (54) or cell expansion container (54) and a control computer system (24), include means of capturing and recording data and intended to regulate the culture conditions in the chamber and the pilot fluid distribution valves (20) according to a pre-defined sequence, due to the fact that it has a device (16) for supporting and agitating the culture or cell expansion vessel, which is controlled by the informed system and housed in the chamber, and still be the container formed by a pouch (54) that includes at least one inlet hole (68) connected to the aforementioned reservoirs, and an outlet hole (64) connected to recovery (46) means and storage of cells after culture , as such storage means as well as the reservoirs are located outside the chamber, being connected to the holes of the cell expansion bag by conduits (66, 70, 72, 74) that form, with the cell expansion bag, a pre- mounted placed in the chamber and that pass through a wall element of the chamber, in order to allow the feeding of the cell expansion bag in culture medium, growth factors and cells to be cultured, as well as the recovery of the contents of the bag. cellular expansion in the storage means, keeping the chamber closed, said support and agitation device (16) presenting a plate (110) supporting the cell expansion bag (54), mounted in rotation around a first horizontal axis ( A) and movable around that axis between a substantially horizontal cell culture position and a substantially vertical cell recovery position after culture.
[0002]
2. Automated device, according to claim 1, characterized by the fact that it has a cell expansion bag (54) that also comprises an extraction orifice (58) connected, by conduit (60), to extraction means (62) located outside the chamber (14), said conduit going through the aforementioned part of the incubator and being part of the pre-assembled module.
[0003]
3. Automated device, according to claim 1 or 2, characterized by the fact that it has an incubator (12) that has a cabinet with an opening and equipped with a door (50) for hermetically closing that opening, means (80) of passage of the aforementioned conduits (60, 66, 70) being mounted on the peripheral edge of this opening, presenting parallel coupling throats (82) of these conduits, such throats being intended to be covered by the door, when it is in a closed position.
[0004]
4. Automated device, according to any of the previous claims, characterized by the fact that it has reservoirs of growth factors and cells to be cultivated that are formed by pockets (36, 38) located above the entrance hole (68) of the bag cell expansion (54), so that the contents of each growth factor bag and cells to be cultured can drain, by gravity, into the cell expansion bag.
[0005]
5. Automated device according to any of the preceding claims, characterized by the fact that it presents storage means that comprise one or two bags (46) that are, at least in part, located below the outlet hole (64) of the bag. cell expansion (54), so that, after culture, the contents of the cell expansion bag can drain, by gravity, to the storage bag (s).
[0006]
6. Automated device, according to any of the preceding claims, characterized by the fact that it has a cell expansion bag (54) that comprises flexible walls impermeable to liquids and permeable to gases, and in particular to CO2, and equipped, preferably , of properties that limit the adherence of the cells to be cultured to the walls of the bag as much as possible.
[0007]
7. Automated device, according to any of the previous claims, characterized by the fact that it has a peristaltic pump (22) for feeding control in culture medium of the cell expansion bag (54), as well as reservoirs (36, 38) of growth factors and cells to be cultivated, with a view to washing these reservoirs.
[0008]
8. Automated device, according to any of the previous claims, characterized by the fact that it has two pockets (40, 56) forming vapor traps, one of which is connected to the reservoirs (36, 38) of growth factors and cells to be cultivated and the other connected to the cell expansion bag (54), and destined to the collection and stock of air contained in the conduits (70, 72, 74), in the cell expansion bag (54) and / or in the reservoirs (36 , 38).
[0009]
9. Automated device, according to any of the previous claims, characterized by the fact that it has conduits formed by flexible pipes (60, 66, 70, 72, 74, 76, 78), among which at least some cross valves (86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108) which are intended, in the closed position, to clamp such pipes.
[0010]
10. Automated device according to any one of claims 1 to 9, characterized in that it has a plate (110) mounted in rotation around a second horizontal axis (B), around which the plate is intended to oscillate to promote agitation and homogenization of the contents of the cell expansion bag.
[0011]
11. Automated device according to any one of claims 1 to 10, characterized by the fact that it has a plate (110) that contains valves (86, 88, 90) for controlling the supply of the cell expansion bag (54), retrieving the contents of that bag and extracting samples from that bag.
[0012]
12. Automated device according to any one of claims 1 to 11, characterized in that it has a support and stirring device (16) which has a vertical arm (128) comprising, at its upper end, means (130) coupling a bag (56) forming an air collector connected to the cell expansion bag (54).
[0013]
13. Automated device according to any one of the preceding claims, characterized by the fact that it has storage means (46) mounted in rotation around a horizontal axis, and displaceable around that axis between a vertical position and a horizontal position, in which such means are entirely located below the cell expansion bag.
[0014]
14. Sterile, single-use cell culture kit for an automated cell culture device (10) as defined in any one of claims 1 to 13, characterized in that it has at least one culture bag and tubes (54) flexible (60, 66, 70, 72, 74) for connection between the bag and other bags or reservoirs, with the tubes and cell expansion bag pre-assembled, and the cell expansion bag must have an entry hole (68 ), an exit orifice (64) and, possibly, an extraction orifice (58), and present the entrance orifice (68) of the cell expansion bag (54) which is connected by pipes (70, 72, 74) to inlet and outlet holes (36) of growth factors, and to inlet and outlet holes (38) of cells to be cultured, in which the kit additionally features two bags (40, 56 ) forming an air collector, one of which is connected to the outlet pockets (36, 38) of growth factors and cells to be cultured and the other connected to the entrance hole (68) of the cell expansion bag (54).
[0015]
15. Kit, according to claim 14, characterized by the fact that it presents one or two bags (46) of cell recovery after culture, which are connected, by tubes (66), to the outlet hole (64) of the bag. cell expansion (54).
[0016]
16. Device (16) of support and agitation for an automated device (10) of cell culture, characterized by the fact that it presents a plate (110) supporting a cell expansion bag (54), such plate having three pipes ( 86, 88, 90) and be mounted in rotation around a first horizontal axis (A) to drag the plate from a horizontal position to a vertical position, and a second horizontal axis (B), around which the The plate is intended to oscillate to promote agitation and homogenization of the contents of the cell expansion bag, and the device must also have means (124, 126) controlled for dragging the plate around the aforementioned horizontal axes.
[0017]
17. Device according to claim 16, characterized by the fact that it has a vertical arm (128) that comprises, at its upper end, means (130) for coupling a bag (56) forming steam traps.
[0018]
18. Automated cell culture process using an automated device (10) as defined in any one of claims 1 to 13, characterized by the fact that it presents the steps that consist of: a - feeding the cell expansion bag (54) in culture medium, in growth factors and then in cells to be cultured, keeping the incubator chamber (14) closed (12); b - shake the cell expansion bag in order to homogenize its content; c - keep the cell expansion bag in incubation conditions for several days; and d - recovering the contents of the cell expansion bag in the storage means (46), keeping the chamber closed.
[0019]
19. Process, according to claim 18, characterized by the fact that it presents: - before step a), a step of installing the pre-assembled module, mounting the cell expansion bag (54) on the stirring device (16 ), mounting the conduits (60, 66, 70, 72, 74) in the incubator's passage means (80) (12) and valves (20), and connecting these conduits to the reservoirs or bags (34, 36, 38 ), and / or, - before step a), a step of evacuation of the air contained in the conduits (70, 72, 74), thanks to the passage of the culture medium from the culture medium reservoir (34) to the or the bags (40, 56) that form steam traps; and / or - after feeding the cell expansion bag with growth factors in step a), a step of washing the growth factors reservoir (36), promoting the passage of culture medium in this reservoir and then evacuating its contents to the cell expansion bag (54); and / or - after feeding the cell expansion bag into cells to be cultured in step a), a step of washing the reservoir (38) of cells to be cultured, promoting the passage of culture medium in that reservoir and, then , evacuating its contents to the cell expansion bag (54); and / or - during step c), one or more steps of extracting samples from the contents of the cell expansion bag (54), each of which is preceded by a stage of dragging the support plate (110) from a horizontal position from culture to an inclined position, in which the extraction hole (58) of the bag represents the lowest point of the bag; - before step c), a step of removing the reservoirs (34, 36, 38) of culture medium, growth factors and cells to be cultivated by cutting, soldering or clamping the conduit or pipe (70) connecting such reservoirs to the inlet port (68) of the cell expansion bag (54); and / or - before or during step d), drag the plate (110) in a vertical position, so that the exit hole (64) of the cell expansion bag (54) represents the lowest point of the bag .
[0020]
Use of an automated device as defined in any one of claims 1 to 13, a cell culture kit as defined in claim 14 or 17 or a support or agitation device as defined in claim 18 or 19, characterized in that it is in the culture of CD34 + type stem cells or blood mononuclear cells, such as, for example, lymphocytes.
[0021]
21. Use according to claim 20, characterized by the fact that stem cells come from one or more sources, such as, more specifically, umbilical cord blood, bone marrow and whole blood.

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SG11201405632PA|2014-10-30|

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-18| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-10-29| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-20| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP12305310.0|2012-03-15|

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EP12305310.0A|EP2639294A1|2012-03-15|2012-03-15|Automaton and automated method for cell culture|

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PCT/EP2013/055244|WO2013135817A1|2012-03-15|2013-03-14|Automated device and automated process for cell culture|

---