Cell manufacturing system and cell manufacturing method

Abstract

There has been a demand for an advantageous feature for obtaining a target cell number and cell quality in subculture. The present invention comprises a first cartridge (2), a second cartridge (2), and a culture device (33) to which the first cartridge and the second cartridge can be attached. Each of the first cartridge and the second cartridge includes a culture container (100), a supply container (4d) for supplying cells to the culture container, and a recovery container (4r) that recovers cells cultured in the culture container. The first cartridge (2) is attached to the culture device (33), cells are cultured in the culture container (100) of the first cartridge (2), the cultured cells are recovered in the recovery container (4r) attached to the first cartridge (2), the recovery container (4r) is detached from the first cartridge (2) and attached to the second cartridge (2) as the supply container (4d), the second cartridge (2) is attached to the culture device (100), and the cells are cultured in the culture container (100) of the second cartridge (2).

Description

Cell Manufacturing System and Cell Manufacturing Method

[0001] The present disclosure relates to, for example, a cell manufacturing system for manufacturing iPS cells, etc.

[0002] Induced pluripotent stem cells (iPS cells) are expected to play an important role in realizing regenerative medicine as new pluripotent stem cells having the ability to differentiate into various cells. Currently, clinical research using cells differentiated from iPS cells into various cells is actively underway.

[0003] iPS cells are created by introducing genes into cells such as skin collected from donors such as patients. However, in the conventional method, a series of operations such as culturing are performed by manual techniques. The same is true when differentiating iPS cells into specific cells or tissues, and the manufacturing process that requires complex and delicate operations is performed manually. For this reason, in some processes, skilled work may be required, and quality control takes time and cost. In addition, since human operation is involved in the manufacturing process, it is essential to maintain a sterile environment for the manufacturing process including raw materials, materials, equipment, etc., so as not to contaminate the cells to be manufactured with microorganisms, fine particles, etc., and for this purpose as well, a huge cost is incurred.

[0004] Therefore, it has been proposed to solve such problems by automating the cell manufacturing process. For example, Patent Document 1 proposes an apparatus for easily culturing adherent cells without depending on the skill of an operator.

[0005] Japanese Unexamined Patent Application Publication No. 2005 - 198626

[0006] As mentioned above, when manufacturing cells such as iPS cells and differentiated cells, the rate of cell proliferation and the ease with which cells differentiate can vary depending on the source cells used. Therefore, it is difficult to manufacture cells under a single set of conditions, and adjustments are made, for example, by monitoring the state of each source cell and adjusting the number of cell culture cycles. These differences are thought to be due to differences in the donor or cell line of the source cells, but there are multiple causes, and none have yet been identified. Therefore, it is difficult to predict in advance the manufacturing conditions such as the time and number of culture cycles, and it is necessary to repeatedly continue culturing until the target quality and number of cells are obtained. On the other hand, the inventors have found that variations in the technique of each operator during the cell transplantation process into a new culture vessel (hereinafter referred to as "passaging") greatly affect the quality of the cells.

[0007] In view of the above problems, the inventors conducted extensive research and found that it is important to eliminate the effects of variations in technique among operators by automating the subculturing process, while also being able to flexibly change the number of subculturing steps, in order to ensure the quality of the cells being manufactured. As a result, they have completed the technology described herein.

[0008] This disclosure is based on the above findings, and a first aspect of this disclosure is a cell manufacturing system comprising a first cartridge, a second cartridge, and a culture device to which the first cartridge and the second cartridge can be attached, wherein each of the first cartridge and the second cartridge has a culture vessel, a liquid container for storing a liquid used for culturing cells, a detachable supply container for supplying cells to the culture vessel, a detachable recovery container for recovering cells cultured in the culture vessel, and a liquid channel connected to the culture vessel, wherein the first cartridge is attached to the culture device, cells are cultured in the culture vessel of the first cartridge, the cultured cells are recovered into the recovery container attached to the first cartridge, the recovery container is removed from the first cartridge and attached to the second cartridge as the supply container, the second cartridge is attached to the culture device, cells are cultured in the culture vessel of the second cartridge, and the cultured cells are recovered into the recovery container attached to the second cartridge.

[0009] Furthermore, a second aspect of the present disclosure is a cell manufacturing method using a cell manufacturing system comprising a first cartridge, a second cartridge, and a culture device to which the first cartridge and the second cartridge can be attached, wherein each of the first cartridge and the second cartridge has a culture vessel, a liquid container for storing a liquid used for cell culture, a detachable supply container for supplying cells to the culture vessel, a detachable recovery container for recovering cells cultured in the culture vessel, and a liquid channel connected to the culture vessel, and the cell manufacturing method involves attaching the first cartridge to the culture device, culturing cells in the culture vessel of the first cartridge, recovering the cultured cells in the recovery container attached to the first cartridge, removing the recovery container from the first cartridge and attaching it to the second cartridge as the supply container, attaching the second cartridge to the culture device, culturing cells in the culture vessel of the second cartridge, and recovering the cultured cells in the recovery container attached to the second cartridge.

[0010] This disclosure provides a technology that is advantageous for achieving target cell numbers and cell quality in a cell manufacturing system capable of automated cell production. Other features and advantages of this disclosure will become apparent from the following description with reference to the attached drawings.

[0011] A block diagram showing an example of the configuration of the iPS cell manufacturing system according to the embodiment. A perspective view of the external appearance of the iPS cell manufacturing system according to the embodiment. A plan view of the external appearance of the iPS cell manufacturing apparatus 3 according to the embodiment. A schematic perspective view of the inside of the cartridge according to the embodiment. An exploded view illustrating the flow path configuration of the cartridge according to the embodiment. The first half of a flowchart for explaining a series of processes for producing iPS cells. The second half of a flowchart for explaining a series of processes for producing iPS cells. A schematic diagram showing a simplified view of the inside of the cartridge according to the embodiment. A perspective view showing the configuration of the culture vessel used in the embodiment. A cross-sectional view showing the internal configuration of the culture vessel used in the embodiment. A schematic diagram illustrating the configuration of the imaging unit. A schematic diagram showing a first modified example of the cartridge according to the embodiment. A schematic diagram showing a second modified example of the cartridge according to the embodiment. A schematic diagram showing a third modified example of the cartridge according to the embodiment. A diagram illustrating the connection part of a container that attaches to and detaches from the cartridge.

[0012] The iPS cell manufacturing system and the like according to the embodiments of this disclosure will be described with reference to the drawings. The embodiments shown below are illustrative, and for example, the detailed configurations can be modified as appropriate by those skilled in the art without departing from the spirit of this disclosure.

[0013] In the drawings referenced in the following description of embodiments, elements indicated by the same reference numeral have the same function unless otherwise specified. If multiple identical elements are shown in a drawing, the assignment of the reference numeral and its description may be omitted. Furthermore, for the sake of illustration and explanation, drawings may be schematic; therefore, the shape, size, and arrangement of elements shown in the drawings may not strictly correspond to those of actual objects.

[0014] In the following explanation, for example, when we refer to the "X-plus direction," it refers to the same direction as the X-axis arrow in the illustrated Cartesian coordinate system, and when we refer to the "X-minus direction," it refers to the direction 180 degrees opposite to the direction indicated by the X-axis arrow in the illustrated Cartesian coordinate system. Furthermore, when we simply refer to the "X direction," it refers to the direction parallel to the X-axis, regardless of whether it is the same as or different from the direction indicated by the illustrated X-axis arrow. The same applies to directions other than X.

[0015] [Embodiment] Below, as a preferred embodiment, we illustrate the case in which iPS cells are produced using a cell production cartridge. Note that the cell production cartridge can be used not only to produce iPS cells, but also to produce any type of cell from iPS cells.

[0016] Figure 1 is a block diagram showing an example configuration of the iPS cell manufacturing system 1 according to an embodiment. Figure 2 is a perspective view of the external appearance of the iPS cell manufacturing system 1. Figure 3 is a plan view of the external appearance of the iPS cell manufacturing apparatus 3. Here, we introduce an XYZ coordinate system based on the cartridge 2. The X direction is defined as the longitudinal direction of the cartridge 2, the Y direction as the height direction, and the Z direction as the short direction. The X, Y, and Z axes are orthogonal to each other.

[0017] The iPS cell manufacturing system 1 is a mechanical system that can automatically manufacture iPS cells from a cell solution containing the donor's target cells, by attaching a removable cartridge 2 with a built-in flow path. For example, blood can be used as the cell solution. In this case, cells contained in peripheral blood mononuclear cells (PBMCs) are suitable as raw material cells, and one example is CD34-positive cells.

[0018] As shown in Figure 1, the iPS cell manufacturing system 1 comprises a cartridge 2 and an iPS cell manufacturing device 3. The cartridge 2 is a disposable container equipped with a channel for manufacturing iPS cells from target cells derived from a donor's sample, and is detachably attached to the iPS cell manufacturing device 3. The cartridge 2 will be described in detail later with reference to Figures 4 and 5.

[0019] The iPS cell manufacturing apparatus 3 performs the following steps in cartridge 2 (first cartridge): a blood cell separation step to separate PBMCs from the donor's blood; a reprogramming factor introduction step to introduce reprogramming factors into target cells (e.g., CD34-positive cells) contained in the PBMCs; an iPS cell culture step to culture the iPS cells established by the action of the reprogramming factors on the target cells; and a detachment step to detach the iPS cells from the culture vessel.

[0020] Furthermore, the iPS cell manufacturing system 1 performs a recovery step to collect the detached iPS cells into a recovery container. The recovery step may also be a dispensing step in which the cells are divided and injected into multiple cryopreservation containers for subculturing. Furthermore, the iPS cells stored in the cryopreservation containers are injected into the culture vessel of cartridge 2 (second cartridge) and subculturing is performed. Furthermore, a detachment step is performed to detach the subculturated iPS cells from the culture vessel. Furthermore, a recovery step is performed to collect the detached subculturated iPS cells into a recovery container. The recovery step may also be a dispensing step in which the cells are divided and injected into multiple cryopreservation containers for subculturing.

[0021] The iPS cell manufacturing apparatus 3 is a machine that manufactures iPS cells from target cells in a cartridge 2. The iPS cell manufacturing apparatus 3 includes a liquid delivery mechanism 31, a culture device 33 (incubator), a valve opening / closing mechanism 35, a cooling device 37, a moving mechanism 39, an imaging unit 16, an analysis unit 17, and a control device 30. In the iPS cell manufacturing apparatus 3, the liquid delivery mechanism 31, the culture device 33, the valve opening / closing mechanism 35, the cooling device 37, the moving mechanism 39, the imaging unit 16, and the analysis unit 17 are each connected to the control device 30 by wired or wireless means, enabling signal communication.

[0022] The fluid delivery mechanism 31 is a mechanical mechanism that applies energy to deliver various liquids to a flow path provided in the cartridge 2. As an example, the fluid delivery mechanism 31 applies energy to deliver a cell solution containing the donor's target cells and reagents used for processing the cell solution to the flow path in the cartridge 2. The cell solution and reagents are delivered by the application of energy. Reagents delivered by the fluid delivery mechanism 31 may include, for example, reagents containing reprogramming factors for reprogramming peripheral blood mononuclear cells, culture media used for culturing peripheral blood mononuclear cells and iPS cells, and phosphate-buffered saline (PBS) used for washing peripheral blood mononuclear cells and iPS cells. Examples of energy include air, magnetic force, electric power, electromagnetic waves, and mechanical action. For example, as a fluid delivery method using air, a syringe pump method, a pressure delivery method, or any other method can be used. In the following description, the fluid delivery method by the fluid delivery mechanism 31 will be assumed to be a pressure delivery method. In this case, the liquid delivery mechanism 31 is equipped with liquid delivery pads 311 that deliver various liquids into the cartridge 2 using a pressure delivery method. In Figure 2, the liquid delivery mechanism 31 is equipped with two liquid delivery pads 311 that can be driven independently of each other. However, the number of liquid delivery pads 311 equipped in the liquid delivery mechanism 31 may be one or three or more. It is preferable that the number of liquid delivery pads 311 be less than the number of ports 21 provided in the cartridge 2.

[0023] The culture device 33 is a mechanical device (incubator) that controls the culture environment within the cartridge 2 in order to culture iPS cells established in the cartridge 2 by the action of reprogramming factors on target cells within the cartridge 2. The cartridge 2 is equipped with a culture vessel for culturing iPS cells. The culture device 33 controls the temperature, humidity, and / or carbon dioxide gas concentration in the culture vessel as the culture environment. As shown in Figures 2 and 3, the culture device 33 has a storage chamber 331 with an opening 332. The storage chamber 331 is positioned in the cartridge 2's movement path P39 so that the cartridge 2 can be inserted through the opening 332.

[0024] The valve opening / closing mechanism 35 is a mechanical mechanism that opens and closes a valve that switches the flow path within the cartridge 2. The valve opening and closing method can be appropriately selected according to the valve type of the cartridge 2. The valve opening and closing method is not particularly limited, but as an example, a magnetic valve method is used. As will be described in detail later, the magnetic valve is composed of a combination of a first magnet and a magnetic material such as an iron plate, which are arranged on either side of the flow path within the cartridge 2. In this case, the valve opening / closing mechanism 35 has one or more cylinders 351 that extend and retract in relation to the first magnet. A second magnet (not shown) is attached to the tip of each cylinder 351. The magnetic valve is opened and closed by the extension and retraction of the cylinders 351. In Figure 3, the valve opening / closing mechanism 35 is equipped with two cylinders 351 that can be driven independently of each other. However, the number of cylinders 351 equipped in the valve opening / closing mechanism 35 may be one or three or more. The cylinder 351 may be supported so as to be movable in the XY direction relative to the valve opening / closing mechanism 35, or it may be supported so as to be detachable.

[0025] The cooling device 37 is a mechanical device that cools the culture medium stored in a culture medium storage container (not shown) located inside the cartridge 2. Specifically, the cooling device 37 has a main body installed on the floor and a cooling element such as a Peltier element housed inside the main body. The cooling element is attached to the rear side of the housing of the cartridge 2 and cools the culture medium stored in the culture medium storage container located near the attachment area.

[0026] The moving mechanism 39 to which the cartridge 2 can be mounted is a mechanical mechanism that supports and moves the mounted cartridge 2. Specifically, the moving mechanism 39 has a transport mechanism that supports the cartridge 2 so that it can reciprocate along the moving path P39, and a drive device that drives the transport mechanism. The moving path P39 is along the X direction, which is the longitudinal direction of the cartridge 2. The transport mechanism is composed of a ball screw, rack and pinion, LM guide, linear guide, etc. A servo motor or other motor is used as the drive device. As shown in Figure 3, the moving path P39 is set to a liquid delivery position P31 where liquid is delivered by the liquid delivery mechanism 31, a culture position P33 where culture is performed by the culture device 33, an opening and closing position P35 where the valve is opened and closed by the valve opening and closing mechanism 35, and a mounting position P37 where the cooling body is attached by the cooling device 37. In Figure 3, the liquid delivery position P31 and the opening and closing position P35 are arranged so as not to overlap in the Z direction, but they may overlap in the Z direction. The moving mechanism 39 supports the cartridge 2 so that it can be positioned at the liquid delivery position P31, the culture position P33, the open / close position P35, and the mounting position P37.

[0027] The imaging unit 16 is a device that, in response to imaging commands issued by the control device 30, images the inside of the culture vessel in the cartridge 2 and outputs imaging data. A fluorescence microscope is preferably used as the imaging unit 16, but it is not limited to that; an optical microscope such as a phase-contrast microscope or a bright-field microscope, or an optical camera may also be used.

[0028] The analysis unit 17 determines the cell growth state based on the images captured by the imaging unit 16. For example, by analyzing the images, it calculates the number of cells, the number of colonies, the size of the colonies, confluence, the positive rate of fluorescent protein expression such as GFP (Green Fluorescent Protein), etc. Fluorescent proteins can be introduced as marker genes to detect the expression of genes introduced into raw material cells during iPS cell production. Based on the calculation results, it determines the progress of cell growth and reprogramming and transmits the determination results to the control device 30.

[0029] The control device 30 is a computer that comprehensively controls the operation of the iPS cell manufacturing apparatus 3 and includes a processor. The processor may consist of, for example, a CPU, a GPU, an Application Specific Integrated Circuit (ASIC), a programmable logic device (for example, a Simple Programmable Logic Device (SPLD), a Complex Programmable Logic Device (CPLD), and a Field Programmable Gate Array (FPGA)). The processor performs its functions by reading and executing programs stored in memory circuits. Alternatively, instead of storing the program in a memory circuit, the program may be directly incorporated into the processor's circuitry. In this case, the processor implements its function by reading and executing the program incorporated into the circuitry. On the other hand, if the processor is, for example, an ASIC, the function is directly incorporated into the processor's circuitry as a logic circuit instead of being stored in a memory circuit. The processor can perform control over part or all of the subculturing process by executing a control program recorded on a computer-readable recording medium. The control device 30 can perform control over part or all of the subculturing process by, for example, sequence control, feedback control, or a combination of sequence control and feedback control. The control device 30 controls, for example, the operation of the liquid delivery mechanism 31, the culture device 33, the valve opening / closing mechanism 35, the cooling device 37, the moving mechanism 39, the imaging unit 16, and the analysis unit 17.

[0030] The control device 30 can produce or subculture iPS cells within the cartridge 2 by sequentially controlling the liquid delivery mechanism 31, culture device 33, valve opening / closing mechanism 35, cooling device 37, transfer mechanism 39, etc., according to a control sequence. For example, the control device 30 controls the liquid delivery mechanism 31 to deliver various liquids to the flow path in the cartridge 2 located at the liquid delivery position P31. The control device 30 controls the culture device 33 to culture iPS cells in the culture vessel in the cartridge 2 located at the culture position P33. The control device 30 controls the valve opening / closing mechanism 35 to open and close the valve in the cartridge 2 located at the opening / closing position P35. The control device 30 controls the cooling device 37 to attach a cooling element to the side of the cartridge 2 housing located at the mounting position P37.

[0031] The control device 30 may include a display unit for displaying information to the user, an input unit for the user to input commands and data, and an interface unit for connecting to external devices (e.g., external computers and storage devices) via a network. The control device 30 can, for example, display images captured by the imaging unit 16, imaging condition data, and analysis results from the analysis unit 17 on the display unit or transmit them to external devices.

[0032] As for the control method by the control device 30, for example, an indirect control method and / or a direct control method are possible. In the case of the indirect control method, the control device 30 transmits drive commands and corresponding operation contents to each of the liquid delivery mechanism 31, culture device 33, valve opening / closing mechanism 35, cooling device 37, and moving mechanism 39 in the order determined by the control sequence. The device that receives the drive command supplies a drive signal to its own drive device according to the operation contents, and operates the internal mechanism of the device. In the case of the direct control method, the control device 30 supplies a drive signal to each of the liquid delivery mechanism 31, culture device 33, valve opening / closing mechanism 35, cooling device 37, and moving mechanism 39 in the order determined by the control sequence, and directly operates the internal mechanism of the device. The control method of the control device 30 does not need to be common to all controlled objects; an indirect control method may be adopted for some devices and a direct control method for the remaining devices.

[0033] Next, we will describe cartridge 2. Figure 4 is a schematic perspective view of the inside of cartridge 2. Figure 5 is an exploded view illustrating the flow path configuration of cartridge 2. The flow path configuration shown in Figure 5 is integrated into cartridge 2 shown in Figure 4.

[0034] As shown in Figure 4, the cartridge 2 has a housing 20 that is roughly rectangular in shape. One side in the Z direction is called the main surface, and the side opposite the main surface is called the back surface. A flow path is formed on the main surface side. Various storage containers, etc. (not shown) are arranged on the back surface. One side in the X direction is called the front surface, and the side opposite the front surface is called the rear surface. The front surface faces the culture device 33, and the rear surface faces the cooling device 37. One side in the Y direction is called the top surface, and the side opposite the top surface is called the bottom surface. A port 21 to which the liquid delivery pad 311 of the liquid delivery mechanism 31 is connected is provided on the top surface. The bottom surface is in contact with the moving mechanism 39. The housing 20 is covered by an external housing (not shown) so that the top surface is exposed. The external housing protects the liquid flow path and various containers inside the cartridge 2 from the outside air.

[0035] The housing 20 contains a network of fluid channels for various liquids used in the iPS cell manufacturing process. To reduce contact with the outside air, the cartridge 2 has a closed-system fluid channel. Specifically, the housing 20 is equipped with fluid delivery pipes 23 that form the fluid channels for various liquids. The fluid delivery pipes 23 are made of a flexible material such as vinyl or resin. Both ends of the fluid delivery pipes 23 are connected to various containers or devices to reduce the intrusion of outside air into the fluid delivery pipes 23. Valves are provided at arbitrary points on the fluid delivery pipes 23. By opening and closing the fluid delivery pipes 23 with the valves, the fluid channels formed by the fluid delivery pipes 23 are switched. The valves are opened and closed individually by a valve opening and closing mechanism 35. The fluid delivery pipes 23 may be molded integrally with the housing 20.

[0036] The valve opening and closing method is not particularly limited, but as an example, a magnetic valve system is employed. The magnetic valve is composed of a combination of a first magnet and a magnetic material such as an iron piece, which are arranged on either side of the liquid supply pipe 23. The valve opening and closing mechanism 35 has a cylinder that extends and retracts to approach and move away from the first magnet, and a second magnet is attached to the tip of the cylinder. When opening the valve, the valve opening and closing mechanism 35 moves the second magnet closer to the first magnet by contracting the cylinder. As the second magnet is attracted to the first magnet, the first magnet is pulled away from the magnetic material, and as a result the liquid supply pipe 23 is opened. When closing the valve, the valve opening and closing mechanism 35 moves the second magnet away from the first magnet by extending the cylinder. As a result the first magnet is attracted to the magnetic material, and as a result the liquid supply pipe 23 is closed.

[0037] A container chamber 25 is provided inside the housing 20 for the installation of various containers. In addition to the culture vessel 100, the container chamber 25 may be equipped with various other containers such as tapered containers, sample containers, expanded culture vessels, infection containers, pre-culture containers, recovery containers, and cryopreservation containers. Specifically, it may be equipped with a culture vessel, a liquid container for storing the liquid used for cell culture, a detachable supply container for supplying cells to the culture vessel, and a detachable recovery container for recovering cells cultured in the culture vessel. The culture vessel 100 is positioned near the front side (X-plus direction side) to facilitate its placement inside the culture device 33. It is desirable that the internal space of the container chamber 25 be shielded from the external atmosphere of the cartridge 2.

[0038] As shown in Figure 4, the top surface of the housing 20 is provided with a plurality of ports 21 arranged in a row along the X direction. An air filter is provided at the opening of each port 21. The air filter is a filter that prevents or reduces unwanted substances contained in the outside air from entering the housing 20. Each port 21 communicates with a port chamber 24 provided in the cartridge 2. A suction and discharge device is provided in the port chamber 24. The suction and discharge device is a device having a syringe with a discharge port and a plunger that reciprocates inside the syringe. A liquid delivery pad 311 (Figure 2) equipped with a liquid delivery mechanism 31 is connected to the port 21. In this example, the liquid delivery method by the liquid delivery mechanism 31 is assumed to be a pressure delivery method.

[0039] When using the pressurized feeding method, the liquid delivery pad 311 uses air pressure to deliver various liquids. The liquid delivery pad 311 is branched and connected to an ejector and a compressor via a hollow tube through which gases such as air or gas flow. The ejector depressurizes the port chamber 24, which communicates with the port 21 connected to the liquid delivery pad 311. The depressurization of the port chamber 24 pulls the plunger out of the syringe of the suction dispenser, and various liquids are transported into the syringe. The compressor pressurizes the port chamber 24, which communicates with the port 21 connected to the liquid delivery pad 311. The pressurization of the port chamber 24 pushes the syringe of the suction dispenser into the plunger, and various liquids are pumped out of the syringe.

[0040] Suction and dispensing devices are classified into fluid delivery devices, recovery devices, and waste devices. Fluid delivery devices are connected to blood bags or reagent bags via suction tubes that form the flow paths for various liquids. Blood bags are containers for storing donor blood. Reagent bags are containers for storing reagents such as culture media, PBS, and reprogramming factor solutions. Blood bags and reagent bags are collectively referred to as storage containers. The suction tubes connecting the storage containers and the fluid delivery devices are opened or closed by valves or one-way valves. The valves or one-way valves are opened or closed by valve opening / closing mechanisms 35. Hereinafter, the port 21 where the fluid delivery device is located will be called the fluid delivery port, the port 21 where the recovery device is located will be called the recovery port, and the port 21 where the waste device is located will be called the waste port. Fluid delivery ports include blood delivery ports for delivering blood, reprogramming factor delivery ports for delivering reagents containing reprogramming factors, culture media delivery ports for delivering culture media, and iPS cell delivery ports for delivering iPS cells.

[0041] Here, we will explain the operation of dispensing the liquid to be dispensed (hereinafter referred to as "target liquid"). First, under the control of the control device 30, the moving mechanism 39 moves the cartridge 2 so that the port 21 corresponding to the liquid to be dispensed (hereinafter referred to as "target liquid") is located at the dispensing position P31. Next, under the control of the control device 30, the dispensing pad 311 of the dispensing mechanism 31 connects to the port 21 located at the dispensing position P31, and dispenses the target liquid from the dispensing source to the dispensing destination.

[0042] When the target liquid is transferred from the storage container using a liquid transfer device, a liquid transfer pad 311 is connected to the port 21 to which the liquid should be transferred. The liquid transfer pad 311 transfers the target liquid to the destination via a liquid transfer device located in a port chamber 24 communicating with the port 21, and then via a liquid transfer pipe 23 by depressurizing and pressurizing. Specifically, the liquid transfer pad 311 transports the target liquid from the storage container to the liquid transfer device by depressurizing, and then pressurizes the target liquid from the liquid transfer device to the destination via the liquid transfer pipe 23. Possible destinations for the liquid include various containers such as culture vessels 100, filters, vortex channels, waste liquid containers, tapered containers, sample containers, expanded culture vessels, infection containers, pre-culture containers, recovery containers, and cryopreservation containers.

[0043] When the target liquid is fed from a liquid feed source other than the storage container using a recovery device, the liquid feed pad 311 is connected to the port 21 for liquid feeding. The liquid feed pad 311 transports the target liquid existing in the liquid feed source within the cartridge 2 to the recovery device disposed in the port chamber 24 communicating with the port 21 by decompression, and pressure-feeds the target liquid from the recovery device to the liquid feed destination via the liquid feed pipe 23 by pressurization. As the liquid feed source and the liquid feed destination, there can be various containers such as the culture container 100, filter, spiral flow path, waste liquid container, tapered container, sample container, enlarged culture container, infection container, pre-culture container, recovery container, freeze storage container, etc.

[0044] When the target liquid is discarded into a waste liquid device, the liquid feed pad 311 is connected to the port 21 for discarding. The liquid feed pad 311 transports the target liquid existing in the cartridge 2 to the waste liquid device disposed in the port chamber 24 communicating with the port 21 by decompression. The waste liquid device is removed from the cartridge 2 by an operator or the like and discarded.

[0045] Referring to FIG. 5, the flow path configuration of the cartridge 2 will be described. The cartridge 2 includes a cover 301, a pipe TU, and a plurality of containers 4a to 4r, and is a closed system device in which the liquid flow path is substantially closed from the outside air. The cartridge 2 includes a filter, a valve, a pressure loss member, a drive receiving part, etc., as members constituting the liquid flow path, but the description of these flow path members is omitted with respect to FIG. 5.

[0046] The containers 4a to 4r are used to store various liquids used for the production of iPS cells, liquids generated during the production of iPS cells, and cell suspensions containing the produced iPS cells. Hereinafter, when the containers 4a to 4r are not individually specified, they are simply collectively referred to as the container 4. The liquids used for the production of iPS cells are samples and reagents, and specific examples include blood, cell suspensions, liquid media, and physiological saline, etc. As the physiological saline, for example, PBS (Phosphate-buffered saline) may be used.

[0047] The pipe TU for connecting the flow path members in the liquid flow path is, for example, a pipe member through which various liquids used for the production of iPS cells flow. For the pipe TU, for example, a silicone tube is used.

[0048] The pipe TU, the container 4, and various flow path members constitute a flow path system 300 for feeding the liquid used for the production of iPS cells within the cartridge 2. In the cartridge 2 according to the present embodiment, the flow path system 300 is configured as a closed system flow path closed from the external environment. For this reason, leakage of liquid and substances from the flow path system 300 to the outside of the cartridge and entry of foreign substances from outside the cartridge are prevented.

[0049] The cover 301 covers the outside of the cartridge 2. The cover 301 isolates the inside of the cartridge 2 from the external environment, and suppresses leakage from the cartridge 2 to the outside even if, by any chance, substances such as liquid leak from the closed system flow path within the cartridge 2. That is, the cartridge 2 has a double-closed structure of the cover 301 and the closed system flow path. The double-closed structure is also referred to as a double-sealing structure. Further, the cartridge 2 is discarded after sterilization treatment after use, thereby reducing the risk of occurrence of infection by blood, viruses, etc. used for the production of iPS cells. Among the components included in the iPS cell production system, the cartridge 2 is disposable and is discarded each time iPS cells are produced. Further, the other components (for example, the liquid feeding mechanism 31, the valve opening / closing mechanism 35, the cooling device 37, the culturing device 33, the moving mechanism 39, the imaging unit 16, the analysis unit 17) can be used for multiple productions of iPS cells.

[0050] (iPS Cell Production Process) The iPS cell production process using the iPS cell production system 1 will be described. Figures 6 and 7 are flowcharts illustrating a series of processes for producing iPS cells, with the process in Figure 7 being executed following the process in Figure 6. As shown in the flowchart, iPS cell production includes a reprogramming factor introduction step, a culture step, a subculturing step, and a dispensing step. In this description, each process related to cell production shown in the flowchart will be referred to as a "step". Each step includes one or more single actions, such as flowing a liquid like PBS. Figure 8 is a schematic diagram showing a simplified view of the inside of the cartridge 2 to illustrate the process.

[0051] When iPS cell production begins, in step ST1 shown in Figure 6, a container 4d containing a cell suspension is attached to one cartridge 2 (first cartridge). It is preferable to perform the attachment of the container 4d in a sterile environment, such as a safety cabinet.

[0052] Next, in step ST2, the first cartridge, cartridge 2, is attached to the transfer mechanism 39 (Figures 1 and 2) of the iPS cell manufacturing apparatus 3.

[0053] In the flowchart in Figure 6, the steps after step ST2 are divided into two cases. If the cells contained in the cell suspension are cells that have not undergone reprogramming (step ST2: No), proceed to step ST3. If the cells contained in the cell suspension are iPS cells for subculturing (step ST2: Yes), proceed to step ST4. In this case, the container 4r used as the recovery container when the previous generation of cells was cultured is attached to cartridge 2 as container 4d.

[0054] In step ST3, iPS cells are established by introducing inducing factors (reprogramming factors) into a cell suspension and allowing it to react. Specifically, when reprogramming raw materials, the inducing factors used are Oct3 / 4, Sox2, Klf4, c-Myc, or L-Myc, known as "Yamanaka factors." Other factors such as NANOG, LIN28, and KLF2 are also used. Inducing factors can be supplied in various forms. For example, inducing factors can be supplied incorporated into various vectors. The vector is not particularly limited, but may be a viral vector such as a Sendai virus vector or a retroviral vector, or a non-viral vector such as a plasmid. For example, when using a Sendai virus vector into which the inducing factor has been incorporated, the inducing factor is introduced into the target cells when the Sendai virus vector comes into contact with the target cells. Subsequently, the gene product of the inducing factor is synthesized. The action of this gene product induces the reprogramming of the target cells, and iPS cells, which are undifferentiated cells with pluripotency and proliferative capacity, are established. During the establishment process, cartridge 2 was kept at a temperature of 37 degrees Celsius, humidity of 95%, and CO2. ２ Maintaining a concentration of around 5% is sufficient. While this description focuses on reprogramming factors, when inducing iPS cells into differentiated cells, growth factors, cytokines, specific inducing factors, recombinant proteins, and small molecule compounds are used depending on the target differentiated cell. These can be introduced not only through methods using various vectors, but also by adding components to the culture medium during cultivation.

[0055] The culture process, detachment process, and subculturing process from step ST4 onward are performed using the flow path 300a schematically shown in Figure 8. The flow path 300a is part of the flow path system 300 provided by the cartridge 2. As shown in Figure 8, waste liquid or reagents necessary for cell culture are stored in the liquid delivery containers 4a to 4e. The cartridge 2 also includes a culture vessel 100, which is equipped with a culture dish 120, connectors 130A to 130F, and connector 140. Figure 9 is a perspective view showing the configuration of the culture vessel 100 used in this embodiment, and Figure 10 is a cross-sectional view showing the internal configuration of the culture vessel 100.

[0056] As shown in Figure 8, the culture vessel 100 comprises a plurality of connectors 130 (connectors 130A to 130F, connector 140) and a plurality of tubes 131 (tubes 131A to 131F). For example, the plurality of tubes may include a tube for cell seeding, a tube for waste liquid collection, a tube for PBS supply, a tube for EDTA supply, a tube for cell suspension collection, and a tube for culture medium supply. In the example shown in Figure 8, the culture vessel 100 comprises a connector 130A connected to a waste liquid collection tube 131A, a connector 130B connected to a PBS supply tube 131B, a connector 130C connected to a culture medium supply tube 131C, a connector 130D connected to a cell seeding tube 131D, a connector 130E connected to an EDTA supply tube 131E, and a connector 130F connected to a cell suspension collection tube 131F.

[0057] As shown in Figures 8 to 10, a culture dish 120, which is a dish for culturing cells, is provided at the bottom of the culture vessel 100. The bottom surface of the culture dish 120 is, for example, circular. The diameter of the bottom surface of the circular culture dish 120 is, for example, 35 mmφ or more and 100 mmφ or less. A lid 110 is provided on top of the culture dish 120. As for the height of the culture vessel 100, for example, the height from the bottom surface of the culture dish 120 to the tips of the multiple connectors 130 is 30 mm or more and 120 mm or less. The tips of tubes 131A to 131F are placed inside the culture dish 120.

[0058] Returning to the flowchart in Figure 6, in step ST4, the control device 30 supplies the cell suspension from the liquid delivery container 4d to the culture dish 120 via the seeding tube 131D. The control device 30 then sets the environment inside the cartridge to a temperature of 37.0°C, a humidity of 95%, and CO2. ２ The culture is started after adjusting the concentration to 5%. During the culture, the control device 30 replaces the culture medium in the culture dish 120 as needed. For example, the control device 30 discharges the culture medium (waste liquid) from the culture dish 120 to container 4a via tube 131A, and supplies new culture medium from the liquid supply container 4c to the culture dish 120 via tube 131C. This operation is performed every other day.

[0059] In step ST5, the control device 30 completes the culture. The timing of completion may be determined based on the time the culture environment has been maintained, or it may be determined by taking a picture of the inside of the culture vessel 100 using the imaging unit 16 and checking the state of the cells captured in the image.

[0060] In step ST6, the control device 30 uses the imaging unit 16 to photograph the cells in the culture dish 120. Preferably, the area to be photographed is 70% or more of the bottom area of ​​the culture dish 120. If photography was performed in step ST5, step ST6 may be skipped.

[0061] In step ST6, for example, the cells in the culture dish 120 are photographed using the imaging unit 16 shown in Figure 11. The imaging unit 16 is configured as a fluorescence microscope and irradiates the culture vessel 100 with excitation light from a light source via a dichroic mirror and a translucent culture dish 120. Then, the fluorescence emitted by cells that have not yet completed initialization, for example, green fluorescence, is detected by a photodetector through the dichroic mirror.

[0062] In step ST7, the analysis unit 17 analyzes the progress of cell reprogramming and growth state based on the image captured in step ST6. By analyzing the image, the analysis unit 17 can determine, for example, the number of cells, confluence, colony size, number of colonies, GFP positivity rate, etc. Preferably, the analysis unit 17 calculates at least the number of cells. The analysis unit 17 can analyze the image captured by the imaging unit 16 illustrated in Figure 11, for example, and calculate the GFP positivity rate (virus persistence rate) based on the area of ​​the parts emitting green fluorescence. Here, GFP is due to the expression of a fluorescent protein factor introduced into the cells along with the reprogramming inducer. However, after the establishment of iPS cells, genes programmed to suppress GFP expression are also introduced, and cells showing GFP positivity are determined to be cells whose quality as iPS cells has not been ensured.

[0063] In step ST8, the control device 30 performs an operation to detach the cells in the culture dish 120. In the culture vessel 100 according to the embodiment, in order to detach the cells in a closed state, a nozzle inserted into the culture vessel 100 is used to spray liquid and detach the cells. As shown in Figures 9 and 10, the culture vessel 100 according to the embodiment is equipped with a joint 140 and a detachment nozzle 141 as shown in Figure 10. The joint 140 is provided in the central part of the upper part of the housing 111. The detachment nozzle 141 is provided inside the housing and is connected to the joint 140. An opening is provided in the central part of the light-shielding plate 115, and the tip of the detachment nozzle 141 is positioned in the opening provided in the central part of the light-shielding plate 115. The distance between the tip of the detachment nozzle 141 (the droplet outlet) and the bottom surface of the culture dish 120 is, for example, 10 mm or more and 50 mm or less. Because the bottom surface of the culture dish 120 is circular, the detachment nozzle 141 discharges droplets of liquid that detach cells in a conical shape. The detachment nozzle 141 sprays fine droplets, for example, like a mist. By spraying droplets onto more than 95% of the bottom surface area of ​​the culture dish 120, for example, the detachment nozzle 141 makes it possible to easily detach cells.

[0064] The cell detachment process in step ST8 will now be explained in detail. Since the cells in the culture dish 120 are mixed with waste materials such as the culture medium, a washing process is first performed, followed by a process to detach the cells from the culture dish 120. Specifically, a washing process is performed in which PBS is delivered from the liquid delivery container 4b to the culture dish 120 via tube 131B to wash the cells in the culture dish 120, and the PBS is collected as waste liquid using waste liquid collection tube 131A.

[0065] Subsequently, EDTA is delivered from the liquid delivery container 4e to the culture dish 120 via the EDTA supply tube 131E, thereby weakening the adhesion between cells and between cells and the culture dish 120. The EDTA is then collected as waste liquid in container 4a via the waste liquid collection tube 131A.

[0066] The control device 30 then supplies PBS to the detachment nozzle 141 via the connector 140, and sprays droplet-shaped PBS into the culture dish 120 from the detachment nozzle 141. By discharging droplets into the culture dish 120, physical force is applied to detach cells adhering to the bottom of the culture dish 120. The PBS containing the detached cells is aspirated from the culture vessel 100 and collected into the collection container 4r via the cell suspension collection tube 131F. These processes are performed automatically in a closed state under the control of the control device 30.

[0067] In step ST9, the control device 30 moves cartridge 2, which is the first cartridge for cell production, out of the culture apparatus 33 using the transfer device 15.

[0068] In step ST10, the cell collection container 4r in cartridge 2 is detached from the flow path and removed from cartridge 2. It is preferable to detach the container 4r from the flow path system 300 in a sterile environment such as a safety cabinet. A portion of the suspension may be taken from the cell collection container 4r and used for cell evaluation. Alternatively, the container 4r containing the cell suspension may be temporarily stored in a refrigerator.

[0069] In step ST11, the control device 30 determines whether to continue subculturing based on the cell characteristics analyzed by the analysis unit 17 in step ST7. The control unit compares at least one piece of information, including the cell count, from the calculated confluence, GFP positivity rate, undifferentiated marker positivity rate, or marker positivity rate specific to differentiated cells, and cell count, with predetermined conditions to determine whether subculturing can be carried out.

[0070] For example, if the GFP positivity rate is less than 1%, it is determined that most of the cells in the culture vessel 100 have completed reprogramming, and that an appropriate number and quality of iPS cells have been recovered for use as seed cells for subculturing. If the GFP positivity rate is 1% or higher, it is determined that there are many unnecessary cells that have not completed reprogramming, and that subculturing and continued culture are necessary.

[0071] If, based on the analysis results, it is determined that the appropriate number and quality of iPS cells have been recovered (Step ST11: Yes), proceed to Step ST12. If, it is determined that the appropriate number and quality of iPS cells have not been recovered (Step ST11: No), return to Step ST2 and repeat the process already described in order to obtain cells suitable for subculturing.

[0072] In step ST12 and beyond, as shown in Figure 7, subculturing is performed using a new cartridge (second cartridge). In step ST12, the container 4r containing the cell suspension recovered from the first cartridge is attached to cartridge 2, which will be the second cartridge, as a supply container. To prevent errors in the subculturing operation, it is desirable to verify the container and cartridge using identification information (ID) when attaching container 4r to the new cartridge 2. It is desirable that the attachment of container 4r be performed automatically in a clean environment under the control of the control device 30.

[0073] In step ST13, the control device 30 moves the cartridge 2, which is the second cartridge with the container 4r attached, using the moving device 15 and sets it in the culture device 33.

[0074] In steps ST14 to ST16, the same process as in steps ST4 to ST8 is performed under the control of the control device 30, and subculturing and detachment of subculturized cells are carried out.

[0075] In step ST17, the control device 30 controls the transfer of the cell suspension in the container 4r for retrieving the second cartridge to a dispensing container. There may be one or more dispensing containers. It is also desirable that the dispensing containers be cryopreservation containers that can be frozen. In step ST18, the control device 30 uses the transfer device 15 to remove cartridge 2 as the second cartridge from the culture device.

[0076] In step ST19, the dispensing container is detached from the liquid flow path and removed from cartridge 2. It is preferable to detach the dispensing container from the flow path system 300 within the cartridge in a sterile environment such as a safety cabinet. The removed dispensing container should be frozen for storage.

[0077] This concludes the subculturing process of iPS cells using the iPS cell manufacturing system 1. In this embodiment, subculturing is not limited to one generation; by performing the subculturing process sequentially using the iPS cell manufacturing system 1, it is possible to perform subculturing for any number of generations. Thus, according to this embodiment, it is possible to produce cells of a desired quality in a desired number of cells.

[0078] [Modification 1] Figure 12 schematically shows a first modification of the cartridge 2 used in the iPS cell manufacturing system 1. In the first modification as well, the culture vessel 100 is located on the X-plus side within the cartridge 2 to facilitate its placement within the culture apparatus 33.

[0079] In the first modification, the system includes a container 4s for storing the culture medium used when seeding in the culture vessel 100, a container 4c for storing the culture medium to be replaced during the main culture, and a container 4t for injecting the cells to be cultured into the culture vessel 100. When the cartridge of Modification 1 is used for subsequent subculturing, the container 4r used for recovering the cell suspension in the cartridge used for the previous culture may be attached to the cartridge for subculturing as container 4t.

[0080] [Modification 2] Figure 13 schematically shows a second modification of the cartridge 2 used in the iPS cell manufacturing system 1. In the second modification as well, the culture vessel 100 is located on the X-plus side within the cartridge 2 to facilitate its placement within the culture apparatus 33.

[0081] The second modification, like the first modification, includes a container 4s for storing the culture medium used when seeding in the culture vessel 100, a container 4c for storing the culture medium to be replaced during the main culture, and a container 4t for injecting the cells to be cultured into the culture vessel 100. In the second modification, a removable cryopreservation container 4v and a container 4u for storing cryopreservation solution are further included so that the cells after culture can be collected and frozen for storage. When the cryopreservation solution is injected from container 4u into the culture vessel 100 and the cells after culture are dispersed, the cryopreservation solution containing the cells is injected into the cryopreservation container 4v. In addition, when the cartridge of modification 2 is used for subculturing from the second generation onward, the container 4r used for collecting the cell suspension in the cartridge used for the previous generation of culture may be attached to the cartridge for subculturing as container 4t.

[0082] [Modification 3] Figure 14 schematically shows a third modification of the cartridge 2 used in the iPS cell manufacturing system 1. In this third modification as well, the culture vessel 100 is positioned on the X-plus side within the cartridge 2 to facilitate its placement within the culture apparatus 33.

[0083] The third modification, like the second modification, includes a container 4s for storing the culture medium used when seeding in the culture vessel 100, a container 4c for storing the culture medium to be replaced during the main culture, a container 4t for injecting the cells to be cultured into the culture vessel 100, and a container 4u for storing cryopreservation solution. The third modification 3, like the second modification, also includes a detachably attached cryopreservation container 4v so that the cultured cells can be collected and frozen for storage. However, in the third modification 3, the cultured cells are divided and injected into multiple cryopreservation containers 4v. In order to ensure that the cultured cells are uniformly distributed into the multiple cryopreservation containers 4v, the third modification 3 stirs the liquid containing the cells before injecting them into the cryopreservation containers 4v so that the concentration of cells in the liquid becomes uniform. That is, a stirring container 4w is placed between the culture vessel 100 and the cryopreservation containers 4v. When recovering the cryopreservation solution containing cultured cells from the culture vessel 100, the solution is stirred by reciprocating flow between the culture vessel 100 and the stirring container 4w via the reciprocating flow path 131W to homogenize the cell concentration in the solution. Once the cell concentration is sufficiently homogenized, the solution (cell suspension) is divided and injected from the stirring container 4w into each cryopreservation container 4v. Note that the method for homogenizing the cell concentration in the solution before dividing and injecting it into multiple containers may be different from stirring by reciprocating flow between the culture vessel 100 and the stirring container 4w. For example, after moving the solution containing cells from the culture vessel 100 to the stirring container 4w, the stirring container 4w may be oscillated or a stirrer installed inside the stirring container 4w may be driven to homogenize the cell concentration in the solution. According to the third modification, cells cultured in a culture vessel can be distributed uniformly and quantitatively into multiple containers.

[0084] In the iPS cell manufacturing system 1 according to this embodiment, containers 4d, 4r, 4t, and a cryopreservation container 4v are used to inject cells into culture vessels and to retrieve cells from culture vessels. In order to enable subculturing with ensured cell quality, it is desirable to prevent contamination of cells inside the containers when attaching or detaching these containers to the cartridge. Figure 15 illustrates the connection parts for attaching and detaching these containers to the cartridge 2. These containers are equipped with vents (air vents) to allow liquid inflow and outflow, and tubes that serve as liquid inflow / outflow channels. To prevent foreign matter from entering the container through the vents, for example, a sterile filter can be attached to the vents. Also, to prevent foreign matter from entering the container through the opening when attaching or detaching the tubes to the liquid flow path of the cartridge, tubes made of a heat-sealable material (for example, silicone tubes) are used. When removing a container from the cartridge, the middle of the tube can be clamped with a heat sealer, heat-sealed and sealed, and then cut to detach it. When attaching a container to a cartridge, the tube is fitted into the liquid flow path and then heated with a heat sealer to heat-seal and connect it. Containers with such a connection structure are preferably used for cartridges used for subculturing.

[0085] [Other Embodiments] This disclosure is not limited to the embodiments described above, and many modifications are possible within the technical concept of this disclosure.

[0086] For example, the first cartridge used in steps ST1 to ST10 and the second cartridge used in steps ST12 to ST19 may have the same structure, or they may have different structures.

[0087] An example was shown in which cells cultured using the first cartridge are subcultured using the second cartridge. However, cells subcultured using the second cartridge may be further subcultured (third generation) using the third cartridge. In this way, according to the cell manufacturing apparatus and cell manufacturing method of this disclosure, cells of the target quality can be mass-produced by repeating subculture any number of times.

[0088] This disclosure can also be implemented by supplying a program that implements one or more functions of the embodiments to a system or device via a network or storage medium, and by having one or more processors in the computer of that system or device read and execute the program. It can also be implemented by a circuit (e.g., ASIC) that implements one or more functions.

[0089] The cell manufacturing systems and cell manufacturing methods disclosed herein can be suitably implemented, for example, in the fields of pharmaceuticals and medicine as technologies for manufacturing target cells such as iPS cells.

[0090] The present invention is not limited to the embodiments described above, and various modifications and variations are possible without departing from the spirit and scope of the invention. Accordingly, the following claims are attached to make the scope of the invention public.

[0091] This application claims priority based on Japanese Patent Application No. 2025-018588, filed on February 6, 2025, and all of its contents are incorporated herein by reference.

[0092] 1...iPS cell manufacturing system / 2...cartridge / 3...iPS cell manufacturing apparatus / 4a-4u...container / 4v...cryogenic storage container / 4w...stirring container / 15...transfer device / 16...imaging unit / 17...analysis unit / 20...housing / 21...port / 23...liquid delivery tube / 24...port chamber / 25...container chamber / 30...control device / 31...liquid delivery mechanism / 33...culture apparatus / 35...valve opening / closing mechanism / 37...cooling device / 39...transfer mechanism / 100...culture vessel / 120...culture dish / 130, 130A-130F...connectors / 131, 131A-131F...tubing / 131W...reciprocating flow path / 140...connectors / 300...flow path system / 300a...flow path / 301...cover / 311...liquid delivery pad / 331...storage chamber / 332...opening / 351...cylinder

Claims

1. A cell manufacturing system comprising: a first cartridge; a second cartridge; and a culture device to which the first cartridge and the second cartridge can be attached, wherein each of the first cartridge and the second cartridge has: a culture vessel; a liquid container for storing a liquid used for cell culture; a detachable supply container for supplying cells to the culture vessel; a detachable recovery container for recovering cells cultured in the culture vessel; and a liquid channel connected to the culture vessel, wherein the first cartridge is attached to the culture device, cells are cultured in the culture vessel of the first cartridge, the cultured cells are recovered into the recovery container attached to the first cartridge, the recovery container is removed from the first cartridge and attached to the second cartridge as the supply vessel, and the second cartridge is attached to the culture device, cells are cultured in the culture vessel of the second cartridge, and the cultured cells are recovered into the recovery container attached to the second cartridge.

2. The cell manufacturing system according to claim 1, wherein the supply container and the collection container are equipped with tubes that can be connected to and / or disconnected from the liquid flow path by heat welding.

3. The cell manufacturing system according to claim 1 or 2, wherein the supply container and the collection container are provided with air vents to allow liquid inflow and / or liquid outflow.

4. The cell manufacturing system according to any one of claims 1 to 3, wherein the first cartridge and / or the second cartridge can be fitted with a plurality of the recovery containers, and the cells cultured in the culture vessel are collected in the plurality of the recovery containers in separate portions.

5. The cell manufacturing system according to any one of claims 1 to 4, wherein the first cartridge and / or the second cartridge are fitted with a cryopreservation container as the collection container.

6. The cell manufacturing system according to any one of claims 1 to 5, wherein the first cartridge and / or the second cartridge comprises a stirring container positioned between the culture vessel and the collection container.

7. The cell manufacturing system according to claim 6, wherein the first cartridge and / or the second cartridge, after culturing cells in the culture vessel, stir the liquid containing the cells by causing it to flow back and forth between the culture vessel and the stirring container, and then recover the liquid containing the cells from the stirring container into the recovery container.

8. The cell manufacturing system according to any one of claims 1 to 7, wherein the culture apparatus has an imaging unit for imaging the inside of the culture vessel of the attached cartridge.

9. The cell manufacturing system according to claim 8, wherein the imaging unit comprises a light source for irradiating the inside of the culture vessel with excitation light and a photodetector for receiving fluorescence emitted inside the culture vessel.

10. The cell manufacturing system according to claim 8 or 9, wherein the culture apparatus has an analysis unit that analyzes the state of cells present inside the culture vessel based on an image captured by the imaging unit.

11. The cell manufacturing system according to claim 10, wherein the analysis unit obtains at least one of the following based on the image captured by the imaging unit: cell count, confluence, colony size, number of colonies, and GFP positivity rate.

12. The cell manufacturing system according to claim 10 or 11, wherein cells are cultured in the culture vessel of the first cartridge, and when the results obtained by the analysis unit based on an image taken by the imaging unit of the inside of the culture vessel satisfy predetermined conditions, the recovery container from which the cells have been recovered is removed from the first cartridge and attached to the second cartridge as the supply container.

13. The cell manufacturing system according to claim 12, wherein the predetermined condition is that the GFP positivity rate is less than 1%.

14. A cell manufacturing method using a cell manufacturing system comprising a first cartridge, a second cartridge, and a culture device to which the first cartridge and the second cartridge can be attached, wherein each of the first cartridge and the second cartridge comprises a culture vessel, a liquid container for storing a liquid used for cell culture, a detachable supply container for supplying cells to the culture vessel, a detachable recovery container for recovering cells cultured in the culture vessel, and a liquid channel connected to the culture vessel, wherein the first cartridge is attached to the culture device, cells are cultured in the culture vessel of the first cartridge, the cultured cells are recovered into the recovery container attached to the first cartridge, the recovery container is removed from the first cartridge and attached to the second cartridge as the supply vessel, and the second cartridge is attached to the culture device, cells are cultured in the culture vessel of the second cartridge, and the cultured cells are recovered into the recovery container attached to the second cartridge.

15. The cell production method according to claim 14, wherein the supply container and the collection container are equipped with tubes for carrying liquid, and connection to and / or disconnection from the liquid flow path is performed by heat welding the tubes.

16. The cell production method according to claim 14 or 15, wherein the first cartridge and / or the second cartridge can be fitted with a plurality of the recovery containers, and the cells cultured in the culture vessel are collected in the plurality of the recovery containers in separate portions.

17. The cell manufacturing method according to any one of claims 14 to 16, wherein the first cartridge and / or the second cartridge are fitted with a cryopreservation container as the recovery container.

18. The cell production method according to any one of claims 14 to 17, wherein the first cartridge and / or the second cartridge comprises a stirring container positioned between the culture vessel and the recovery container, and after culturing cells in the culture vessel, the liquid containing the cells is stirred by reciprocating flow between the culture vessel and the stirring container, and then the liquid containing the cells is recovered from the stirring container into the recovery container.

19. The cell manufacturing method according to any one of claims 14 to 18, wherein the culture apparatus comprises an imaging unit for imaging the inside of the culture vessel of a mounted cartridge, and an analysis unit for analyzing the state of cells present inside the culture vessel based on the image captured by the imaging unit, and the analysis unit obtains at least one of the following based on the image captured by the imaging unit: cell number, confluence, colony size, number of colonies, and GFP positivity rate.

20. The cell manufacturing method according to claim 19, wherein cells are cultured in the culture vessel of the first cartridge, and when the results obtained by the analysis unit based on an image taken by the imaging unit of the inside of the culture vessel satisfy predetermined conditions, the recovery container from which the cells have been recovered is removed from the first cartridge and attached to the second cartridge as the supply container.

21. The cell production method according to claim 20, wherein the predetermined condition is that the GFP positivity rate is less than 1%.

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