Method for producing induced pluripotent stem cells

Abstract

Provided are a method and an apparatus for producing induced pluripotent stem cells (iPS cells). Cells are sequentially transferred in one direction by feed mechanisms between n (n≥3) containers (sealed containers) connected in series via a connection conduit, from a first container (A1) to an n-th container (An), and steps for producing iPS cells are performed in sequence in the containers. In the container (A1), a step (s1) is performed for bringing an initializing factor into contact with somatic cells in a liquid medium; in the (n-1)th sealed container (A(n-1)) from a second container (A2), a step (s2) is performed for reducing the concentration of the initializing factor in the liquid medium; and in the n-th container (An), a step (s3) is performed for culturing the somatic cells in the liquid medium to establish iPS cells.

Description

Method for producing induced pluripotent stem cells

[0001] The present invention relates to a method for producing induced pluripotent stem cells and a method for producing differentiated cells. The present invention also relates to a cell production device for carrying out the production method, and a cell production method using the cell production device.

[0002] In recent years, research into regenerative medicine using differentiated cells derived from induced pluripotent stem cells (iPS cells) has been actively conducted. In particular, a therapy in which iPS cells are established from a patient's somatic cells (e.g., peripheral blood mononuclear cells) and various differentiated cells or organoids induced to differentiate from the iPS cells are transplanted into the patient (autotransplantation) has attracted attention as a therapy that can reduce the risk of rejection (Patent Document 1, Non-Patent Documents 1 and 2).

[0003] When autologous transplantation is intended, obtaining iPS cells from cells (somatic cells) obtained from a living organism (e.g., a human) requires a long-term, multi-step process, such as contacting somatic cells with reprogramming factors in a liquid medium, reducing the concentration of the reprogramming factors in the liquid medium, and further culturing the cells to establish iPS cells. Furthermore, obtaining various differentiated cells from the established iPS cells also requires a long-term, multi-step process, such as inducing differentiation into desired cells and testing the quality of the cells.

[0004] Patent Publication No. 2021-72818

[0005] Shinsuke Yoshida., et al., CLINICAL AND TRANSLATIONAL RESOURCE AND TECHNOLOGY INSIGHTS VOLUME 4, ISSUE 1, P51-66.E10, JANUARY 13, 2023Madrid, M., et al., Current Protocols,1, e88. doi: 10.1002 / cpzl.88

[0006] To accommodate the multi-stage, time-consuming processes and adjustment of production conditions, manufacturers have traditionally proposed cell production devices designed to automatically perform all processes in sequence, and some clinical cell production has been reported. A common feature of such cell production devices is that, as shown in Figure 42(a), materials required for each process are sequentially supplied from multiple material supply bags X20 into a single culture vessel X10, and all processes are sequentially performed within the single culture vessel X10. Flexible tubes (hereinafter also referred to as "tubes") X30 extend from each of the multiple material supply bags X20. Each tube X30 passes through a pinch valve X40 that can be controlled to open and close, then repeatedly merges to form a single tube, which then passes through a peristaltic pump X50 and is connected to the culture vessel X10. The pinch valve X40 is an electromagnetic valve that operates in response to commands from a control unit (not shown). When the pinch valve compresses the tube from the outside, the flow path within the tube is closed. The pinch valve and peristaltic pump are controlled by a computer program, and the materials required for each process to be performed are sequentially sent into the culture vessel X10, and each process is performed sequentially. The flow path for waste liquid is also configured so that waste liquid (such as old culture medium) that flows through the peristaltic pump passes through the tube, passes through the pinch valve, and is then discharged into a waste liquid bag. While waste liquid may be disposed of as a liquid, using a hygroscopic waste liquid bag containing a hygroscopic material (e.g., water-absorbent resin, more specifically, polyacrylic acid, sodium polyacrylate, polyacrylic acid copolymer, etc.) or an absorbent article containing such a material (e.g., absorbent pad, absorbent sheet, etc.) allows for incineration without separating the liquid components, facilitating disposal. This configuration allows all processes to be performed within the single culture vessel X10.

[0007] In the conventional cell manufacturing device described above, the manner in which multiple tubes merge into one tube and are connected to a single sealed container X10 is as shown schematically in Figure 42(b). As shown in Figure 42(b), the closer the flow path to the sealed container X10, the more shared the flow path is for multiple material supply bags. In Figure 42(b), pinch valves, peristaltic pumps, and waste fluid flow paths are not shown.

[0008] Conventional cell production devices such as those described above are extremely expensive due to the complex valves and control circuits required for switching flow paths. Furthermore, detailed studies by the inventors have revealed the following problems with such conventional cell production devices: Conventional cell production devices require tubing that merges in multiple stages, and the process of setting each section of the tubing to a pinch valve is complex and time-consuming. Because there are many shared sections through which multiple flow paths pass, it is necessary to not only manage the flow rate using a motor (power source), but also to install sensors that detect air bubbles in the flow paths and sensors that confirm that the liquid has reached a designated location in the tubing. Furthermore, the length of the flow path from the supply bag to the culture vessel X10 is necessarily long due to the need to secure the tube X30 section, making it difficult to transport small volumes of liquid, such as 100 ml or less. Because all processes are performed within a single sealed vessel, the cell production device cannot begin producing other cells until the final process is completed.

[0009] The object of the present invention is to provide a new manufacturing method (a method for manufacturing iPS cells, a method for manufacturing differentiated cells), a new cell manufacturing device, and a cell manufacturing method using the same, which can suppress or eliminate the above-mentioned problems in conventional cell manufacturing devices.

[0010] The main configuration of the present invention is as follows. [1] A method for producing induced pluripotent stem cells, comprising: sequentially transferring cells in one direction between n (n≧3) sealed containers connected in series via connecting pipelines, from a first sealed container (A1) to an nth sealed container (An), using a transfer mechanism; and sequentially carrying out a production process for induced pluripotent stem cells in each sealed container; each of the sealed containers (A1 to An) has one or more inlet / outlet ports that can be opened or closed; the connecting pipelines are configured to be switchable between a connected state and a non-connected state; and the transfer mechanism is a mechanism for transferring contents from each sealed container to the next sealed container through the connecting pipeline that has been switched to a connected state; and the production process for induced pluripotent stem cells comprises: a step (s1) of contacting somatic cells with reprogramming factors in a liquid medium in the first sealed container (A1); and a step (s2) of reducing the concentration of reprogramming factors in the liquid medium in the second sealed container (A2) to the (n-1)th sealed container (A(n-1)). and (s3) culturing the somatic cells in a liquid medium in the n-th sealed container (An) to establish induced pluripotent stem cells. [2] The method for producing induced pluripotent stem cells according to [1], further comprising, after step (s3), a step (s4) of expanding the induced pluripotent stem cells p times (p≧1), wherein p sealed containers (B1 to Bp) corresponding to the number of times the expansion culture is to be performed are connected in series after the nth sealed container (An) via connecting pipelines, the induced pluripotent stem cells are sequentially moved in one direction from the sealed container (An) to the sealed container (Bp) by each feed mechanism, and one expansion culture is performed inside each of the sealed containers (B1 to Bp), thereby performing a total of p expansion cultures, each of the sealed containers (B1 to Bp) has one or more inlet / outlet ports that can be opened or closed, the connecting pipelines are configured to be switchable between a communicating state and a non-communicating state, and the feed mechanism is a mechanism that moves the contents from each sealed container to the next sealed container through the connecting pipeline that has been switched to the communicating state.[3] The method for producing artificial pluripotent stem cells according to [1] or [2], wherein the sealed container is selected from the group consisting of a sealed container having a container body made of a flexible material, a sealed container having a container body made of a hard material, and a sealed container having a container body made of a composite material of a flexible material and a hard material. [4] The method for producing induced pluripotent stem cells according to any one of [1] to [3], wherein the feeding mechanism is selected from the group consisting of: a mechanism for adding fluid to the source sealed container to push out and move the contents of the source sealed container to the next sealed container; a mechanism for reducing the volume of the source sealed container to push out and move the contents of the source sealed container to the next sealed container; a mechanism for moving the contents of the source sealed container to the next sealed container by a pump device provided in the connecting pipeline; a mechanism for moving the contents of the source sealed container to the next sealed container by applying suction force from the next sealed container to the source sealed container; a mechanism for moving the contents of the source sealed container to the next sealed container by utilizing gravity; and a mechanism for adhering cells to magnetic microcarriers and applying an external magnetic force to the microcarriers to move the microcarriers in the source sealed container and the cells adhered thereto to the next sealed container.[5] A method for producing differentiated cells, comprising: a step of producing induced pluripotent stem cells by the production method according to any one of [1] to [4]; and a step (s5) of inducing differentiation of the produced induced pluripotent stem cells, wherein a sealed container (C1) for carrying out the step (s5) is further connected via a connecting pipeline to the rearmost sealed container (X1) among the sealed containers used in the method for producing induced pluripotent stem cells, the sealed container (C1) has one or more openable / closable inlet / outlet ports, and materials necessary for the differentiation induction in the step (s5) are supplied into the sealed container (C1) through the inlet / outlet ports, the induced pluripotent stem cells are moved from the sealed container (X1) to the sealed container (C1) by a feed mechanism, and the step (s5) is carried out in the sealed container (C1), and the connecting pipeline is configured to be switchable between a communicating state and a non-communicating state, [6] The method for producing differentiated cells according to [5], further comprising a step (s6) of removing undifferentiated cells after the differentiation inducing step, wherein a sealed container (D1) for carrying out the step (s6) is further connected via a connecting pipeline to the rearmost sealed container (X2) among the sealed containers used in the differentiation inducing step, the sealed container (D1) having one or more openable / closable inlet / outlet ports, the differentiated cells are moved from the sealed container (X2) to the sealed container (D1) by the feeding mechanism, and the step (s6) is carried out in the sealed container (D1), the connecting pipeline is configured to be switchable between a communicating state and a non-communicating state, and the feeding mechanism is a mechanism for moving the contents from the sealed container (X2) to the sealed container (D1) through the connecting pipeline switched to the communicating state.[7] A cell manufacturing apparatus comprising: a sealed container (A1) for carrying out a step (s1) of contacting somatic cells with a reprogramming factor in a liquid medium; a sealed container (A2) for carrying out a step (s2) of reducing the concentration of the reprogramming factor in the liquid medium; and a sealed container (A3) for carrying out a step (s3) of culturing the somatic cells in the liquid medium to establish induced pluripotent stem cells, wherein each of the sealed containers (A1) to (A3) has one or more openable / closable inlet / outlet ports, and the sealed containers (A1) to (A3) are connected in series in the order of the steps via connecting pipelines that can be switched between a communicating state and a non-communicating state, or are capable of being connected in series in the order of the steps, and the cell manufacturing apparatus comprises: a feed mechanism that moves the contents of the sealed container (A1) to the sealed container (A2) via the connecting pipelines that have been switched to a communicating state, and The cell manufacturing apparatus further comprises a transfer mechanism that transfers the contents of the sealed container (A2) to the sealed container (A3) through the connecting pipeline that has been switched to a communicating state.[8] The cell manufacturing apparatus further comprises p sealed containers (B1 to Bp) for carrying out a step (s4) of expanding the induced pluripotent stem cells in a liquid medium p times (p≧1), each of the sealed containers (B1 to Bp) having one or more openable / closable inlet / outlet ports, (i) when the number of times of expansion culture, p, is one, the p sealed containers are one sealed container (B1), and the sealed container (B1) is connected to or connectable to the sealed container (A3) via a connecting pipeline that can be switched between a communicating state and a non-communicating state, and the cell manufacturing apparatus has a transfer mechanism that moves the contents of the sealed container (A3) to the sealed container (B1) through the connecting pipeline that has been switched to a communicating state, and (ii) when the number of times of expansion culture, p, is two or more, the p sealed containers are two or more sealed containers (B1 to Bp), The cell manufacturing apparatus described in [7], wherein the sealed container (B1) is connected to or connectable to the sealed container (A3) via a connecting pipeline that can be switched between a communicating state and a non-communicating state, the sealed containers (B1) to (Bp) are connected in series or connectable in the order of step (s4) via connecting pipelines that can be switched between a communicating state and a non-communicating state, and the cell manufacturing apparatus has a feed mechanism that moves the contents of the sealed container (A3) to the sealed containers (B1) to (Bp) in sequence via the connecting pipeline that has been switched to the communicating state.[9] The cell manufacturing apparatus further comprises q (q≧1) sealed containers (C1) to (Cq) for carrying out the step (s5) of inducing differentiation of the induced pluripotent stem cells, each of the sealed containers (C1) to (Cq) having one or more openable / closable inlet / outlet ports, (i) when the q sealed containers are one sealed container (C1), the sealed container (C1) is connected or connectable to the sealed container (A3) or the last sealed container (Bp) among the sealed containers (B1) to (Bp) via a connecting pipeline that can be switched between a communicating state and a non-communicating state, and the cell manufacturing apparatus comprises a feed mechanism that moves the contents of the sealed container (A3) or the sealed container (Bp) to the sealed container (C1) through the connecting pipeline that has been switched to a communicating state, and (ii) when the q sealed containers are two or more sealed containers (C1) to (Cq), The sealed containers (C1) to (Cq) are connected in series or are connectable in the order of step (s5) via a connecting pipe that can be switched between a communicating state and a non-communicating state, and the sealed container (C1) is connected or is connectable to the sealed container (A3) or the last sealed container (Bp) among the sealed containers (B1) to (Bp) via a connecting pipe that can be switched between a communicating state and a non-communicating state, and the cell manufacturing apparatus has a feed mechanism that moves the contents of the sealed container (A3) or the last sealed container (Bp) to the sealed containers (C1) to (Cq) in sequence via the connecting pipe that has been switched to the communicating state. [7] or [8] The cell manufacturing apparatus has a feed mechanism that moves the contents of the sealed container (A3) or the last sealed container (Bp) to the sealed containers (C1) to (Cq) in sequence via the connecting pipe that has been switched to the communicating state.

[10] A cell manufacturing apparatus according to [9], further comprising a sealed container (D1) for carrying out a step (s6) of removing undifferentiated cells from the contents of the last sealed container (Cq) among the sealed containers (C1) to (Cq), wherein the sealed container (D1) has one or more openable / closable input / output ports, and the sealed container (D1) is connected to or connectable to the last sealed container (Cq) via a connecting pipeline that can be switched between a communicating state and a non-communicating state, and the cell manufacturing apparatus has a feed mechanism that moves the contents of the last sealed container (Cq) to the sealed container (D1) via the connecting pipeline that has been switched to a communicating state.

[11] The cell manufacturing device according to any one of [7] to

[10] , wherein the sealed container is a sealed container selected from the group consisting of: a sealed container having a container body made of a flexible material; a sealed container having a container body made of a hard material; and a sealed container having a container body made of a composite material of a flexible material and a hard material.

[12] The cell manufacturing apparatus of any one of [7] to

[11] , wherein the feeding mechanism is a mechanism selected from the group consisting of: a mechanism for pushing out and moving the contents of the source sealed container to the next sealed container by adding fluid into the source sealed container; a mechanism for pushing out and moving the contents of the source sealed container to the next sealed container by reducing the volume of the source sealed container; a mechanism for moving the contents of the source sealed container to the next sealed container by a pump device provided in the connecting pipeline; a mechanism for moving the contents of the source sealed container to the next sealed container by applying suction force from the next sealed container to the source sealed container; a mechanism for moving the contents of the source sealed container to the next sealed container by utilizing gravity; and a mechanism for adhering cells to magnetic microcarriers and applying a magnetic force to the microcarriers from the outside to move the microcarriers in the source sealed container and the cells adhered thereto to the next sealed container.

[13] A cell manufacturing apparatus according to any one of [7] to

[12] , further comprising a substrate for arranging all of the sealed containers, the sealed containers being arranged on the substrate and each sealed container being fixed to the substrate, and the sealed containers being connected or connectable in the order of the steps via the connecting pipeline which can be switched between a communicating state and a non-communicating state.

[14] The cell manufacturing apparatus described in

[13] , wherein the substrate can be folded in two around the folding center line, (i) in one region (e1) of the two regions (e1), (e2) on the substrate surface separated by the folding center line, a predetermined number of the sealed containers are arranged in order in one direction (d1) along the folding center line, (ii) in the other region (e2) of the two regions (e1), (e2) on the substrate surface separated by the folding center line, the remaining sealed containers are arranged in order in an opposite direction (d2) to the direction (d1) along the folding center line, and (iii) the rearmost sealed container of the sealed containers in the one region (e1) and the frontmost sealed container of the sealed containers in the other region (e2) are connected or connectable by the connecting pipeline that can be switched between a communicating state and a non-communicating state.

[15] The device further comprises two flexible sheets superimposed on each other, wherein the two flexible sheets are joined to each other so that all of the regions that will become the sealed containers, the region that will become one or more inlet / outlet ports, and the region that will become the connecting pipeline are formed at predetermined positions between the two flexible sheets, leaving these regions as non-jointed regions; and pressing actuators for opening and closing the connecting pipelines are provided on the outer surfaces of the two flexible sheets, wherein the pressing actuators operate to take a pressing position in which the region that will become the connecting pipeline is pressed from the outside of the flexible sheets to bring it into a non-communicating state, and a non-pressing position in which the region that will become the connecting pipeline is not pressed to bring it into a communicating state, and the region that will become the connecting pipeline functions as a connecting pipeline that can be switched between a communicating state and a non-communicating state by the operation of the pressing actuators. A cell manufacturing device described in any one of [7] to

[14] , wherein the areas that become one or more input / output ports extend from the areas that become each sealed container to the outer peripheral edges of the two flexible sheets to form open ends, and the open ends are provided with a structure for an openable / closable input / output port.

[16] The outer peripheral shape of the two flexible sheets that are superimposed and joined together is a shape that can be folded in two around a folding center line, (i) in one region (e3) of the two regions (e3), (e4) separated by the folding center line, regions that will become a predetermined number of the sealed containers are formed to be lined up in one direction (d3) along the folding center line, from the outer peripheral portion of each of the regions that will become the predetermined number of sealed containers that is located farther from the folding center line, regions that will become the one or more inlet / outlet ports extend in a direction away from the folding center line and extend to the outer peripheral edge of the two flexible sheets to form an open end, and between the regions that will become the predetermined number of sealed containers, a region that will become the connecting pipeline that connects the regions that will become the sealed containers is formed, and (ii) in the other region (e4) of the two regions (e3), (e4) separated by the folding center line, The cell manufacturing device described in

[15] , wherein the regions that will become the remaining sealed containers among the above-mentioned sealed containers are formed to be lined up in order along the folding center line in a direction (d4) opposite to the one direction (d3), and from the outer periphery of each of the regions that will become the remaining sealed containers that is located farther from the folding center line, the regions that will become the one or more input / output ports extend in a direction away from the folding center line and extend to the outer periphery of the two flexible sheets to form open ends, and between the regions that will become the remaining sealed containers, a region that will become a connecting pipeline that connects the regions that will become the sealed containers is formed, and (iii) the last sealed container among the sealed containers in one region (e3) and the first sealed container among the sealed containers in the other region (e4) are connected by a region that will become a connecting pipeline that crosses the folding center line.

[17] A cell production method using the cell production apparatus described in any one of [7] to

[16] , comprising at least the steps of: a step (s1) of contacting somatic cells with reprogramming factors in a liquid medium in the sealed container (A1); a step (s2) of, after completion of the step (s1), transferring the contents of the sealed container (A1) into the sealed container (A2) through the connecting pipeline that has been switched to a communicating state, and reducing the concentration of the reprogramming factors in the liquid medium in the sealed container (A2); and a step (s3) of, after completion of the step (s2), transferring the contents of the sealed container (A2) into the sealed container (A3) through the connecting pipeline that has been switched to a communicating state, and establishing induced pluripotent stem cells in the liquid medium in the sealed container (A3).

[18] The method for producing differentiated cells according to [6], wherein the differentiation-inducing step (s5) is a step (s5a) of inducing differentiation of induced pluripotent stem cells into ectodermal cells, mesodermal cells, or endodermal cells.

[19] The method further comprises, after the step (s5a), a step (s5b) of inducing differentiation of the ectodermal, mesodermal, or endodermal cells, wherein a sealed container (C2) for carrying out the step (s5b) is further connected to the sealed container (C1) via a connecting pipeline, the sealed container (C2) has one or more openable / closable inlet / outlet ports, and materials necessary for the differentiation induction in the step (s5b) are supplied to the inside of the sealed container (C2) via the inlet / outlet ports, the ectodermal, mesodermal, or endodermal cells are transferred from the sealed container (C1) to the sealed container (C2) by a transfer mechanism, and the step (s5b) is carried out in the sealed container (C2), the connecting pipeline is configured to be switchable between a communicating state and a non-communicating state, and the transfer mechanism is a mechanism for transferring the contents from the sealed container (C1) to the sealed container (C2) via the connecting pipeline switched to the communicating state.

[18] A method for producing differentiated cells.

[20] A method for producing differentiated cells according to [6], further comprising a step (s7) of removing a sample for testing differentiated cells after the step (s6) of removing the undifferentiated cells, wherein a sealed container (E1) for carrying out the step (s7) is further connected to the sealed container (D1) via a connecting pipeline, the sealed container (E1) has one or more openable / closable input / output ports, and the sample is taken out to the outside through the one or more input / output ports, the contents are moved from the sealed container (D1) to the sealed container (E1) by a feed mechanism, the connecting pipeline is configured to be switchable between a communicating state and a non-communicating state, and the feed mechanism is a mechanism for moving the contents from the sealed container (D1) to the sealed container (E1) through the connecting pipeline switched to the communicating state.

[0011] Hereinafter, the sealed container will also be referred to simply as a "container." In the manufacturing method and manufacturing apparatus of the present invention, multiple containers are used to process cells, rather than a single container. Each of the multiple steps required to produce iPS cells or differentiated cells is associated with one of the multiple containers, and each step is performed in the associated container. Once a process is completed in each container, the cells processed there are aseptically transported by a transport mechanism through a connecting pipeline to the next container, where they undergo the next process. For example, if all the processes include steps s1 to s3, in a preferred embodiment, each step is associated with a single container, with step s1 being performed in the first container A1, step s2 being performed in the second container A2, and step s3 being performed in the third container A3, resulting in the production of target cells.

[0012] The above-described configuration provides the following advantages: The complex piping and control mechanisms of the conventional manufacturing apparatus described above can be simplified, making it possible to construct a manufacturing apparatus at lower cost. Furthermore, since each manufacturing process is completed by a corresponding container, and the order in which the containers are connected determines the order of the manufacturing processes, it is possible to freely configure a series of manufacturing processes by changing the order in which the containers are connected.

[0013] Furthermore, the following effects are achieved: Multistage merging tubes are no longer necessary, eliminating the need for complex setup procedures. The reduction in the number of converging flow paths also reduces the effort required to clean shared areas. Using dedicated containers for each specific process facilitates visual confirmation and monitoring of cells and the environment in each process. Using containers whose internal conditions can be visually observed from the outside makes it easy to determine which process (i.e., which container) a problem occurred in. With conventional single-container devices, determining the process in which a problem occurred requires identifying the time when the problem occurred, which is time-consuming. According to the present invention, for example, after a first process is completed in a first container, cells are transferred to a second container for a second process. This clarifies the management classification for in-process testing, such as sterility testing, and allows a new first process to be started in the first container. This increases the parallelism of manufacturing processes and improves throughput per manufacturing device. Because reaction vessels and processes can be assigned one-to-one, vessels can be easily inserted to add or remove processes depending on the type of cells to be produced (e.g., differentiated cells), and each process can be increased or decreased like blocks. This allows for assembly of vessel connections according to the contents of the manufacturing plan during the planning stage of the manufacturing process. Furthermore, because the cells are moved from vessel to vessel for each process, the cells at each process can be moved separately from the device along with the vessel. As a result, the movable vessels containing cells can be made more flexible, suitable for assembly line operations on a manufacturing line.

[0014] FIG. 1 is a block diagram illustrating a method for producing iPS cells according to the present invention. In this example, each container has a container body and a stopper, and the container is shown in cross-section. FIG. 1 is also a block diagram illustrating an example of the configuration of a cell production apparatus according to the present invention. For ease of understanding, a gap is depicted between the opening of the container body and the stopper, but the stopper actually seals the opening of the container body (this also applies to other figures). FIG. 2 is a diagram illustrating an example of the configuration of an inlet / outlet port according to the present invention. FIG. 3 is a block diagram illustrating an example of the configuration of an inlet / outlet port according to the present invention. The container is shown in cross-section (this also applies to other block diagrams). FIG. 4 is a block diagram illustrating an example of the configuration of a connecting pipeline according to the present invention. FIG. 5 is a block diagram illustrating an example of the configuration of a feed mechanism according to the present invention. FIG. 6 is a block diagram illustrating an example of the configuration of a feed mechanism according to the present invention, showing an example using gravity. FIG. 7 is a block diagram illustrating another example of the configuration of a feed mechanism according to the present invention, showing an example using magnetic force. FIG. 8 is a block diagram illustrating step s4 of expanding iPS cells in the method for producing iPS cells according to the present invention. In the example shown in the figure, each container has a container body and a stopper, and the containers are shown in cross-section. FIG. 8 is also a block diagram showing an example of the configuration of the portion of the cell manufacturing apparatus according to the present invention where iPS cells are expanded and cultured. To clearly show the main parts, reference numerals have been omitted for input / output ports and the like. FIG. 9 is a block diagram for explaining the method for producing differentiated cells according to the present invention. FIG. 9 is also a block diagram showing an example of the configuration of the cell manufacturing apparatus according to the present invention. FIG. 10 is a block diagram for explaining a preferred embodiment of the method for producing differentiated cells according to the present invention, illustrating step s6 of removing undifferentiated cells and step s7 of inspecting differentiated cells. FIG. 10 is also a block diagram showing an example of the configuration of the cell manufacturing apparatus according to the present invention. FIG. 11 is a block diagram for explaining the portion of the cell manufacturing apparatus according to the present invention where iPS cells are produced. FIG. 12 is a diagram showing the portion of the cell manufacturing apparatus according to the present invention where iPS cells are differentiated. To clearly show the main parts, reference numerals have been omitted for input / output ports and the like. FIG. 13 is a diagram showing an example of a preferred embodiment of the cell manufacturing apparatus according to the present invention.FIG. 14 is a diagram illustrating a state in which the substrate of the cell production apparatus shown in FIG. 13 is folded. FIG. 15 is a diagram illustrating another preferred embodiment of the cell production apparatus according to the present invention. FIG. 16 is a diagram illustrating an example of a preferred embodiment for arranging and fixing multiple containers on a substrate. FIG. 17 is a diagram illustrating an example of the substrate shown in FIG. 16 and its state of use. FIG. 18 is a diagram illustrating another preferred embodiment for arranging and fixing multiple containers on a substrate. FIG. 19 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 20 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 21 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 22 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 23 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 24 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 25 is a diagram illustrating the cell production apparatus of the present invention produced in Example 1 and its state of use. FIG. 26 is a diagram relating to the evaluation of iPS cells after expansion culture in Example 1 of the present invention. FIG. 27 is a diagram relating to the evaluation of iPS cells after establishment in Example 2 of the present invention. FIG. 28 is a diagram relating to the evaluation of iPS cells after expansion culture in Example 2 of the present invention. FIG. 29 is a diagram showing the cell manufacturing device of the present invention produced in Example 3 and its usage state. FIG. 30 is a diagram showing the cell manufacturing device of the present invention produced in Example 3 and its usage state. FIG. 31 is a diagram showing the cell manufacturing device of the present invention produced in Example 3 and its usage state. FIG. 32 is a diagram showing the cell manufacturing device of the present invention produced in Example 3 and its usage state. FIG. 33 is a diagram showing the cell manufacturing device of the present invention produced in Example 3 and its usage state. FIG. 34 is a diagram showing the cell manufacturing device of the present invention produced in Example 3 and its usage state. FIG. 35 is a diagram relating to the evaluation of iPS cells after expansion culture in Example 3 of the present invention. FIG. 36 is a diagram relating to the evaluation of iPS cells after expansion culture in Example 4 of the present invention. FIG. 37 is a diagram relating to the confirmation of expression of a cardiomyocyte marker protein (troponin T (TNNT2)) and the like in Example 5 of the present invention.FIG. 38 is a diagram showing the induction of expression of a marker protein in cardiomyocytes and cardiac progenitor cells in Example 5 of the present invention. FIG. 39 is a diagram showing confirmation of expression of a marker protein (PDX-1) in pancreatic progenitor cells in Example 6 of the present invention. FIG. 40 is a diagram showing confirmation of expression of a marker protein (NKX6.1) in pancreatic progenitor cells in Example 6 of the present invention. FIG. 41 is a diagram showing the induction of expression of a marker protein in pancreatic progenitor cells in Example 6 of the present invention. FIG. 42 is a diagram explaining the configuration of a conventional cell manufacturing apparatus, where FIG. 42(a) is a diagram showing an example of a commercially available cell manufacturing apparatus, and FIG. 42(b) is a block diagram showing the features of the apparatus in FIG. 42(a). Pinch valves and peristaltic pumps are omitted from FIG. 42(b).

[0015] FIG. 1 is a block diagram illustrating the iPS cell production method of the present invention, and also a schematic block diagram illustrating an example of the configuration of a cell production apparatus of the present invention. The input / output ports of each sealed container and the connecting pipes between the sealed containers are configured to be openable and closable, allowing each sealed container to be sealed. Therefore, the production method and production apparatus of the present invention enable cell processing and cell culture in a closed system. Hereinafter, the sealed containers used in the production method and production apparatus of the present invention will be referred to simply as "containers" or, as appropriate, as "sealed containers." The thick black lines (h1, h2, h3, etc.) in FIG. 1 indicate piping lines such as flexible tubing. To simplify the illustration of the main components of the invention, connectors, on-off valves, and other components installed on the piping lines are omitted from FIG. 1. The same applies to other examples. While FIG. 1 shows only one material supply source (G1 to Gn in FIG. 1) connected to each container, the number is not limited. The feeding mechanism for sending materials from the material supply sources to the input / output ports is not limited and may be determined according to the material to be supplied. For example, for gas, the internal pressure of the cylinder that supplies the material or any pump can be used. For liquids or cell suspensions, various pumps such as syringe pumps and peristaltic pumps can be used. The same applies to the examples in other figures. In this specification, the terms "cell suspension" and "culture medium containing cells" are used interchangeably. Each container may be connected to an external container (sealed or open) for material disposal depending on the process operation, but this is not shown in Figure 1 . The same applies to the examples in other figures. In the present invention, a feeding mechanism is provided to move cells from one container to the next container through a connecting pipeline. For the sake of explanation, Figure 1 shows a feeding mechanism (designated F1 to Fn in Figure 1) installed as a pump on the connecting pipeline, but as described below, various feeding mechanisms can be used. The same applies to the examples in other figures.

[0016] 1. Method for producing induced pluripotent stem cells First, the method for producing iPS cells according to the present invention (hereinafter also referred to as "production method (I)") will be described in detail with reference to an example configuration of a cell production apparatus according to the present invention. The description of each part of the apparatus, such as the sealed container, also includes the description of each part of the cell production apparatus described below.

[0017] As shown in FIG. 1 , the manufacturing method (I) uses n containers (A1 to An) connected in series via connecting pipelines J1 to J(n-1). Here, n is an integer equal to or greater than 3 (i.e., n≧3). Each container is provided with an openable / closeable input / output port (e.g., symbol h1 for container A1), and each input / output port is connected to a necessary material supply source (G1 to Gn) so that the process associated with each container can be performed. The content K1 contained in the container is a cell suspension, and the cells or the composition of the cell suspension change each time the content K1 moves to the next container. The connecting pipelines (the configuration of which will be described in more detail later) can be switched between a connected state and a disconnected state. The key point of this production method (I) is that cells are sequentially moved in one direction from the first container A1 to the nth container An through connecting pipelines J1 to J(n-1) by means of feed mechanisms F1 to F(n-1), and the following steps s1 to s3 are sequentially carried out in each container: Step s1: In the first container A1, contacting somatic cells with reprogramming factors in a liquid medium; Step s2: In the second container A2 to the (n-1)th container A(n-1), reducing the concentration of the reprogramming factors in the liquid medium; Step s3: In the nth container An, culturing the somatic cells in a liquid medium to establish iPS cells.

[0018] By transferring the cells from one container to another and carrying out the corresponding steps in each container, the final step is completed in the final container An, where iPS cells are established. By carrying out the steps sequentially in multiple containers in this manner, the above-mentioned effects are achieved, and conventional problems are suppressed or alleviated.

[0019] In the example of Figure 1, step s2 is divided into multiple processing stages (multiple steps), and these multiple steps are performed sequentially in containers A2 to A(n-1) until step s2 is completed. After step s2 is completed, the cells are transferred to container An, where step s3 is performed. In this manner, the steps associated with each container are performed sequentially in each container, and the final step s3 is completed in the last container An, where iPS cells are established.

[0020] (n containers (A1 to An)) There is no particular limitation on the number of stages into which step s2 is divided, but in a normal treatment operation, about 1 to 3 stages are preferable, and even a single-stage treatment operation can preferably reduce the concentration of the reprogramming factor in the liquid medium. Therefore, the number n of containers in production method (I) is not particularly limited, but is preferably about 3 to 5. When step s2 is a single-stage treatment operation, only container A2 is used in step s2, and n is 3, and production method (I) uses three containers (A1 to A3) connected in series via connecting pipelines J1 and J2.

[0021] (Serial Connection of Containers) In the present invention, multiple containers are connected in series via connecting pipelines. However, even connections that arbitrarily include parallel connections as described below are included in the serial connection of the present invention, as long as each step is associated with multiple containers and performed sequentially for the containers A1 to An. (i) A connection that includes a parallel connection at the beginning, such as multiple containers (A'11, A'12, A'13, ...) joining together into one container (A2). (ii) A connection that includes a parallel connection midway, such as branching in parallel from one container A1 to multiple containers (A2a, A2b, A2c, ...) and then joining from those multiple containers to one container A3. (iii) A connection that includes a parallel connection at the end, such as branching in parallel from one container A(n-1) to multiple containers (A'n1, A'n2, A'n3, ...).

[0022] When the total number of steps for cell production is two, each step is carried out in two containers. However, when the number of steps is three or more, the number of containers used does not necessarily have to be the same as the number of steps (i.e., each step does not have to correspond to one container), and the number of containers may be fewer than the number of steps. For example, in the case of the three steps s1 to s3 described above, two containers may be used, and steps s1 and s2 may be carried out sequentially in container A1 and step s3 may be carried out in container A2, or step s1 may be carried out in container A1 and steps s2 and s3 may be carried out in container A2. The number of containers into which three or more steps are divided can be selected appropriately. From the viewpoint of significantly demonstrating the effects of the present invention described above, it is preferable to use the same number of containers as the number of steps and carry out each step in one container. In the example of FIG. 1, one step s2 is further divided into multiple steps, and each divided step is associated with one container.

[0023] (Sealed Container) A container capable of containing cells together with a liquid medium and processing and culturing the cells in a sterile environment can be used. Various sealed containers for culture used in conventional cell culture and cell production can be used. The containers used in the present invention may be different from one another. However, from the viewpoints of cost reduction, modularization, use of standardized and unified containers for tissue culture engineering evaluation, temperature control stability of the culture container, specification of the amount of medium supplied, management of the number of cultured cells, evaluation of medium components, correction of lot-to-lot variations, setting of oxygen and carbon dioxide supply values ​​for the medium, speeding up container acceptance testing, standardization of gas sterilization, gamma ray sterilization, ultraviolet sterilization, and packaging form for connected containers, and standardization of container packaging, transportation, and storage methods, it is preferable that all containers have the same shape and specifications. Standardization of containers can be easily achieved by providing a sufficient number of inlet / outlet ports for each container and closing unnecessary inlet / outlet ports.

[0024] (Preferred Embodiments of Container) Preferred embodiments of the container include: (i) a sealed container having a container body made of a flexible material; (ii) a sealed container having a container body made of a hard material; and (iii) a sealed container having a container body made of a composite material of a flexible material and a hard material. The container body has an opening (mouth), and the opening is closed with a lid, a stopper, or the like to form a sealed container. Alternatively, a cylindrical connector or the like may be attached to the opening, and the conduit of the connector may be closed with a stopcock or the like to seal the container body. The materials of the lid, stopper, and inlet / outlet port that close the opening of the container body may be selected appropriately with reference to conventionally known sealed containers for cell culture, etc.

[0025] The structure of the sealed container having a container body made of the flexible material (i) above is not particularly limited, but typical examples include soft bags made of flexible film, such as conventionally known cell culture bags and infusion bags (MACS (registered trademark) GMP Cell Culture Bags (Miltenyi Biotec), Flexboy (registered trademark) bags (SARTORIUS)). The flexible film is a film that is flexible enough to be deformed according to the amount of contents contained in the bag. The structure of the bag is not limited, but examples include a bag structure in which two rectangular or square flexible films are overlapped and the outer edges, excluding the parts that will become the openings and inlet / outlet ports, are fused (thermally fused, high-frequency fused, etc.) or glued together. The flexible film that makes up the bag is suitable for the O2 required for cell culture. ２ YaCO ２ When the flexible film is gas impermeable, O necessary for cell culture can be passed through the inlet / outlet port described later. ２ YaCO ２and supply the appropriate amount of the flexible film. Furthermore, the flexible film is preferably made of a material that is industrially excellent in moldability, can withstand gamma ray sterilization, and is transparent enough to allow observation of the state of the culture medium inside. Additional structures, such as holes for suspending the bag, can be provided as appropriate, with reference to conventionally known cell culture bags. The above-mentioned sealed container used in the production method of the present invention is preferably non-cell-adhesive, so as to enable suspension culture in the production method of the present invention described below. For example, the sealed container may be one that has not been artificially treated (e.g., coated with an extracellular matrix, etc.) in order to improve adhesion to cells.

[0026] The flexible film constituting the bag can be made of known materials used for cell culture bags, etc., such as polyethylene, polyethylene terephthalate, polypropylene, ethylene vinyl acetate copolymer (EVA), ultra-low density polyethylene (ULDPE) / ethyl vinyl alcohol (EVOH), ultra-low density polyethylene (ULDPE), polyolefin (PO), tank liner, polyvinylidene fluoride, polyethersulfone, etc. The flexible film may be a single-layer film or a multi-layer film made of these materials.

[0027] Examples of the sealed container having a container body made of the above-mentioned hard material (ii) include, but are not limited to, flasks, tissue culture flasks, vials, tubes (test tubes), bioreactors, jar fermentors, hollow fiber membrane bioreactors, and other containers that are resistant to deformation due to the weight of the cell suspension they contain. Hard materials include known materials conventionally used in cell culture containers, such as glass and plastic materials (e.g., polycarbonate, polyester, polyamide, polystyrene, acrylonitrile-butadiene-styrene copolymer (ABS resin), polyethylene, polypropylene, polyvinyl chloride, and biodegradable resins). The opening of the container body is sealed with a lid or stopper, thereby forming a sealed container. The inlet / outlet ports preferably have the required number of tubes penetrating the lid or stopper from the outside to the inside. The sealed container having a container body made of a hard material may be one designed and manufactured specifically for the present invention, or a commercially available cell culture container equipped with inlet / outlet ports may also be used. For example, G-Rex (registered trademark) 10N-CS and G-Rex 100N-CS manufactured by Wilson Wolf Co., Ltd. are provided with a sufficient number of inlet and outlet ports on the lid, and the bottom of the vessel body is provided with an O2 port necessary for cell culture. ２ YaCO ２ The cell culture device is provided with a gas-permeable membrane through which the gas can pass, and can be preferably used as the sealed container in the present invention.

[0028] An example of the sealed container having a container body made of a composite material of a flexible material and a hard material as described above in (iii) is one in which the framework is made of a hard material and the walls are made of a flexible film. Such a container has the three-dimensional characteristics of a hard container, but is inexpensive and disposable.

[0029] (Sealability of Sealed Container) A sealed container usable in the present invention is a container that, when the inlet / outlet ports, including the openings for the connecting pipelines, are closed, provides a sealed internal space that allows cell culture in a closed system. The "sealed internal space that allows cell culture in a closed system" is a space in which the walls, openings, and inlet / outlet ports are closed to the extent that microorganisms and viruses cannot enter from the outside, i.e., to the extent that sterility within the container is maintained. In addition, it is preferable that the space be airtightly sealed so that gas or contents do not leak from unintended locations when, for example, gas is pumped into the container and the contents are pushed out through a specified inlet / outlet port. For example, a container having an inlet / outlet port equipped with a porous filter that does not allow bacteria or viruses to pass through but allows fluids (especially gases) to pass through can allow outside air to flow into the container through the porous filter, while maintaining sterility within the container. Therefore, the interior of the container is the "sealed internal space that allows cell culture in a closed system." The inlet / outlet port equipped with such a porous filter may be configured so as to be airtightly closed by a valve, stopcock, etc. In addition, the containers such as the above-mentioned G-Rex (registered trademark) 10N-CS and G-Rex 100N-CS manufactured by Wilson Wolf have a gas-permeable membrane at the bottom wall of the container body, which does not allow bacteria or viruses to pass through, and the O 2 required for cell culture is maintained. ２ Gas molecules and CO ２ Gas molecules can pass through the container, and the inside of the container can be said to be an airtight sealed space. Therefore, the inside of the container can be kept sterilized, and it is also possible to send gas into the container through an inlet / outlet port and push the contents out through a specified inlet / outlet port. Therefore, the inside of the container is the aforementioned "sealed internal space that allows culture in a closed system."

[0030] (Volume of sealed container) The volume of the container is not particularly limited and may be large enough to industrially produce a large amount of iPS cells at once, but for applications such as transplantation therapy using differentiated cells derived from autologous iPS cells, the volume is preferably about 1 to 1000 ml, and more preferably about 10 to 100 ml.

[0031] (Input / Output Ports) Input / Output ports are provided for introducing substances into and removing substances from containers. A required number of input / output ports may be provided for each container for the following purposes: (i) as a supply port for sending materials (somatic cells, liquid culture medium, various reagents, etc.) required for the process performed in each container from the outside into the container; (ii) as a pressure adjustment hole (gas vent hole, air supply hole, liquid outflow hole) for eliminating pressure changes when sending materials from the outside into each container or when removing materials from the container; (iii) as a discharge port for discharging materials (liquid culture medium containing reprogramming factors or undifferentiated cells, as described below, old liquid culture medium, etc.) that are no longer needed in the process within each container to the outside; (iv) as an outlet for removing samples for testing or harvested products; (v) as an opening for connecting a connecting pipe to the container; (vi) as a fluid injection port or suction port for configuring the feeding mechanism described below. The inlet / outlet port may be provided as various inlets and outlets as required, in addition to the above uses.

[0032] (Number of Inlet / Outlet Ports) The number of inlet / outlet ports provided in one container does not necessarily have to be the same as the number of applications described above. One inlet / outlet port may be used for multiple applications by using a valve or branch pipe for switching flow paths, or by attaching and detaching external conduits. For example, the inlet / outlet port for supplying gas necessary for cell culture (i) above can also be used as an inlet / outlet port for supplying gas when pushing the contents out of the container (vi) above. Furthermore, the inlet / outlet port serving as a pressure adjustment hole (ii) above can also be used as an inlet / outlet port for discharge (iii) above. When replacing a liquid culture medium, two inlet / outlet ports are used when supplying new liquid culture medium into a container and pushing out the old liquid culture medium from the container. Using one inlet / outlet port for multiple applications can be cumbersome, requiring the attachment, detachment, and switching of multiple external conduits to a single inlet / outlet port, or can result in complex piping. Therefore, the number of inlet / outlet ports may be the same as the number of applications, or one inlet / outlet port may be used for an appropriate number of applications. From this point of view, the number of inlet / outlet ports provided in one container is generally about 3 to 10.

[0033] The number of inlet / outlet ports may vary from container to container, but in order to standardize the container specifications to ensure compatibility and reduce costs, it is possible to provide the same number of inlet / outlet ports in all sealed containers and close unused inlet / outlet ports when using the container.

[0034] (Inlet / Outlet Port Connectors) The inlet / outlet port may be a simple through-hole provided in the wall or lid of the container, or may be a short pipe for a fitting airtightly inserted through the through-hole. It is preferable that the inlet / outlet port be equipped with an appropriate connector so that external piping, the tip of an external syringe, or the like can be securely and leak-freely connected to the inlet / outlet port, and that the connection can be easily detached. However, directly fixing multiple connectors to the container stopper or the like can be difficult due to interference between the connectors. Therefore, the embodiment shown in Figure 2 provides an inlet / outlet port configuration that solves this problem. In the example shown in the figure, each container 10 is provided with three inlet / outlet ports 11. Each container 10 has a configuration in which the opening of the container body 10a is closed with a stopper 10b. Each inlet / outlet port 11 in the figure has a tubular member 11a that penetrates the stopper 10b in an inward / outward direction. In the example shown in the figure, each tubular member is a soft tube, but the tubular member at the penetration portion may be made of a hard material. A luer connector 11b is provided at the distal end (the end farthest from the stopper) of the tubular member 11a on the external side. This prevents the connectors of the three input / output ports from interfering with each other, facilitating attachment and detachment to each connector, and preventing the force applied during attachment and detachment from significantly affecting the stopper. A clip for closing the conduit is attached to the central tubular member of the three tubular members.

[0035] As the connector for the input / output port, any pipe joint (also called a connector or a coupling) can be used, and examples thereof include a luer connector (old ISO 594-1, old ISO 594-2, etc.), a misconnection-proof connector (ISO 80369), a sterile connection joint, a one-touch sterile disconnect joint, a push-in joint for tubing, a one-touch joint, etc. Also included in the openable / closable connector of the present invention is a combination of a stopper configured to be pierced by a syringe needle, such as a rubber stopper for a vial, and a syringe needle or chemoclaving bag spike that pierces it.

[0036] (Configuration for Opening and Closing an Inlet / Outlet Port) The configuration for opening and closing an inlet / outlet port is not particularly limited, and examples include a cap that closes the connection end of a disengaged connector, a pinch valve that closes the conduit of a tube provided as an inlet / outlet port, a clip, a stopcock, and the like. Switching of flow paths using a three-way stopcock may also be used as a mechanism for opening and closing an inlet / outlet port. Furthermore, a configuration for opening and closing an inlet / outlet port also includes a configuration in which an external conduit that remains connected to the inlet / outlet port is configured to be openable and closable. Furthermore, a combination of the above-mentioned stopper and an injection needle that pierces it is also included in a configuration for opening and closing an inlet / outlet port.

[0037] (Configuration of Inlet / Outlet Ports Inside the Container) FIG. 3 is a block diagram illustrating the configuration of an inlet / outlet port inside a container, with the container shown in cross section for illustrative purposes. In the example of FIG. 3, similar to FIG. 2, the container 10 has a configuration in which the opening of the container body 10a is closed with a stopper 10b, and the container 10 is provided with three inlet / outlet ports 111, 112, and 113. Each inlet / outlet port has a tube member (soft tube) penetrating the stopper 10b in both the inward and outward directions, and the tube member also extends inside the container. A tube fitting 11b1 is provided at the distal end of the tube member on the external side. In addition, a device 12 for opening and closing the conduit is provided on the external side of the tube member. The external sides of the other inlet / outlet ports 112 and 113 are similarly configured but are not shown.

[0038] In the example of Fig. 3, the tubular members of the inlet / outlet ports 111, 112, and 113 extend into the container, but their lengths are characteristic. In the examples of Figs. 3(a) and (b), the portion of the tubular member of the inlet / outlet port 111 inside the container does not reach the liquid level of the contents (such as a cell suspension). This portion inside the container is preferable because it allows gas to be introduced into the container without stirring the contents, making it possible to adjust the volume pressure using gas without being affected by the amount of liquid inside the container. The gas is used to introduce O necessary for cell culture. ２ YaCO ２ Alternatively, air may be used to push out the contents of the container. In the examples of Figures 3(a) and (b), the internal portion of the tubular member of the inlet / outlet port 112 extends from the liquid surface to a suitable depth within the contents. This type of internal portion of the container is preferable because it can supply liquid medium to the container without generating strong convection in the liquid medium or the like and thus without stirring up settled cells. Therefore, this type of internal portion of the container is suitable for operations such as replacing liquid medium in cell culture, diluting to reduce the concentration of reprogramming factors, and washing cells. In the example of Figure 3(a), the internal portion of the tubular member of the inlet / outlet port 113 extends from the liquid surface to a suitable depth within the liquid contents, similar to the inlet / outlet port 112. This type of internal portion of the container is preferably used when gently removing the liquid (supernatant) near the liquid surface of the liquid medium from the container. Furthermore, by using two inlet / outlet ports 112 and 113 as in the example of Figure 3(a), depending on the orientation of these inlet / outlet ports, a vortex can be generated in the liquid culture medium, enabling rotational culture. In the example of Figure 3(b), the internal portion of the tubing of inlet / outlet port 113 reaches near the bottom of the container body. If this internal portion of the container is used as a supply port for liquid culture medium or gas, it can generate strong vertical convection and send new liquid culture medium or gas to the settled cells. Furthermore, if it is used as an outlet for the contents of the container or a port for a connecting pipe (described below), the contents can be discharged until the liquid level of the contents reaches near the bottom of the container.

[0039] (Connecting Pipeline) In the present invention, the connecting pipeline is used to connect containers and has a configuration that can be switched between a communicating state and a non-communicating state. The communicating state refers to a state in which fluid can move from one container to the next through the connecting pipeline, and the non-communicating state refers to a state in which fluid cannot move from one container to the next. The connecting pipeline may be configured in any manner, including, but not limited to, the following: (Mode i) As illustrated in FIG. 4( a), a connecting pipeline J1 is provided to connect container A1 and container A2. Various on-off valves (including shutters installed to open and close the pipeline), stopcocks, clips, and other on-off devices J1a are provided on the connecting pipeline J1, and the connecting pipeline J1 can be switched between a communicating state and a non-communicating state by opening and closing the on-off device J1a. (Mode ii) As illustrated in FIG. 4( b), a connecting pipeline J1 is provided to connect container A1 and container A2. A connector J1b is provided on the line or at both ends of the connecting pipe J1. The connector J1b is not a so-called opening / closing device, but when the connector J1b is connected, the connecting pipe J1 switches to a connected state, and when the connector J1b is disconnected, the connecting pipe J1 switches to a non-connected state as shown in FIG. 4(b). Such a connector may be a connector for connecting to an input / output port. (Aspect iii) As shown in FIG. 4(c), a connecting pipe J1 for connecting the container A1 and the container A2 is provided on the container A1. A syringe needle J1c is attached to the tip of the connecting pipe J1. The syringe needle J1c is not pierced into the stopper of the container A2, so that the containers A1 and A2 are not connected by the connecting pipe J1 but are in a connectable state (i.e., a pierceable state). When the contents are transferred from container A1 to container A2, the connecting pipe J1 is switched from a non-communicating state to a communicating state by piercing the stopper of container A2 with the injection needle J1c. In the example of Fig. 4(c), the opening of the container body is sealed with a single stopper, but a stopper for piercing the injection needle J1c may be provided locally at a predetermined position on the lid that seals the opening.The inner diameter of the injection needle J1c may be any diameter that allows cells or microcarriers to pass through, and an injection needle with an inner diameter of about 23 gauge (G) or larger is preferably used, and more preferably about 18 G is used, for example.

[0040] In the present invention, when a container is "connectable" to another container via a connecting pipeline, this means a state before connection, such as the relationship between containers via the injection needle and stopper (where the containers are not connected because the injection needle has not been pierced into the stopper, but will be connected when the injection needle is pierced into the stopper) or the relationship between containers via disengaged connectors (where the connectors are not connected because they are not engaged, but will be connected when the connectors are engaged), in which the disconnected connecting pipelines are in a connectable state and the containers are connected when the connecting pipelines are joined.

[0041] 4(a) to 4(c) are for illustrative purposes only, and the position and configuration of the inlet / outlet port to which the connecting conduit is connected can be designed as appropriate. Furthermore, even if the connecting conduit itself does not have a configuration that can be opened and closed, it is sufficient that the inlet / outlet port connected to the connecting conduit can be opened and closed, thereby allowing the connecting conduit to be switched between a communicating state and a non-communicating state.

[0042] (Feeding Mechanism) The feeding mechanism in the present invention is a mechanism that transfers the contents from each container to the next container through the above-mentioned connecting pipe line that has been switched to the communicating state. Here, "each container" refers to a source container that has a container to which the contents should be transferred next (i.e., a container other than the last container).

[0043] When transferring contents, pressure adjustment (pressure release, such as air venting) within the next-stage container can be performed using an appropriate inlet / outlet port. If the next-stage container is a flexible, empty bag such as a cell culture bag, the next-stage container will expand as the contents are transferred, and pressure adjustment may not be necessary. The same applies to pressure adjustment (pressure release, such as air inflow) within the source container when the contents are removed by suction from the outside, and an appropriate inlet / outlet port can be used. If the source container is a flexible bag such as a cell culture bag, the bag will shrink as the contents are removed, and pressure adjustment may not be necessary.

[0044] Specific embodiments of the feed mechanism include a mechanism selected from the group consisting of the following mechanisms (i) to (vi), and may also be a mechanism that combines two or more mechanisms selected from the group.

[0045] (i) Mechanism for pushing out contents from a container (1) This mechanism is a mechanism for pushing out and moving the contents of the sealed source container A1 to the next container A2 by adding (supplying through an inlet / outlet port) a fluid (gas, liquid culture medium, etc.) into the source container A1, for example, in the configuration of FIG. 4(a). In an embodiment in which a feed gas is injected into the source container, the inlet / outlet port for injecting the gas may be dedicated to the feed mechanism, or may also serve as an inlet / outlet port for injecting gas for cell culture. The feed gas may be air (preferably clean air passed through a filter (oxygen concentration of about 20%, carbon dioxide concentration of about 5%)) or O2 necessary for cell culture. ２ YaCO ２Any gas that does not adversely affect cells, such as acetone, can be used. It is also possible to supply liquid culture medium into the source container A1 and then push the contents into the next container A2. In this case, the contents in container A1 are mixed with the supplied liquid culture medium and then pushed out, so it can be considered that the dilution and movement of the contents in container A1 are carried out simultaneously. Any external pump device can be used to supply fluid into the source container A1, and the delivery mechanism of the material supply source can also be used. While the above explanation shows an example of movement from container A1 to A2, it may also be applied to mechanisms for moving contents to subsequent containers A3 to An.

[0046] (ii) Mechanism (2) for Extruding Contents from a Container. As shown in FIG. 5( a), this mechanism reduces the volume of the source container A1, thereby extruding and transferring its contents K1 to the next container (not shown). In the example shown in FIG. 5( a), the container A1 is a flexible cell culture bag lying on a surface. A pressure head member 1 attached to the tip of a pressure device such as an air cylinder presses and compresses the container A1 from the outside, reducing its volume. The contents K1 are then extruded into the next container (not shown) through the input / output port 11 and the connecting pipeline J1. While the example shown in the figure illustrates the transfer from container A1 to the next container, the mechanism may also be applied to mechanisms for transferring contents to subsequent containers A3 to An. The dashed arrows in the figure indicate the contents being extruded. The pressure on the container may be performed automatically using a pressure device or manually. In the example of FIG. 5( a ), a flexible bag is used as the container, but a container whose volume can be changed and whose contents can be pushed out, such as a container having a syringe structure as a whole, may also be used.

[0047] (iii) Mechanism for moving contents via a pump device. As illustrated in FIG. 1 , this mechanism uses a pump device F1 installed in a connecting pipe J1 between two containers A1 and A2 to move contents K1 from a source container A1 to a subsequent container A2. The pump device usable in this mechanism is not particularly limited, but is preferably a pump device that can transfer contents (suspension) without damaging cells, such as a peristaltic pump or a syringe pump. The pump device may be manually operated or may be an automated device equipped with a drive source and a control unit. In the example shown in FIG. 5( b), a syringe pump is connected as the pump device F1 to a T-shaped pipe F1c inserted into the connecting pipe J1. Check valves F1a and F2b are connected before and after the T-shaped pipe F1c so that the contents are moved in one direction from container A1 to container A2 by the suction and discharge operations of the syringe pump. While the illustrated example shows the transfer of contents from container A1 to A2, the present invention may also be applied to a mechanism for transferring contents to subsequent containers A3, A4, etc. A stopcock may be used instead of a check valve. The arrows on check valves F1a and F2b indicate that flow is restricted to one direction. If a three-way stopcock or a three-way solenoid valve is used instead of the T-shaped pipe F1c in FIG. 5(b), the check valves F1a and F2b can be omitted, and the contents will flow from container A1 to the syringe pump when the syringe pump is suctioning, and from the syringe pump to container A2 when the syringe pump is discharging.

[0048] (iv) Mechanism for Aspirating Contents from a Subsequent Container This mechanism, for example, in the configuration shown in FIG. 4( a), applies suction force from a subsequent container A2 to a source container A1, sucking and transferring the contents K1 of the source container A1 into the subsequent container A2. To apply the suction force, for example, air inside the container A2 is sucked out of the container A2 through an inlet / outlet port of the container A2, thereby applying suction force to the contents K1 of the container A1 through the connecting pipe J1. When sucking the air inside the container A2 out of the container, the inlet / outlet port for sucking the air out of the container A2 may be dedicated to the feeding mechanism, or may also serve as an inlet / outlet port for injecting gas for cell culture. Alternatively, a container capable of aspirating the contents K1 of the container A1 through the connecting pipe J1 may be used, such as a container in which the entire subsequent container A2 has a syringe structure. In this explanation, the transfer from container A1 to container A2 is shown as an example, but the present invention may also be applied to a mechanism for transferring contents to subsequent containers A3 to An.

[0049] (v) Mechanism for Moving Contents Using Gravity This mechanism is configured by the positional relationship between two containers and the connection of connecting pipelines so that gravity can be utilized. For example, as shown in FIG. 6( a), this mechanism places a next-stage container A2 below a source container A1, and uses gravity to move the contents of the source container down to the next-stage container A2. In the example of FIG. 6( a), container A2 is placed directly below container A1, and a connecting pipeline J1 is connected to the bottom of container A1, with an opening / closing device J1a provided on the connecting pipeline J1. When step s1 is completed in container A1, the opening / closing device J1a opens, switching the connecting pipeline J1 to a connected state, and the contents in container A1 fall through gravity into the next-stage container A2. In addition to the example shown in FIG. 6(a), a configuration in which the contents flow from the container A1 into the next container A2 by the principle of a siphon is also a mechanism for moving the contents by utilizing gravity.

[0050] In the example shown in FIG. 6(b), vessel A1 and vessel A2 are adjacent to each other, and the internal spaces of vessel A1 and vessel A2 are separated from each other by an openable partition J1a1 of a shutter device J1a. The region J1 opened and closed by the partition J1a1 corresponds to the connecting pipeline switchable between a connected state and a disconnected state in the present invention. In the figure, the partition J1a1 is in the closed position. When the partition J1a1 is completely retracted into the main body of the shutter device J1a and moved to the open position, vessel A1 and vessel A2 become one integrated internal space. As shown in FIG. 6(b), vessel A2 is located below vessel A1, and the bottom surfaces of vessel A1 and vessel A2 form a single inclined surface so that cells M can slide down from vessel A1 to vessel A2.

[0051] The partition plate J1a1 can be stopped in a state where it is retracted by any amount into the main body of the shutter device J1a. When the partition plate J1a1 is retracted a small amount, the connecting pipeline is slightly open. When step s1 is performed in the container A1, the partition plate J1a1 is in the closed position. When step s1 is completed, the partition plate J1a1 opens (i.e., the connecting pipeline J1 switches to a connected state), and the contents of the container A1 move by gravity into the next container A2. Furthermore, as shown in FIG. 6(b), if a liquid medium is also contained in the container A2, the cells M in the container A1 slowly descend through the liquid medium by gravity and enter the next container A2. At this time, the partition plate J1a1 may be fully opened; however, as shown in FIG. 6(b), in order to primarily move the settled cells, it is preferable to open the partition plate J1a1 less than halfway, or to an extent that allows the settled cells to move. This prevents the liquid medium in container A1 from mixing with the liquid medium in container A2. This type of cell migration also falls under the category of "content migration" in the present invention. While FIG. 6 shows an example of migration from container A1 to A2, the present invention may also be applied to a mechanism for transferring contents to subsequent containers.

[0052] (vi) Mechanism for Moving Cells Using Magnetic Force This mechanism moves cells using magnetically imparted microcarriers and magnets. Cells are attached to magnetically imparted microcarriers, and the microcarriers are magnetically attracted by a magnet. This allows the microcarriers and the cells attached thereto to be moved to the next container A2. In the example of Figure 7, similar to the example of Figure 6(b), containers A1 and A2 are adjacent to each other, and an openable / closable partition plate J1a1 of the shutter device J1a separates and connects containers A1 and A2. The bottom of container A2 does not need to be located below the bottom of container A1. In the example of Figure 7, the bottoms of containers A1 and A2 are a single horizontal surface at the same level. If gravity is also used to move cells, it is preferable that the bottoms of containers A1 and A2 form a single inclined surface, similar to the example of Figure 6(b).

[0053] When step s1 is performed in container A1, the partition plate J1a1 is in the closed position. Upon completion of step s1, as shown in FIG. 7, the partition plate J1a1 opens an appropriate amount, connecting container A1 and container A2. FIG. 7 shows the state in which the external magnet F1 moves, attracting the microcarriers with attached cells toward container A2. As shown in FIG. 7, if liquid medium is also contained in container A2, the liquid medium does not move violently even when the partition plate J1a1 opens. Therefore, the microcarriers M1 with attached cells move gently through the liquid medium due to magnetic force and enter the next container A2. At this time, a portion of the liquid medium in container A1 mixes with the liquid medium in container A2, but the entire liquid medium in container A1 does not move to container A2. While FIG. 7 illustrates the example of transfer from container A1 to A2, the mechanism may also be applied to a mechanism for transferring contents to subsequent containers.

[0054] (Magnetic Microcarriers) The magnetic microcarriers that can be used in the present invention are not particularly limited, but include, for example, the well-known γFe ２ O ３ and Fe ３ O ４The magnetic particles may be made of a magnetizable material such as cellulose, coated with carboxydextran or cross-linked agarose, and functional groups such as amino, carboxyl, hydroxyl, or epoxy groups may be introduced thereto. A physiologically active substance such as an antibody that recognizes the cells may be bound to the magnetic particles via these functional groups, thereby exhibiting adsorptivity to surface antigens on target cells. Examples of commercially available microcarrier substrates that can be made magnetic include Corning Synthemax vitronectin substrate (Corning) and Atelocollagen Microspheres (KOKEN). Examples of commercially available microcarriers include Global Eukaryotic Microcarriers. TM (Global Cell Solutions) is an example.

[0055] (Magnet) The magnet may be a permanent magnet or an electromagnet. Electromagnets are preferable for moving magnetically imparted microcarriers because they can generate a magnetic field only when necessary (particularly when moving in the feed direction). The shape of the magnet (particularly the outer shape and area of ​​the surface facing the container), the magnetic field strength, and the moving speed can be appropriately determined so as to attract the microcarriers. The magnet moves along a predetermined course from container to container so that the microcarriers move from container to container. Alternatively, the magnet may be stationary and the containers A1 and A2 may be moved to move the magnet relative to the containers. The magnet may be moved manually or automatically (moved by a driving source). While a single magnet may be moved, multiple electromagnets may be arranged along the direction in which the microcarriers are moved, and the electromagnets may be operated in sequence in the moving direction to move (shift) the magnetic field as if the magnet were moving, thereby attracting the microcarriers.

[0056] (Cell movement in the present invention) Cells may move as a cell suspension together with a liquid medium or microcarriers, or the cells may move mainly in the liquid medium, or the microcarriers and the cells attached thereto may move mainly in the liquid medium.

[0057] The processes and materials used to produce iPS cells in the production method (I) will be described in detail below.

[0058] (Production method (I) of the present invention) As described above, the production method of induced pluripotent stem cells of the present invention comprises the following steps: Step s1: A step of contacting somatic cells with reprogramming factors in a liquid medium in a first container A1. Step s2: A step of reducing the concentration of reprogramming factors in a liquid medium in the second container A2 to the (n-1)th container A(n-1). Step s3: A step of culturing the somatic cells in a liquid medium in an nth container An to establish iPS cells.

[0059] The culture in production method (I) of the present invention (typically, steps s1 to s4) is suspension culture. As used herein, "suspension culture" refers to culture carried out under conditions that maintain cells or cell aggregates suspended in a culture solution, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell aggregates and the culture vessel.

[0060] As used herein, "induced pluripotent stem cells (iPS cells)" are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells through the introduction of specific factors (reprogramming factors). Induced pluripotent stem cells have the ability to differentiate into various tissues and cells with different morphologies and functions in the body, and into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm).

[0061] Currently, there are various types of induced pluripotent stem cells, including human iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872.), Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) etc. can also be used. In addition, iPS cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), iPS cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), iPS cells produced by Sakurada et al. (JP Patent Publication No. 2008-307007), etc. can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797), or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008 / 2336610 A, US 2009 / 047263 A, WO 2007 / 069666 A, WO 2008 / 118220 A, WO 2008 / 124133 A, WO 2008 / 151058 A, WO 2009 / 006930 A, WO 2009 / 006997 A, WO 2009 / 007852 A) and known in the art can be used.

[0062] As induced pluripotent stem cell lines, various iPSC lines established by the NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPSC lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's AdiPS cells, 253G1 line, 253G4 line, 1201C1 line, 1205D1 line, 1210B2 line, 1383D2 line, 1383D6 line, 201B7 line, 409B2 line, 454E2 line, 585A1 line, 585B2 line, 606A1 line, 610B1 line, 648A1 line, 1231A3 line, and FfI-01s04 line.

[0063] In this specification, induced pluripotent stem cells may be cells derived from a patient with a genetic disease. Cells induced to differentiate from pluripotent stem cells derived from a patient with a genetic disease can serve as disease models that reflect the pathology of the disease, and are therefore suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from a patient with a genetic disease can be genetically repaired by genome editing using a CRISPR-Cas system or the like, and then differentiated into the desired cells, making it possible to use the cells as a therapeutic drug for the disease.

[0064] As used herein, "somatic cells" refers to cells other than germ cells that constitute an animal. Somatic cells are not particularly limited, but include fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells may be, for example, floating cells (e.g., blood cells, etc.) or adhesive cells, with floating cells being preferred. Examples of somatic cells used in the production method of the present invention include, but are not limited to, mesenchymal stem cells derived from skin fibroblasts, etc., skin cells, visual cells, brain cells, hair cells, oral mucosa, lung cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells, kidney cells, neural stem cells, wisdom teeth, etc., tissue stem cells, tissue progenitor cells, blood cells (e.g., hematopoietic stem cells, peripheral blood mononuclear cells (PBMCs) (including T cells and non-T cells), umbilical cord blood cells, etc.), epithelial cells, endothelial cells (e.g., vascular endothelial cells), muscle cells, and the like.

[0065] In one embodiment, when blood cells (e.g., peripheral blood mononuclear cells) are used as somatic cells, the cells are obtained by centrifuging whole blood (density gradient centrifugation). In this case, by using a centrifuge tube equipped with a syringe mechanism and by using a centrifuge that can remove a predetermined fraction obtained by centrifugation while maintaining a closed system, the fraction layer obtained by centrifugation, that is, the layer containing peripheral blood mononuclear cells, can be directly transferred to the first container A1 without contacting the outside air.

[0066] In the present specification, the species of origin of the somatic cells is not particularly limited, and may be, for example, cells from rodents such as rats, mice, hamsters, guinea pigs, etc.; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, sheep, etc.; carnivores such as dogs and cats; tarsiers, long-tailed monkeys, cynomolgus monkeys, rhesus monkeys, Japanese macaques, gibbons, spider monkeys, capuchin monkeys, orangutans, gorillas, chimpanzees, humans (all of which belong to the Platyrhini order); and primates such as lemurs, aye-ayes, and lorises (all of which belong to the Platyrhini order). Humans are preferred as the species of origin of the somatic cells.

[0067] In this specification, unless otherwise specified, the term "cell" includes a "cell population." A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

[0068] As used herein, examples of "reprogramming factors" include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, ESrrb, Nr5a2, Tbx3, and Glis1, and these reprogramming factors may be used alone or in combination.Combinations of reprogramming factors include those described in WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO20 10 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0 68955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 11142 2, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Nat Biotechnol,2008.26.795-797, Cell Stem Cell,2008.2.525-528, Stem Cells,2008.26.2467-2474, Nat Biotechnol,2008.26.1269-1275, Cell Stem Cell,2008.3.568-574, Cell Stem Cell,2008.3.475-479, Cell Stem Cell,2008.3.132-135, Nat Cell Biol,2009.11.197-203, Nat Biotechnol,2009.27.459-461, Proc Natl Acad Sci Examples of combinations include those described in USA, 2009.106.8912-8917, Nature, 2009.461.643-649, Cell Stem Cell, 2009.5.491-503, Cell Stem Cell, 2010.6.167-74, Nature, 2010.463.1096-1100, Stem Cells, 2010.28.713-720, and Nature, 2011.474.225-229.

[0069] Examples of combinations of reprogramming factors include the following: (1) Oct3 / 4, Klf4, Sox2, c-Myc (wherein Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18. Klf4 can be replaced with Klf1, Klf2, or Klf5. Furthermore, c-Myc can be replaced with T58A (active mutant), N-Myc, or L-Myc. L-Myc is particularly preferred for clinical use.) (2) Oct3 / 4, Klf4, Sox2 (3) Oct3 / 4, Klf4, c-Myc (4) Oct3 / 4, Sox2, Nanog, Lin28 (5) Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Lin28 (6) Oct3 / 4, Klf4, Sox2, bFGF (7) Oct3 / 4, Klf4, Sox2, SCF (8) Oct3 / 4, Klf4, c-Myc, Sox2, bFGF (9) Oct3 / 4, Klf4, c-Myc, Sox2, SCF

[0070] In step s1 of the present invention, the reprogramming factor is introduced into the somatic cells by contacting the reprogramming factor with the somatic cells in a liquid medium in a first container A1. The reprogramming factor introduced into the somatic cells may be in the form of a protein, a nucleic acid (RNA or DNA) encoding the protein, or an expression vector containing the nucleic acid. When the reprogramming factor is introduced in the form of RNA, immunogenic RNA introduced into the cells may activate the cellular defense mechanism, so RNA (e.g., E3 mRNA, K3L mRNA, B18 mRNA, or a combination of these mRNAs derived from vaccinia virus) to circumvent the defense mechanism may be introduced into the somatic cells. Furthermore, in order to improve the efficiency of establishing pluripotent stem cells, various miRNAs or mimics thereof (e.g., miR-302a-3p or mimic thereof, miR-302b-3p or mimic thereof, miR-302c-3p or mimic thereof, miR-302d-3p or mimic thereof, miR-367-3p or mimic thereof, combinations of these miRNAs or mimics, etc.) may be introduced into somatic cells.

[0071] When the miRNA described above is introduced into somatic cells, it may be in the form of natural miRNA, miRNA in which the nucleic acid has been chemically modified, miRNA mimic, or DNA encoding the miRNA or an expression vector containing the DNA, but is preferably a miRNA mimic. A miRNA mimic is in the form of double-stranded RNA and typically consists of a guide strand made of natural RNA and a passenger strand chemically modified (e.g., 2'-O methyl modified). The guide strand exhibits RNAi activity, while the passenger strand does not, and therefore mimics natural miRNA in cells.

[0072] Examples of expression vectors include viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, as well as plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors (piggyBac, piggyBat, TolII). Examples of promoters used in expression vectors include the EF1α promoter, ACTB promoter, UbqC promoter, PGK promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (moloney murine leukemia virus) LTR, HIV LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter. On the other hand, in an expression vector containing DNA encoding miRNA, the promoter is preferably a pol III promoter (e.g., SNR6, SNR52, SCR1, RPR1, U3, U6, H1 promoter, etc.).

[0073] RNAs such as mRNA and miRNA can be produced by chemical synthesis or by in vitro transcription (IVT). Alternatively, they can be expressed in an organism (Escherichia coli or cultured mammalian cells) and then purified. Examples of chemical synthesis methods include methods using nucleoside phosphoramidites and solid-phase supports. Furthermore, modified nucleotides can be introduced into any base position in an RNA sequence by using chemically modified nucleoside phosphoramidites (e.g., 2'-O-methylated phosphoramidites of the sugar moiety).

[0074] Nucleic acids (e.g., the above-mentioned mRNA, miRNA, DNA encoding them, etc.), expression vectors containing the nucleic acids, or proteins (e.g., reprogramming factors) can be introduced into cells by various known methods, including calcium phosphate-mediated transfection, electroporation, liposome transfection, lipofection, gene guns, microinjection, viral vector methods, virus-like particle methods, Agrobacterium methods, agroinfiltration methods, PEG-calcium methods, sonoporation methods, and lipid nanoparticle methods.

[0075] The culture in production method (I) of the present invention (typically, steps s1 to s4) may be cultured under feeder-free conditions and / or xeno-free conditions for all or part of the period. From the viewpoint of clinical use, the production method of the present invention is preferably carried out under feeder-free and xeno-free conditions for the entire period. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to prepare the culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms different from the biological species of the cells to be cultured.

[0076] The basal medium used in the production method (I) of the present invention is not particularly limited, but may be, for example, Essential 8 medium (CTS TM Essential 8 TMMedium, Essential 8 TM Medium, Essential 8 TM Flex Medium, Essential 6 TM Examples of suitable media include StemFit® AK02 Medium (Thermo Fisher Scientific), StemFit® AK03 Medium (Ajinomoto Co., Inc.), StemFit® Basic03 Medium, CTS® KnockOut SR XenoFree Medium (Gibco), mTeSR1 Medium, TeSR1 Medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), and Improved MEM (Thermo Fisher Scientific). These media can also be used for culture under feeder-free and xeno-free conditions. In addition, RPMI-1640 medium, Eagle MEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, DMEM, Hybridoma Serum-free medium, KnockOut ＴＭ DMEM, Advanced ＴＭMedium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g., Waymouth's MB752 / 1), CMRL medium (e.g., CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34, MesenPRO RS (all Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (both ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (both Lonza), EX-CELL 302 Medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed ＴＭ Examples of suitable medium include, but are not limited to, iPSC Medium (ReproCell Inc.) and mixtures thereof.

[0077] The basal medium used in the production method (I) of the present invention can be supplemented with physiologically active substances and nutritional factors necessary for cell survival or proliferation, as needed. These additives may be added to the medium in advance or may be added during cell culture. The method for adding additives during culture may be in any form, such as a single solution or a mixed solution of two or more types, and may be added continuously or intermittently.

[0078] Examples of physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.). Examples of nutritional factors include sugars, amino acids, vitamins, hydrolysates, lipids, etc. Examples of sugars include glucose, mannose, fructose, etc., and these may be used alone or in combination of two or more. Amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and may be used alone or in combination of two or more. Vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, and DL-α-tocopherol, and may be used alone or in combination of two or more. Hydrolysates include those obtained by hydrolysis of soybeans, wheat, rice, peas, corn, cottonseed, yeast extract, etc. The lipids include cholesterol, linoleic acid, and linolenic acid.

[0079] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the medium as needed. When an acidic substance such as sialic acid is added to the medium, it is desirable to adjust the pH of the medium to a neutral range suitable for cell growth, that is, pH 5 to 9, preferably pH 6 to 8.

[0080] As used herein, the medium may be a serum-containing medium (e.g., fetal bovine serum (FBS), human serum, or horse serum) or a serum-free medium. From the viewpoint of preventing contamination with components derived from different animal species, it is preferable that the medium does not contain serum, or that serum derived from the same animal species as the cells to be cultured is used. Here, serum-free medium refers to a medium that does not contain unconditioned or unpurified serum. The serum-free medium may contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0081] As used herein, the medium may or may not contain serum substitutes, as well as serum, such as albumin, lipid-rich albumin, and recombinant albumin, plant starch, dextran, protein hydrolysates, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thioglycerol, or equivalents thereof. Specific examples of serum substitutes include those prepared by the method described in WO98 / 30679, commercially available products such as Knockout Serum Replacement [KSR] (Life Technologies), Chemically-defined Lipid Concentrated (Life Technologies), and Glutamax (Life Technologies). Examples of biologically derived factors include platelet-rich plasma (PRP) and culture supernatant components of human mesenchymal stem cells.

[0082] As used herein, the medium may contain a scaffold material (hereinafter also referred to as "scaffold") used in cell suspension culture, and the scaffold material refers to a material or substrate that functions as a scaffold for cells in cell culture. The scaffold material is not particularly limited as long as it can be used for cell suspension culture as described above (in other words, it may be free in the medium), but examples include those containing or made of synthetic resin, and those made of flexible materials such as collagen. Furthermore, as an example, the scaffold material may contain or consist of atelocollagen, specifically atelocollagen molded into a shape suitable for use as a scaffold material. Typically, the scaffold material is a material other than nanofibers.

[0083] The term "synthetic resin" refers to a material whose main component is a polymer (hereinafter simply referred to as "polymer") obtained by polymerizing (including polycondensation) polymerizable monomers (hereinafter simply referred to as "monomers"). The polymer also includes copolymers of one or more polymerizable monomers. Alternatively, the scaffold material may be primarily composed of an inorganic material such as glass or silicone.

[0084] Examples of the polymer include polymers composed of one or more polymerizable monomers of (un)saturated hydrocarbons, aromatic hydrocarbons, (un)saturated fatty acids, aromatic carboxylic acids, (un)saturated ketones, aromatic ketones, (un)saturated alcohols, aromatic alcohols, (un)saturated amines, aromatic amines, (un)saturated thiols, aromatic thiols, and organosilicon compounds.

[0085] Specific examples of the polymer include polystyrene, polyolefin, polyether, polyvinyl alcohol, polyvinyl acetal, polyester, poly(meth)acrylic acid ester, epoxy resin, polyamide, polyimide, polyurethane, polycarbonate, cellulose, dextran, polypeptide (e.g., gelatin, etc.). These polymers may be used alone or in combination of two or more. When two or more polymers are combined, the two or more polymers may be mixed and used, or the skeletons of the two or more polymers may be chemically bonded to each other.

[0086] The scaffold material may be prepared by a known method, or a commercially available product such as Cytodex-1 (GE Healthcare) or Corning® Low Concentration Synthemax® II Microcarrier (Corning).

[0087] Typically, among the above scaffold materials, a scaffold material containing atelocollagen can be prepared by coating all or part of the surface of the scaffold material with atelocollagen to obtain a scaffold material containing the desired atelocollagen. The purity of the atelocollagen used for coating is not particularly limited, but high purity (e.g., 90% or more, more preferably 95% or more, and most preferably 100%) is preferred. In order to improve the adhesiveness between the surface of a free scaffold material such as a microcarrier and cells, the scaffold material may be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), in addition to atelocollagen. Furthermore, it is preferable that the cell support matrix is ​​substantially free of native collagen (e.g., 10% or less, more preferably 5% or less (e.g., 4%, 3%, 2%, 1%, 0%). The cell support matrix can be any material intended for the attachment of stem cells or feeder cells (if used). Examples of such cell support matrices include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin (or a partial structure of laminin), and fibronectin, as well as mixtures thereof, such as Matrigel, and dissolved cell membrane preparations (Lancet, 2005.365.9471.1636-1641). Unless otherwise specified, the term "purity" used herein refers to the mass percent concentration (hereinafter, "mass percent concentration" will be simply referred to as "concentration") as an indicator of high quality (i.e., low level of impurity contamination). However, it can also refer to the concentration of a specific component (e.g., atelocollagen) in a mixture (e.g., a mixture of atelocollagen and another cell support matrix).

[0088] The shape of the scaffold material is not particularly limited, but examples include cylindrical, spheroidal, and spherical shapes, with spherical shapes being preferred. Specific examples of such spherical scaffold materials include microcarriers. The inventors previously discovered that pluripotent stem cells can be grown using microcarriers with diameters of 105 μm or less, 105-250 μm, 250-425 μm, or 425-600 μm in bioreactor culture. The size of the scaffold material is also not particularly limited, but when using spherical scaffolds such as microcarriers, the particle size (diameter) of the scaffold material is typically 50-1000 μm, may be 70-700 μm, and preferably 100-400 μm. From the perspective of cell proliferation rate, a preferred embodiment has a particle size of 600 μm. Particle size can be measured using the Coulter counter method described in International Standard ISO 13319, "Measurement of Particle Size Distribution—Electronic Sensing Zone Method."

[0089] Type I collagen molecules consist of approximately 95% helical (helical) and approximately 5% non-helical (telopeptide) regions. This non-helical region is highly antigenic and is cleaved by proteases (proteolytic enzymes). Atelocollagen, the natural polymeric material containing scaffold materials, is highly purified after digestion and removal of the highly antigenic telopeptide regions with proteases such as pepsin, resulting in extremely low antigenicity (Matrix, 1992, 12, 274-281). On the other hand, collagen present in living organisms is an insoluble fibrous protein with a "triple helix structure" in which three polypeptide chains are wound helically, also known as "native collagen." The origin of the atelocollagen used in the present invention is not limited, and examples include those derived from mammals (e.g., humans, mice, rats, monkeys, cows, horses, pigs, dogs, etc.). To prevent contamination with components derived from different animals, it is preferable to use atelocollagen derived from the same source as the cells to be cultured. Such atelocollagen may be produced by known methods, or commercially available products may be used. For example, atelocollagen can be purified by treating collagen extracted from cells or tissues containing atelocollagen or collagen secreted from cultured cells with protease.

[0090] The concentration of atelocollagen in the scaffold material ((mass of atelocollagen / mass of scaffold material containing atelocollagen) × 100) is not particularly limited, as long as it is a concentration that exhibits a cell death inhibitory effect on cells. Such a concentration can be appropriately determined by those skilled in the art using the methods described in the Examples or conventionally known methods. The concentration of atelocollagen in the scaffold material is, for example, 0.1% or more (e.g., 0.1%, 1%, 3%, 5%, 10%, 20%, 25%, 30% or more) and 100% or less. When the scaffold material is coated with a cell-support matrix containing atelocollagen, the concentration of atelocollagen in the cell-support matrix is ​​90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 97%, 98%, 99%, or 100%). In one aspect of the present invention, the scaffold material is substantially composed of atelocollagen, and "substantially composed of atelocollagen" does not mean that the atelocollagen concentration is 100%, but rather that it is close to 100% (e.g., 95% or more, preferably 95.5% or more (e.g., 96%, 97%, 98%, 99% or 100%)).

[0091] The concentration of atelocollagen in the medium is not particularly limited, and by appropriately adjusting the atelocollagen concentration, it is possible to control the cell proliferation rate. The atelocollagen concentration in the medium is, for example, 0.01 to 20%, preferably 0.05 to 5%, and more preferably 0.1 to 2%. The atelocollagen concentration in the medium is also preferably 0.5% to 20%, 1% to 15%, or 5% to 10%.

[0092] The conditions for culturing (typically, steps s1 to s4) in the production method (I) of the present invention are not particularly limited, but include a temperature of about 30 to 40°C, preferably about 37°C, and CO ２ The culture was carried out under an atmosphere of CO ２ The concentration is preferably about 2 to 5%. For example, the temperature inside each container can be controlled by referring to a conventionally known temperature control method, such as controlling the temperature at room temperature, providing a heater to each container, or controlling the temperature of the material supplied into the container.

[0093] The cell culture density in the culture (typically, steps s1 to s4) in the production method (I) of the present invention is not particularly limited as long as the cells can grow. ２ ~1.0 x 10 ７ cells / cm ２ , preferably 1.0×10 ３ ~1.0 x 10 ６ cells / cm ２ , more preferably 1.0 × 10 ４ ~1.0 x 10 ５ cells / cm ２ is.

[0094] The period for which step s1 is performed in production method (I) of the present invention is not particularly limited, as long as iPS cells are established in step s3. Typical periods include, for example, 30 minutes to 24 hours, 2 hours to 6 hours, etc.

[0095] In step s2 of the production method (I) of the present invention, the method for reducing the concentration of reprogramming factors in the liquid medium is not particularly limited, and can be achieved by replacing the medium used in step s1 with a medium containing no added reprogramming factors. The medium replacement may involve replacing all or part of the medium used in step s1, as long as the concentration of the reprogramming factors in the medium is reduced by dilution, thereby reducing or eliminating the contact (frequency) between the somatic cells and the reprogramming factors. As used herein, "reducing the concentration of reprogramming factors" may also include a state in which no reprogramming factors are present in the medium. Typically, the concentration of reprogramming factors in the medium in step s2 is, for example, 1 / 10 or less, preferably 1 / 100 or less, more preferably 1 / 300 or less, even more preferably 1 / 1000 or less, and even more preferably 1 / 3000 or less, compared to that in step s1. Furthermore, steps s2 to s3 may proceed as long as the reprogramming factors in the medium are reduced to the desired concentration as described above.

[0096] Step s3 in production method (I) of the present invention is a step of culturing the somatic cells in a liquid medium to establish iPS cells. The establishment of induced pluripotent stem cells can be appropriately confirmed by the expression of reprogramming factors introduced by methods known per se (e.g., Oct3 / 4, SOX2, Nanog, TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase, etc.). The period for performing step s3 in production method (I) of the present invention is not particularly limited as long as iPS cells are established, but is typically, for example, 10 days or more, preferably 14 days or more. There is also no particular upper limit, but it is typically 40 days or less, preferably 30 days or less.

[0097] The materials supplied from each material supply source (G1 to Gn) to each vessel are the materials necessary for carrying out each of the above-mentioned steps (e.g., somatic cells, culture medium, reprogramming factors, etc.). For the materials themselves necessary for obtaining iPS cells from somatic cells other than those mentioned above, reference can be made to the prior art. O necessary for cell culture ２ YaCO ２ However, if the wall of the container is gas permeable, the supply of the gas can be omitted. The same applies to the other containers described below.

[0098] From the viewpoint of the yield of iPS cells, the production method (I) preferably further comprises, after the step s3 of establishing iPS cells, a step s4 of expanding the iPS cells, the number of times of expansion is p (p is an integer of 1 or greater, i.e., p≧1).

[0099] The expansion culture step s4 will be described in detail below. The configurations of the vessel, inlet / outlet port, connecting pipeline, and feed mechanism used in the expansion culture step s4 can be referenced from the configurations shown in the explanation of steps s1 to s3 above, and therefore will not be described here.

[0100] In step s4, part or all of the contents (i.e., the medium containing iPS cells) in the n-th sealed container (An) established in step s3 are transferred to container B1 and cultured until the desired cell density is reached. When expansion culture is continued through steps s5 and s6, the contents transferred at each step are the same as those from steps s3 to s4.

[0101] As shown in an example in Figure 8, in step s4 of expanding iPS cells, p containers (B1 to Bp) corresponding to the number of expansion cultures p are connected in series via connecting pipelines after the nth container An in which step s3 is performed. The necessary material supply sources (Gb1 to Gbp) are connected to the input / output ports of each container (B1 to Bp) so that the process associated with each container can be performed. While only one material supply source is shown connected to each container in the figure, the number is not limited. In Figure 8, input / output ports that do not reach the liquid level are not shown. From the last container (An) for step s3 to the last container (Bp) for step s4, iPS cells are sequentially moved in one direction by the feed mechanisms Fn and Fb1 to Fb(p-1), respectively, and one expansion culture is performed in each of the containers (B1 to Bp). Thus, a total of p expansion cultures are performed, completing step s4 in container Bp, yielding the desired number of iPS cells.

[0102] (Number of times of expansion culture p) In step s4, the number of times of expansion culture is performed is not particularly limited as long as the desired number of iPS cells can be obtained, but in a typical processing operation, it is preferably about 1 to 5 times, more preferably about 2 to 5 times. The number of times of expansion culture p (= the number of containers) in production method (I) is typically preferably about 1 to 5 times, more preferably about 2 to 5 times.

[0103] The period for performing step s4 in production method (I) of the present invention is not particularly limited as long as the desired number of iPS cells can be obtained, but is typically, for example, 1 to 40 days, 3 to 20 days, or 5 to 10 days.

[0104] In the expansion culture, the materials supplied to each vessel from each material supply source (Gb1 to Gbp) are those necessary for the expansion culture, and are mainly the medium. ２ YaCO ２ However, if the wall of the container is gas permeable, the supply of the gas can be omitted.

[0105] 2. Method for Producing Differentiated Cells Next, the method for producing differentiated cells according to the present invention (hereinafter also referred to as production method (II)) will be described in detail with reference to an example of the configuration of a cell production apparatus according to the present invention. The description of each part of the apparatus also includes the description of each part of the cell production apparatus described below.

[0106] In one embodiment, the production method (II) comprises steps s1 to s3 in the above-described production method (I) and step s5 of inducing differentiation of iPS cells. In a preferred embodiment of the production method (II), step s4 of expansion culture is added after steps s1 to s3, and thus production method (II) comprises steps s1 to s4 and step s5 of inducing differentiation of iPS cells.

[0107] Furthermore, when differentiated cells are produced using iPS cells that have already been prepared, production method (II) may be an independent cell production method that does not have production method (I) as a preceding step. In this case, if there are multiple differentiation induction steps s6 and multiple containers associated with each step, this corresponds to the cell production method of the present invention in which "multiple cell production steps are divided into multiple containers, cells are moved from container to container in the order of the steps, and the steps associated with each container are thereby carried out sequentially."

[0108] FIG. 9 is a block diagram illustrating the manufacturing method (II). As shown in FIG. 9(a), in manufacturing method (II), a container C1 for performing step s5 is connected via a connecting pipeline after the last container X1 used in manufacturing method (I). Container X1 may be the last container An in steps s1 to s3 in FIG. 1 or the last container Bp in step s4 in FIG. 8. Container C1 has one or more openable / closeable input / output ports, through which materials necessary for differentiation induction in step s5 are supplied to the interior of container C1 (external material supply sources are not shown). In FIG. 9, input / output ports that do not reach the liquid surface are not shown. iPS cells are transferred from container X1 to container C1 by a feed mechanism Fx1, and step s5 is performed in container C1.

[0109] The configurations of the containers, input / output ports, connecting pipelines, and feed mechanisms used in the differentiation induction step s5 (including steps s5a and s5b described below) can be referenced from the configurations shown in the explanation of steps s1 to s4 above, and will not be explained here.

[0110] The materials supplied to each vessel are those necessary for differentiation induction. For the materials necessary for differentiation induction, reference can be made to the prior art, including those described below. ２ YaCO ２ However, if the wall of the container is gas permeable, the supply of the gas can be omitted.

[0111] (Differentiated cells) In the production method (II) of the present invention, induced pluripotent stem cells can be induced to differentiate to produce desired cells or organoids. The cells obtained may be undifferentiated cells such as stem cells or progenitor cells, or may be terminally differentiated cells. In this specification, the undifferentiated cells to be removed in the production method (II) of the present invention refer to cells other than the stem cells and progenitor cells intended to be produced by the production method (II) of the present invention. Hereinafter, the term "differentiated cells" may be used as a term that encompasses both undifferentiated cells and terminally differentiated cells that can be produced by the production method (II) of the present invention. They may also be germ cells. In this specification, "undifferentiated cells" refers to cells that have not reached terminal differentiation in a cell lineage, and examples of undifferentiated cells include stem cells excluding pluripotent stem cells, progenitor cells, and the like. Examples of stem or progenitor cells include epiblast-like cells, primordial germ cells (also referred to as "primordial germ cell-like cells"), germline stem cells, ectodermal (row) cells such as neural crest cells, neural stem cells, neural progenitor cells, glial progenitor cells, retinal stem cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary stem cells, mesodermal (row) cells such as hematopoietic progenitor cells, myeloid stem cells, lymphoid stem cells, B progenitor cells, T progenitor cells, mesenchymal stem cells, cardiac stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, vascular pericytes, platelet progenitor cells (e.g., megakaryocyte progenitor cells, megakaryoblasts, promegakaryocytes, mature megakaryocytes, etc.), skeletal muscle stem cells, adipose stem cells, kidney progenitor cells, endodermal (row) cells such as hepatic stem cells, liver progenitor cells (e.g., hepatoblasts, hepatic progenitor cells, hepatic stellate cell progenitor cells, hepatic stem cell progenitor cells, etc.), intestinal stem cells, and airway stem cells.

[0112] As used herein, the term "terminally differentiated cells" refers to cells that have reached terminal differentiation in a cell lineage. Examples of terminally differentiated cells include, but are not limited to, osteoblasts, chondrocytes, adipocytes, hepatocytes, hepatic mesothelial cells, bile duct epithelial cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, Kupffer cells, pit cells, vascular endothelial cells, blood cells (e.g., erythrocytes, platelets, leukocytes, mast cells, dendritic cells, etc.), pancreatic duct epithelial cells, pancreatic duct cells, acinar centrocells, acinar cells, Langevin cells, and the like. Examples of cells include islets of Luhans, cardiac muscle cells, fibroblasts, smooth muscle cells, type I alveolar epithelial cells, type II alveolar epithelial cells, Clara cells, ciliated epithelial cells, basal cells, goblet cells, neuroendocrine cells, Kruczykki cells, renal tubular epithelial cells, urothelial cells, columnar epithelial cells, glomerular epithelial cells, glomerular endothelial cells, octopus podocytes, mesangial cells, nerve cells, glial cells (e.g., astrocytes, microglia, oligodendrocytes, ependymal cells, Schwann cells, etc.), etc. Examples of white blood cells include lymphocytes (e.g., B cells, T cells, NK cells, etc.), granulocytes (e.g., neutrophils, eosinophils, basophils, etc.), monocytes, etc.

[0113] In one embodiment of the present invention, the cells or organoids (target cells or organoids) obtained by production method (II) of the present invention are neural crest cells, neural progenitor cells, neurons, cerebral cortical organoids, hematopoietic progenitor cells, platelets, T cells, epiblast-like cells, primordial germ cells, or cardiomyocytes. They may also be inner enamel epithelium, ameloblasts, stratum intermedium cells, stellate reticulum cells, outer enamel epithelium, dental papilla cells, or odontoblasts.

[0114] Furthermore, as used herein, the term "organoid" refers to a structure formed by the accumulation of cells, and typically has a structure and function similar to that of an organ in a living body. Organoids obtainable by the differentiation induction method of the present invention include, for example, neural organoids (e.g., cerebral cortex organoids, cerebellar organoids, spinal cord organoids, midbrain organoids, choroid plexus organoids, hippocampal organoids, hypothalamic organoids, anterior pituitary organoids and basal ganglia organoids, etc.), lung organoids, liver organoids, respiratory epithelial organoids, intestinal organoids, pancreatic organoids, kidney organoids, respiratory tract organoids, stomach organoids, thyroid organoids, thymus organoids, testicular organoids, esophageal organoids, skin organoids, fallopian tube organoids, ovarian organoids, salivary gland organoids, optic vesicle organoids, optic cup organoids, bladder organoids, prostate organoids, cartilage organoids, cardiac organoids, bone tissue organoids, muscle tissue organoids, cancer organoids, and the like. In one embodiment, the organoids are neural organoids such as cerebral cortex organoids.

[0115] Whether a structure is an organoid can be confirmed, for example, by microscopic observation to confirm the presence or absence of layer structure formation or by examining the expression of marker proteins. Specifically, in the case of cerebral cortical organoids, a dome-shaped neuroepithelium in which Foxg1 is expressed throughout is observed, and the neuroepithelium forms a layer structure. In this structure, a layer of neural progenitor cells corresponding to the Pax6- and Sox2-positive ventricular zone is observed inside the epithelium, and on the outside thereof, a first layer consisting of Reelin / Carletinin-positive Cajal-Retzius cells and a Ctip2 / Tbr1-positive deep layer are observed. In addition, markers for liver organoids include HHEX, SOX2, HNF4A, AFP, and ALB; for pancreatic organoids, markers include PDX1, SOX17, and SOX9; for organoids that differentiate into the intestine, markers include CDX2 and SOX9; and for kidney organoids, markers include Pax2 and Six2.

[0116] Known methods can be used to induce differentiation to obtain cells or organoids of interest. For example, differentiation of pluripotent stem cells into neural crest cells can be performed by the methods described in Fukuta M. et al., PLoS One, 2014, 9(12): e112291 or Kamiya D, et al., NPJ Regen Med., 2022 Sep 15;7(1):47. Specifically, pluripotent stem cells can be seeded in a culture vessel and subjected to adhesion culture (suspension culture using a scaffold material), followed by adhesion culture (suspension culture using a scaffold material) in a medium containing a TGFβ inhibitor and a GSK3β inhibitor, thereby differentiating them into neural crest cells.

[0117] It is also possible to produce cells such as mesenchymal stem cells, neural progenitor cells, neurons, glial cells, bone cells, chondrocytes, corneal cells, and melanocytes from neural crest cells. For example, differentiation into these cells can be performed based on the methods described in Fukuta M. et al., PLoS One, 2014, 9(12): e112291, Horikiri T. et al., PLoS One, 2017, 12(1): e0170342, and Kamiya D, et al., NPJ Regen Med., 2022 Sep 15;7(1):47. Specifically, for example, neural crest cells are seeded on a fibronectin-coated plate, and the medium is replaced with DMEM / F12 supplemented with N-2 Supplement, BDNF, GDNF, NT-3, and NGF, and then incubated at 37°C and 5% CO ２ Alternatively, neural crest cells can be plated and cultured in CDM medium containing SB431542 and CHIR99021 for 1 day, after which the medium is replaced with neurobasal medium supplemented with B-27 supplement, N-2 supplement, L-glutamine, penicillin / streptomycin, BDNF, GDNF, NT-3, and NGF, and the medium is incubated at 37°C in 5% CO. ２ By culturing the cells under the conditions described above for about 35 days, neural progenitor cells and neural cells can be obtained.

[0118] Differentiation into mesenchymal stromal cells can be induced, for example, by the following method: Neural crest cells are seeded in a culture vessel and cultured for one day in CDM medium containing SB431542 and CHIR99021. After one day, the medium is replaced with αMEM containing FBS. Mesenchymal stromal cells can be obtained approximately 14 days after the start of differentiation induction.

[0119] Methods for differentiating pluripotent stem cells into T cells include, for example, methods comprising (1) differentiating pluripotent stem cells into hematopoietic progenitor cells, and (2) differentiating the hematopoietic progenitor cells into T cells. Step (1) can be, for example, culturing pluripotent stem cells in a hematopoietic progenitor cell induction medium, as described in WO 2013 / 075222, WO 2016 / 076415, Liu S. et al., Cytotherapy, 17 (2015); 344-358, etc. Step (2) can be, for example, (2-1) inducing CD4 / CD8 bipositive T cells from hematopoietic progenitor cells, or (2-2) inducing CD8 / positive T cells from CD4 / CD8 bipositive T cells, as described in WO 2016 / 076415, etc.

[0120] Examples of the step of inducing differentiation of pluripotent stem cells into platelets include (1) a method comprising a step of differentiating pluripotent stem cells into hematopoietic progenitor cells and a step of differentiating hematopoietic progenitor cells into platelets. Step (2) can be, for example, a step of culturing hematopoietic progenitor cells in a medium containing TPO and / or SCF for approximately 7 to 15 days, as described in WO 2012 / 157586, US 2014 / 127815, Nakamura, Eto, et al. Cell Stem Cell 14, 535-548 (2014), etc. This step can yield a cell population containing megakaryocytes and platelets.

[0121] As described in WO 2017 / 002888, for example, the step of inducing differentiation of pluripotent stem cells into primordial germ cells includes the steps of: (1) culturing pluripotent stem cells in a culture medium containing activin A and a GSK3β inhibitor for approximately 40 to 60 hours to differentiate them into epiblast-like cells; and (2) culturing the epiblast-like cells in a culture medium containing BMP for approximately 4 to 8 days to differentiate them into primordial germ cells (primordial germ cell-like cells).

[0122] Examples of methods for inducing differentiation of pluripotent stem cells into cardiomyocytes include those described in WO2015 / 141827, Laflamme MA and Murry CE, Nature. 473(7347):326-35 (2011), etc. Other methods include, for example, a method for producing cardiomyocytes by forming embryoid bodies through suspension culture of induced pluripotent stem cells, a method for producing cardiomyocytes in the presence of a substance that suppresses BMP signaling (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), and a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical (classical) Wnt signaling pathway (WO2007 / 126077). Typically, for example, marker proteins for cardiomyocytes include NKX2.5 (a cardiac muscle-specific transcription factor) and TNNT2 (troponin T), and marker proteins for cardiac progenitor cells include KDR (a receptor for vascular endothelial growth factor (VEGF)) and ISL1 (a LIM homeodomain transcription factor).

[0123] In addition, organoid can be produced by using multiple kinds of cells.For example, in the case of hepatic organoid, as described in WO2013 / 047639 etc., hepatic progenitor cells (organ cells), mesenchymal stem cells and vascular endothelial cells are induced from pluripotent stem cells, and these mixtures are subjected to suspension culture, thereby hepatic organoid can be produced.

[0124] In the production method (II) of the present invention, the culture may be performed under feeder-free conditions and / or xeno-free conditions for all or part of the period. From the viewpoint of clinical use, the differentiation induction method of the present invention is preferably performed under feeder-free and xeno-free conditions for the entire period.

[0125] In another aspect, there is provided a cell or organoid obtained by the production method (II) of the present invention (hereinafter, also referred to as "the differentiated cell or organoid of the present invention").

[0126] The manufacturing method (II) of the present invention may also include a step of recovering the obtained target cells or organoids. The recovered cells may be cryopreserved using a cell cryopreservation solution. In addition, the cells recovered in the container may be subjected to cell counting using a cell counter, or may be labeled with an antibody against a cell surface marker and purified by flow cytometry, mass cytometry, magnetic cell separation, etc.

[0127] In the differentiation induction method of the present invention, specific examples and definitions of the cells used, the culture method and culture conditions including the culture period and type of medium used, etc. are all incorporated by reference in the above section "1. Method for producing induced pluripotent stem cells."

[0128] In a preferred embodiment of production method (II), step s5 shown in Figure 9(a) may be step s5a of inducing differentiation of iPS cells into ectodermal cells, mesodermal cells, or endodermal cells. In another preferred embodiment of production method (II), step s5a may be followed by step s5b of further differentiation induction, as shown in Figure 9(b). Step s5b is a step of inducing differentiation of the ectodermal cells, etc. obtained in step s5a to obtain further other cells.

[0129] As shown in Figure 9(b), container C2 for carrying out a further differentiation induction step s5b is connected via a connecting pipeline Jc1 after container C1 in which step s5a is carried out. Container C2 has one or more openable / closeable input / output ports (reference numerals omitted). Materials necessary for differentiation induction in step s5b are supplied into container C2 through the input / output ports. Cells (the ectodermal, mesodermal, or endodermal cells) are transferred from container C1 to container C2 by a feed mechanism Fc1, and step s5b is carried out in container C2.

[0130] After step s5b, a further differentiation induction step may be added as necessary, and in that case, the containers associated with the added step are connected in the same manner as described above.

[0131] (Removal of Undifferentiated Cells) In a preferred embodiment of the production method (II), step s5 is followed by step s6 of removing undifferentiated cells, and a container D1 associated with step s6 is connected in the same manner as described above. As shown in FIG. 10 , a container D1 for performing step s6 is further connected via a connecting pipeline Jx2 after the last container X2 (e.g., container C1 or C2) among the containers used in the differentiation induction step s5 (including steps s5a and s5b). Differentiated cells are transferred from container X2 to container D1 by a feed mechanism Fx2. Container D1 has one or more openable input / output ports, and materials necessary for step s6 are supplied into container D1 through the input / output ports, and step s6 is performed. As a result, differentiated cells remain in container D1 from which undifferentiated cells have been removed.

[0132] The configurations of the container, inlet / outlet port, connecting pipeline, and feed mechanism used in step s6 can be referenced from the configurations shown in the explanation of steps s1 to s5 above.

[0133] (Method for Removing Undifferentiated Cells) In the production method (II) of the present invention, the method for removing undifferentiated cells is not particularly limited as long as it can remove cells other than cells produced by the production method, and can be performed by adding a known agent for removing undifferentiated cells to the medium (e.g., Di Mao., et al., Angewandte Chemie International Edition; 9 January 2017; Ben-David, U., et al., Cell Stem Cell, 12, 167 (2013); WO2019 / 187918; JP 2016-93178 A; Yoshiki Nakashima, et al., Molecular Therapy Vol. 26 No. 7 July 2018, etc.).

[0134] (Quality Inspection) In a preferred embodiment of the production method (II), step s6 is followed by step s7 of removing a sample for quality inspection of the differentiated cells, and a container E1 associated with step s7 is connected in the same manner as described above. As shown in FIG. 10 , a container E1 for performing step s7 is further connected to the container D1 via a connecting pipeline Jd1. The container E1 has one or more openable and closable input / output ports. A feed mechanism Fd1 moves the contents from the container D1 to the container E1, and the sample required for inspection is removed to the outside through the input / output port of the container E1. The sample for quality inspection may also be removed from the container D1 after step s6.

[0135] The configurations of the container, the inlet / outlet port, the connecting pipeline, and the feed mechanism used in step s7 can be referenced from the configurations shown in the explanation of steps s1 to s6 above.

[0136] The purpose of the quality test is to confirm whether the cells, organoids, etc. produced by the production method (II) of the present invention are the desired ones. The test items for the quality test are not particularly limited, but include basic tests such as the morphology of the cells or organoids, the presence or absence of expression of cell surface markers, sterility tests, endotoxin tests, and evaluation of cell viability, and various test devices are available for each test item.

[0137] (Provision of differentiated cells to user) After step s7 is completed and the cells are certified as non-defective, the connecting conduits and piping connected to container E1 are removed, and container E1 and the differentiated cells (in the form of a cell suspension) therein may be provided (shipped, etc.) as is to a user (such as a patient undergoing cell therapy), or they may be transferred to a syringe-type container, vial, etc. and provided to the user. Alternatively, the differentiated cells may be provided to the user with a syringe connected to the inlet / outlet port of container E1 for removing the differentiated cells. Alternatively, the differentiated cells may be provided as a medicine, etc., as described below.

[0138] 3. Uses of Differentiated Cells or Organoids The differentiated cells or organoids of the present invention can be suitably used in immunotherapy and regenerative medicine. Therefore, in another aspect, a pharmaceutical comprising the differentiated cells or organoids of the present invention (hereinafter, sometimes referred to as the "pharmaceutical of the present invention") is provided. The pharmaceutical of the present invention is provided, for example, in the form of an immunotherapeutic agent or a cell transplant agent. The present invention also encompasses a method for treating a disease in which an effective amount of the differentiated cells or organoids of the present invention is administered or transplanted into a primate to be treated. Specific examples of primates are as described above in "1. Method for producing pluripotent stem cells," but preferably humans.

[0139] The differentiated cells or organoids of the present invention can be administered or transplanted into the body of a subject in need thereof. Transplantation is preferably performed in a region of the body where the cells can be fixed at a fixed position, such as subcutaneously, intraperitoneally, into the peritoneal epithelium, omentum, adipose tissue, muscle tissue, or under the capsule of various organs such as the pancreas and kidney. Subcutaneous transplantation, which is less invasive, is preferred. The cells to be transplanted should be administered in a therapeutically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the recipient, and are not particularly limited, but may be, for example, 10 x 10 ４ Cell ~10×10 １１ It can be as small as a cell.

[0140] When the differentiated cells or organoids of the present invention are used as pharmaceuticals, it is desirable to use cells or organoids derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to prevent rejection. Here, "substantially the same" means that the HLA genotype is identical to that of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, for example, somatic cells with an HLA type that matches the three loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C. If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers such as polyethylene glycol or silicone.

[0141] The differentiated cell or organoid of the present invention can be prepared as parenteral preparations such as injections, suspensions, infusions, etc., by mixing with pharmaceutically acceptable carriers according to conventional methods.Therefore, in one aspect, there is also provided a method for preparing immunotherapeutic agents or cell transplantation therapeutic agents, which comprises the step of formulating the differentiated cell or organoid of the present invention.This method can also comprise the step of preparing the differentiated cell or organoid of the present invention.Furthermore, it can also comprise the step of preserving the differentiated cell or organoid of the present invention.

[0142] Pharmaceutically acceptable carriers that can be contained in such parenteral formulations include, for example, aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The cells of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0143] The immunotherapeutic agent or cell transplantation therapeutic agent of the present invention is provided in a frozen state under conditions typically used for cryopreserving cells, and can be thawed at the time of use. In such cases, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0144] The differentiated cells or organoids of the present invention can also be used in methods for screening candidate drugs that are useful in treating or preventing disease.

[0145] 4. Cell Manufacturing Apparatus Next, the configuration of the cell manufacturing apparatus (hereinafter also referred to as the apparatus) according to the present invention will be described. The overall features of the apparatus and the configurations of each vessel, input / output port, connecting pipeline, and feed mechanism have been described in detail in the explanation of manufacturing methods (I) and (II) above, and therefore detailed description will be omitted here.

[0146] The device can be divided into a section that produces iPS cells (hereinafter also referred to as the iPS cell production section), a section that expands and cultures iPS cells (hereinafter also referred to as the expansion and culture section), a section that induces differentiation of iPS cells to form differentiated cells (hereinafter also referred to as the differentiation induction section), a section that removes undifferentiated cells (hereinafter also referred to as the undifferentiated cell removal section), and a section that examines differentiated cells (hereinafter also referred to as the examination section).

[0147] (iPS Cell Production Unit) The iPS cell production unit in the device is the part that performs the step in the above-mentioned production method (I) when n = 3. As shown in Figure 11, the device has, as its iPS cell production unit, a container A1 for performing the above-mentioned step s1, a container A2 for performing the above-mentioned step s2, and a container A3 for performing the above-mentioned step s3. The containers A1 to A3 are connected in series in the order of the steps via connecting pipelines J1 and J2, respectively, or are capable of being connected in series in the order of the steps. Each container has a feed mechanism F1 that moves the contents to the next container through connecting pipelines J1 and J2 that are switched to a communicating state. Each material supply source G1 to G3 is selected appropriately depending on the step to be performed in the respective container.

[0148] The steps s1 to s3 are carried out in order using the iPS cell production unit, and the procedure for producing iPS cells from somatic cells is as described in the production method (I) above.

[0149] (Number of Containers) In the above-described production method (I), step s2, which reduces the concentration of the reprogramming factor in the liquid medium, may be divided into multiple processing steps (multiple steps), in which case multiple containers A2 to A(n-1) can be used for step s2. In contrast, in one preferred embodiment of the apparatus, step s2 is a single step, and a single container A2 is provided for performing step s2. However, in the apparatus, step s2 may also be divided into multiple processing steps (multiple steps), in which case multiple containers A2 to A(n-1) can be added and inserted as many times as necessary for step s2, similar to the configuration shown in FIG. 1 . Even if an additional container is inserted for step s2, or even if another container for an additional step is inserted between the series-connected containers A1 to A3, the containers A1 to A3 are still considered to be connected in series.

[0150] (Expansion Culture Section) The expansion culture section in the device is a section for carrying out step s4 in the above-mentioned production method (I). As shown in Figure 8, the device further has p containers B1 to Bp as expansion culture sections for carrying out step s4 of expanding iPS cells p times in a liquid medium. Here, p is an integer of 1 or greater, i.e., p ≥ 1. Each of the containers B1 to Bp has one or more openable and closable inlet / outlet ports.

[0151] The connection of the containers when the number of expansion cultures (p) is one and when it is two or more is as follows: (i) In the example of FIG. 8 , when the number of expansion cultures (p) is one, only container B1 is used. Container B1 is connected to or connectable to container A3 via a connecting pipeline Jn. The device has a feed mechanism Fn that moves the contents of container A3 to container B1 through the connecting pipeline Jn that has been switched to a communicating state. (ii) In the example of FIG. 8 , when the number of expansion cultures (p) is two or more, two or more containers B1 to Bp are used. Container B1 is connected to or connectable to container A3 via a connecting pipeline. The sealed containers B1 to Bp are connected in series or connectable to container A3, respectively, via connecting pipelines Jb1 to Jb(p-1), in the order of step s4. The device has a connecting pipe line Jn, which is switched to a communicating state, and a feed mechanism Fn, which moves the contents of container A3 sequentially to containers B1 to Bp through Jb1 to Jb(p-1), respectively.

[0152] The procedure for carrying out step s4 in the expansion culture section is as described in the above production method (I). The iPS cells may be those obtained by carrying out steps s1 to s3, or may be iPS cells that have already been prepared (for example, commercially available).

[0153] (Differentiation Induction Section) The differentiation induction section of the device is a section for carrying out step s5 in the above-mentioned manufacturing method (II). In the explanation of the above-mentioned manufacturing method (II), an example using containers C1 and C2 was specifically shown with reference to FIG. 9, but the differentiation induction section of the device further uses q sealed containers C1 to Cq for carrying out step s5, as shown in FIG. 12. Here, q is an integer of 1 or greater, i.e., q≧1. Each of the containers C1 to Cq has one or more openable / closable inlet / outlet ports.

[0154] The connection of each container is as follows when the number of containers for forming differentiated cells (i.e., the number of steps for sequentially inducing differentiation) is one or two or more times. (i) In the example of Figure 12, if the q containers are one container C1, container C1 is connected or connectable to container A3 (or to the last container Bp among containers B1 to Bp) via connecting pipeline Jn (or Jbp). The device has a feed mechanism Fn (or) Fbn that moves the contents of container A3 (or container Bp) to container C1 through connecting pipeline Jn (or Jbp) that has been switched to a communicating state. (ii) In the example of Figure 12, if the q containers are two or more containers C1 to Cq, containers C1 to Cq are connected or connectable in series in the order of step s5 via connecting pipelines. Container C1 is connected or connectable to container A3 (or the last container Bp among containers B1 to Bp) via connecting pipeline Jn (or Jbp). The differentiation induction unit of the device has a feed mechanism Fn (or Fbp) and feed mechanisms Jc1 to Jc(q-1) that move the contents of container A3 (or the contents of container Bp) to containers C1 to Cq in order via connecting pipeline Jn (or Jbp) that has been switched to a communicating state and via Jc1 to Jc(q-1).

[0155] (Number of containers q) In the differentiation induction section of the device, the number of containers (= number of differentiation induction stages) q is not particularly limited, but considering the number of differentiation induction stages to obtain the desired differentiated cells from iPS cells, a useful number is approximately 1 to 6 (for example, approximately 3 to 6 in the case of autologous transplantation).

[0156] The operation of carrying out step s5 in the differentiation induction section of the device is as explained in the above production method (II).

[0157] When differentiation is induced using already prepared (e.g., commercially available) iPS cells, the differentiation induction unit may be an independent cell manufacturing device, and there may be no iPS cell production unit or expansion culture unit in the preceding stage. In this case, if there are multiple differentiation induction steps and multiple containers associated with each step, this corresponds to the cell manufacturing device of the present invention, in which the multiple cell production steps are divided into multiple containers, and cells are moved from container to container through connecting pipelines in the order of the steps, thereby sequentially carrying out the steps associated with each container.

[0158] (Undifferentiated Cell Removal Section) The undifferentiated cell removal section in the device is the section that carries out step 6 in the manufacturing method (II). As shown in FIG. 10, the device further includes a container D1 as an undifferentiated cell removal section for carrying out step s6. In the container D1, undifferentiated cells are removed from the contents of the last container Cq (X2 in FIG. 10) among the containers C1 to Cq in the differentiation induction section (step s5). The container D1 has one or more openable / closable inlet / outlet ports. The container D1 is connected to or connectable to the last container Cq (container X2 in FIG. 10) via a connecting pipeline (Jx2 in FIG. 10). The undifferentiated cell removal section of the device includes a feed mechanism (Fx2 in FIG. 10) that moves the contents of the container Cq to the container D1 through the connecting pipeline Jx2 that has been switched to a communicating state.

[0159] The procedure for carrying out step s6 in the undifferentiated cell removal section is as explained in the above production method (II).

[0160] (Testing Unit) The testing unit in the device is the part that performs step s7 in the manufacturing method (II). As shown in FIG. 10, the device further includes a container E1 as the testing unit. The container E1 is a container for extracting a sample for testing the differentiated cells obtained in step s6. The container E1 has one or more openable and closable input / output ports. The container E1 is connected to or connectable to the container D1 via a connecting pipeline Jd1. The testing unit of the device includes a feed mechanism Fd1 that moves the contents of the container D1 to the container E1 through the connecting pipeline Jd1 that has been switched to a communicating state.

[0161] The operation for carrying out step s7 in the inspection section is as explained in the above manufacturing method (II).

[0162] (Mode in which containers are arranged on a substrate) FIG. 13 is a diagram showing an example of a preferred mode of the device. In the example of FIG. 13, the device further includes a substrate for arranging containers used in the device. As shown in FIG. 13, containers necessary for carrying out the steps are arranged on the substrate Y10, and each container is fixed to the substrate Y10. In the example of FIG. 13, a total of 10 containers (A1-A3, B1, B2, C1-C3, D1, E1) are fixed to the substrate surface in the order of the steps. In the example of the same figure, syringe G1, which is a material supply source for supplying somatic cells, is connected to the input / output port 111 of container A1, and a syringe for removing differentiated cells, the final product, is connected to the input / output port of container E1. Each container has an openable / closable input / output port (e.g., the portions indicated by symbols 111, 112, and 113). Furthermore, each container is connected or connectable in the order of the above steps via the connecting pipeline (e.g., the portion designated by the symbol J1) that can be switched between a communicating state and a non-communicating state. Cells move sequentially in the direction of the arrows in the figure, and undergo processing corresponding to each container in each container. For the sake of explanation, Figure 13 does not show the mechanism that switches the connecting pipeline between a communicating state and a non-communicating state, or the mechanism that sets the connecting pipeline to a connectable state. Also, the above-mentioned feeding mechanism that moves the contents of a container to the next container through the connecting pipeline that has been switched to the communicating state is not shown.

[0163] The configuration in which the containers are arranged on a substrate makes handling easier, even when a large number of containers are used. Furthermore, because the containers are arranged on the substrate in the order of the processes, confusion among multiple tubes is suppressed. Furthermore, arranging the containers on a substrate is also suitable for visually checking the manufacturing process and obtaining position information, and serves as a marker for workers to recognize and identify the work process. Furthermore, the addition of position information makes it easier to process information for manufacturing process management using electronic data, making this a preferable configuration.

[0164] The material of the substrate is not particularly limited, and examples include hard materials such as metals and plastics, and flexible plastic materials. Preferably, the material does not generate dust, fine particles, volatile gases, etc., and can be wiped clean with alcohol.

[0165] (Foldable Substrate) As illustrated in FIG. 13 , in a preferred embodiment of the device, the substrate Y10 is foldable in two around the folding center line Y11. In the example of FIG. 13 , the outer peripheral shape of the substrate Y10 is symmetrical about the folding center line Y11. In one region e1 of the two regions e1 and e2 on the substrate surface separated by the folding center line Y11, a predetermined number of the containers (in the example of FIG. 13 , containers (A1, A2, A3, B1, B2)) are arranged in order along the folding center line Y11 in one direction d1. In the other region e2 of the two regions e1 and e2 on the substrate surface separated by the folding center line Y11, the remaining containers (in the example of FIG. 13 , containers (C1, C2, C3, D1, E1)) are arranged in order along the folding center line Y11 in a direction d2 opposite to the direction d1. The last container among the containers in one area e1 (in the example of Figure 13, container B2) and the first container among the containers in the other area e2 (in the example of Figure 13, container C1) are connected or can be connected by a connecting pipe Jb2.

[0166] When containers are placed on a foldable substrate as described above, as illustrated in FIG. 14 , when the substrate is folded in half, the input / output ports of each container face in the same direction (upward in FIG. 14 ) and approach each other. This minimizes the length of each tube connected to the container, and all tubes are the same (or similar) length. As a result, all containers used in the present invention only require the preparation of a single product with similar specifications and similar tubes, thereby achieving cost reduction. Furthermore, if the tube lengths are the same, the difference in material arrival time from the material source to the container can be reduced. This is preferable because it minimizes differences in cell quality between production lots. Furthermore, minimizing the tube length reduces wasted space within the tube and reduces production losses.

[0167] (Folding the substrate in two) Figure 14 is a diagram illustrating an example of folding the substrate in two. The folding may be performed in a manner that forms a sharp crease with a V-shaped cross section. However, as shown in Figure 14, a curved crease with a U-shaped cross section is preferable because it prevents the containers located in the two regions e1 and e2 from being too close to each other.

[0168] When the substrate is folded in half, the inlet / outlet port of the container may extend away from the fold as shown in Fig. 14(a), or may extend toward the fold as shown in Fig. 14(b). Furthermore, when the substrate is folded in half, the container may be positioned so that it is sandwiched between the folded substrates. From the viewpoint of not covering the container with the substrate and facilitating observation of the inside of the container and access to the container with an external tube, a preferred embodiment is one in which the container is located outside the folded substrate as shown in Fig. 14.

[0169] (Aspect in which multiple containers are formed between two flexible sheets) FIG. 15 shows another preferred aspect of the device. In the example of FIG. 15, the device further includes two overlapping flexible sheets Y31 and T32. In this aspect, the areas that will form all of the device's containers, one or more input / output ports, and connecting pipelines are formed at predetermined positions between the two flexible sheets Y31 and T32. That is, the two flexible sheets are bonded to each other, leaving these areas as non-bonded areas. For the sake of explanation, FIG. 15 only shows the container A1 and the connecting pipeline J1. As shown in FIG. 15(a), the two flexible sheets Y31 and T32 form the container A1 and the connecting pipeline J1 between them, and the two flexible sheets Y31 and T32 are bonded to each other around the outer periphery of these areas to form a single sheet Y30. The area where the flexible sheets Y31 and T32 are joined together may be only a strip-shaped area adjacent to the outline of the area where a container, pipeline, etc. is to be formed, or it may be the entire area other than the area where the container, pipeline, etc. is to be formed.

[0170] FIG. 15(b) is an end view of FIG. 15(a) cut along the cutting plane w1-w1. For illustrative purposes, FIG. 15(b) shows the outline of the container A1 visible in the background with a dashed line. An actuator J1a for opening and closing the connecting pipeline is provided on the outer surfaces of the two flexible sheets Y31 and Y32. The pressing actuator J1a is a direct-acting press device, such as a pinch valve, and is provided to penetrate the sheet Y30 in a region on the side of the connecting pipeline, thereby enabling it to clamp and press the connecting pipeline from both sides. The pressing actuator J1a operates to take two positions: a pressing position (a position in which the region that will become the connecting pipeline is pressed from the outside of the flexible sheets Y31 and Y32 to create a non-communicating state) and a non-pressing position (a position in which the region that will become the connecting pipeline is not pressed to create a communicating state). In the example of Figure 15, the pressing actuator J1a is in a non-pressing position, and the connection conduit J1 is in a connected state. By actuating the pressing actuator J1a, the region that becomes the connection conduit functions as a connection conduit that can be switched between a connected state and a disconnected state. As shown in Figure 13, the region that becomes one or more input / output ports in each container extends from the region that becomes each container (A1, A2, A3, etc.) to the outer periphery of the flexible sheet (substrate Y10 in Figure 13) to form an open end. A structure (not shown) for an openable input / output port is provided at the open end.

[0171] As described above, by providing a number of containers, connecting lines, and inlet / outlet ports corresponding to the number of processes between two flexible sheets, the device can be constructed with a large number of containers with a simple structure. From the viewpoint of cost, such a structure can be disposed of after one use.

[0172] (Foldable Sheet) In a preferred embodiment of the device, the outer peripheral shape of the two overlapping and joined flexible sheets Y31 and Y32 shown in FIG. 15 is foldable in two about the folding center line Y11, as shown in FIG. 13 . In a more preferred embodiment, the outer peripheral shape of the sheet is symmetrical about the folding center line Y11. In one region e3 of the two regions e3 and e4 separated by the folding center line Y11, regions that will become a predetermined number of the sealed containers (in the example of FIG. 13 , containers (A1, A2, A3, B1, B2)) are formed in order along the folding center line Y11 in a single direction d3. Of the outer peripheries of the regions that will become the predetermined number of containers, regions (111, 112, 113) that will become the one or more inlet / outlet ports extend in a direction away from the folding center line Y11 from the outer peripheral portion located farther from the folding center line Y11. The regions (111, 112, 113) that will become the inlet / outlet ports extend to the outer periphery of the joined flexible sheets Y31, Y32 to form open ends. Between the regions that will become the containers, regions that will become connecting pipelines that connect the regions that will become the containers are formed.

[0173] In the other region e4 of the two regions e3, e4 separated by the folding center line Y11, regions that will become the remaining of the containers (in the example of Figure 13, containers (C1, C2, C3, D1, E1)) are formed so as to be lined up in order along the folding center line Y11 in a direction d4 opposite to the one direction d3. Furthermore, from the outer periphery of each of the regions that will become the remaining containers that is located farther from the folding center line Y11, regions that will become the one or more inlet / outlet ports extend in a direction away from the folding center line Y11 and extend to the outer peripheral edges of the two flexible sheets to form open ends. Between the regions that will become the remaining containers, regions that will become connecting pipelines that connect the regions that will become the containers (such as the part indicated by symbol J1 in the example of Figure 13) are formed. The last sealed container among the containers in one region e3 (in the example of Figure 13, container B2) and the first container among the containers in the other region e4 (in the example of Figure 13, container C1) are connected by a region that becomes connecting pipeline Jb2 that crosses the bending center line.

[0174] According to this foldable configuration, as described above in the description of the foldable substrate, the input / output ports of each container face in the same direction and are close to each other, which is preferable because the length of each tube connected to the container is shortest and all tubes are the same (or similar) length.

[0175] FIG. 16 shows an example of a preferred embodiment for arranging and fixing multiple containers on a substrate, and FIG. 17 shows an example of the substrate shown in FIG. 16 and its usage state. In the embodiment shown in FIGS. 16 and 17, pockets Y20 capable of accommodating containers are provided at positions where the containers should be placed on the main surface of a substrate Y10 formed from a bendable, flexible sheet. In the example shown in the figure, the main surface of the substrate Y10 is used so that its main surface is a vertical plane, and five pockets are arranged vertically in each of regions e1 and e2 on both sides of the folding center line Y11. Each pocket is configured to be sized to appropriately accommodate the containers. Containers A1, A2, A3, B1, and B2 are inserted into the five pockets in region e1, in order from top to bottom, and containers (C1, C2, C3, D1, and E1) are inserted into the five pockets in region e2, in order from bottom to top. This arrangement allows the ten containers to be compactly arranged in two rows (containers A1, A2, A3, B1, B2 and containers C1, C2, C3, D1, E1), with container B2 and container C1 close to each other and easily connected to each other, just like connecting other containers to each other.

[0176] The provision of a pocket on the substrate surface for inserting the container is preferable because it allows the container to be quickly attached to and detached from the substrate. Also, as shown in Figure 17, the container is arranged vertically, making it possible to obtain a compact device that does not occupy a large space.

[0177] FIG. 18(a) shows another preferred embodiment for arranging and fixing multiple containers on a substrate. FIG. 18(b) shows an example of the substrate shown in FIG. 18(a) and its usage state. In the embodiment shown in FIG. 18, similar to the embodiments shown in FIGS. 16 and 17, a substrate Y10 formed from a flexible sheet is provided with pockets Y20 capable of holding containers. However, in the example shown in FIG. 18, the openings of the five pockets provided in regions e1 and e2 on both sides of the folding center line Y11 all face the folding center line Y11. The double-headed arrows indicate the direction in which containers are inserted and removed from each pocket. Containers A1, A2, A3, B1, and B2 are inserted into the five pockets in region e1, from right to left in the figure, and containers (C1, C2, C3, D1, and E1) are inserted into the five pockets in region e2, from left to top in the figure. By folding the substrate in two at the folding center line Y11 as shown in Figure 14(b), the input / output ports of each container face in the same direction (upward in Figure 14) and approach each other, as shown in Figure 18(b), which is preferable.

[0178] In the device, the opening and closing of the inlet / outlet ports of the container, the starting and stopping of the supply of material from an external material supply source, the opening and closing of the connecting pipelines, the operation and stopping of the feed mechanism, temperature control, time control, etc. may be performed manually, automatically by a control device (a computer that executes a control program, a sequence circuit, etc.), or semi-automatically by combining these.

[0179] (Cell manufacturing method using the above-mentioned cell manufacturing apparatus) This cell manufacturing method is a method for manufacturing iPS cells using the cell manufacturing apparatus of the present invention, and as described in the above-mentioned manufacturing method (I), it is a method for performing steps s1 to s3 in each of containers A1 to A3 and obtaining iPS cells in container A3.

[0180] As shown in Figure 11, the production method includes at least the following steps s1 to s3: (i) step s1 of contacting somatic cells with reprogramming factors in a liquid medium in container A1 of the cell production device; (ii) step s2 of transferring the contents of container A1 into container A2 through connecting pipeline J1 that has been switched to a communicating state after completion of step s1, and reducing the concentration of the reprogramming factors in the liquid medium in container A2; and (iii) step s3 of transferring the contents of container A2 into container A3 through connecting pipeline J2 that has been switched to a communicating state after completion of step s2, and establishing iPS cells in a liquid medium in container A3.

[0181] The details of steps s1 to s3 and the cell manufacturing apparatus are as described above. The manufacturing method may include a step of manufacturing differentiated cells from iPS cells, a step of removing undifferentiated cells from the suspension containing the differentiated cells, and a step of inspecting the obtained differentiated cells.

[0182] The following describes an actual cell production device of the present invention, how the device was used to carry out the iPS cell production method of the present invention, and an evaluation of the iPS cells obtained. The operations of each part of the cell production device (material supply operations, opening and closing operations of input / output ports and connecting pipes, pump operation of the feed mechanism, etc.) were all performed manually for experimental purposes, but all of these operations can be automated by using computer-controlled opening and closing mechanisms and pump devices.

[0183] Example 1 (Production of iPS Cells from Human Whole Blood) In this example, human whole blood was centrifuged to obtain peripheral blood mononuclear cells (PBMCs), and iPS cells were produced from the PBMCs. The cell production apparatus according to the present invention was constructed using the self-made sealed containers shown in Figure 2. The containers were arranged in series in a bottle rack (similar to a test tube stand) as shown in Figure 19, allowing for serial connection. Each container had a volume of 20 ml. Each container had a first inlet / outlet port for gas, a general-purpose second inlet / outlet port, and a general-purpose third inlet / outlet port. The general-purpose inlet / outlet port was used as a material supply line and as a connecting line.

[0184] The steps for producing iPS cells are as follows: Step s1: A step of contacting PBMCs with reprogramming factors in a liquid medium in a container A1. Step s2: A step of reducing the concentration of the reprogramming factors in the liquid medium in a container A2. Step s3: A step of culturing for 14 days in a container A3 to establish iPS cells. Step s4-1: A step of carrying out a first expansion culture in a container B1. Step s4-2: A step of carrying out a second expansion culture in a container B2. (Step s4-2 is performed in the same manner as step s4-1.)

[0185] (Process for Obtaining PBMCs from Human Whole Blood (Centrifugation)) 4 ml of human whole blood was collected from 40 ml of human whole blood contained in a Terumo blood bag MAP solution (TERUMO BB-QM200J8A), and this 4 ml was placed in a blood collection tube for mononuclear cell isolation (BD Vacutainer (registered trademark) CPT (BD 362760)).

[0186] In a safety cabinet, a Chemoclave® bag spike, a connector for injecting and suctioning drug solutions, was connected to the outlet of a blood collection bag. A 5 ml syringe was connected to the bag spike, and 4 ml of blood was aspirated into the syringe. The syringe was removed from the bag spike, and the bag spike was closed with a cap (Combi-Stopper). In a safety cabinet, a syringe needle (23 G) was attached to the syringe containing 4 ml of human whole blood. The needle was pierced through the rubber stopper of a BD Vacutainer CPT, and the human whole blood was drawn and injected under negative pressure. The BD Vacutainer CPT was placed in a centrifuge, and PBMCs were separated by centrifugation (1500-1800 G, 15 minutes (heparin), 20 minutes (citric acid)).

[0187] (Step s1: Contacting PBMCs with Reprogramming Factors in Container A1) (i) Injection of PBMCs into Container A1 The second input / output port of Container A1 was connected to the third input / output port of Container A2 in a safety cabinet via a locking connector (TS-LC11, manufactured by Terumo Corporation). In actual manufacturing, these may be connected in advance. The gas input / output ports of Container A1 and Container A2 were closed. As shown in FIG. 20 , a suction pump syringe was connected to the second input / output port of Container A2, a BD Vacutainer® Luer Lock Access Device (hereinafter referred to as the Luer Lock Access Device) was connected to the third input / output port of Container A1, and the BD Vacutainer CPT containing the centrifuged PBMC-containing liquid was connected to the Luer Lock Access Device. Air was aspirated from Container A2 using the suction pump syringe connected to the second input / output port of Container A2. By operating the syringe for the suction pump, the PBMCs in the BD Vacutainer CPT were made to flow into the syringe, and then the PBMCs in the syringe were made to flow into the container A1.

[0188] (ii) Injection of reprogramming factors into container A1 Commercially available vectors are filled in vials, so the vector was extracted from the vial using a syringe. In the production of cells for clinical use, the vector is provided sealed in a container equipped with a connector that can be connected aseptically. Using a 2.5 ml syringe (manufactured by Terumo Corporation) and a needle (manufactured by Terumo Corporation, 23G x 1), SRV iPS-2 Vector (4.6 x 10 ７ 0.1 ml (total amount) of SRV iPS-2 vector (0.1 ml of 0.1 CIU / ml) was drawn up. The Luer lock access device connected to the third input / output port of Container A1 was detached, and the syringe with its needle removed was connected to the third input / output port. The entire amount of SRV iPS-2 vector was injected into Container A1 through the third input / output port of Container A1. This allowed the PBMCs to come into contact with the reprogramming factors.

[0189] The entire device including the container A1 was incubated for 2 hours in a safety cabinet equipped with a hot plate at 37°C.

[0190] (Step s2: Step of Reducing the Concentration of Reprogramming Factors in Liquid Medium) Using a lock connector (Terumo Corporation, TS-LC11), the second inlet / outlet port of container A1 and the third inlet / outlet port of container A2 were connected with a connecting line in a safety cabinet (in actual production, these were already connected in advance). Next, the gas inlet / outlet port of container A1 was closed, and the syringe connected to the third inlet / outlet port of container A1 was removed. As shown in FIG. 21, a syringe containing 20 ml or more of StemFit AK03 medium was connected to the third inlet / outlet port of container A1, and 20 ml of StemFit AK03 medium was injected. As a result, containers A1 and A2 each contained half the amount of cells (PBMCs) compared to the original vial and reprogramming vectors diluted 200-fold compared to the original vial.

[0191] (Step s3: Establishment of iPS cells by culturing for 14 days) As shown in Figure 22, 5 mL of atelocollagen bead solution (KOKEN: MIC-00) was injected into the third input / output port of container A3 using a syringe in a safety cabinet. The syringe was removed, and the second input / output port of container A2 and the third input / output port of container A3 were connected in the safety cabinet using a lock connector (in actual production, these were already connected in advance). The first input / output port (for gas) of container A2 was closed, and the lock connector connected to the third input / output port of container A2 was removed in the safety cabinet. For the used container A1, the closure of the first input / output port was confirmed, and the opening of the connected lock connector of the second input / output port of container A1 was closed using a fluid dispensing connector. The third input / output port of container A1 was also closed using the cap of the fluid dispensing connector.

[0192] 23 , a syringe filled with 10 mL or more of liquid medium (StemFit AK03) was connected to the third inlet / outlet port of container A2, and 10 mL of the liquid medium was injected. As a result, the amount of liquid medium in container A3 became 10 mL. At this stage, container A3 contained PBMCs in a volume equal to one-quarter of the original vial's volume and reprogramming vectors diluted 400-fold compared to the original vial.

[0193] The lock connector connected to the third input / output port of container A3 was removed inside a safety cabinet. The third input / output port of container A3 was also closed using the cap of a fluid dispensing connector (BRAUN: 415080). For the used container A2, the closure of the first input / output port (for gas) was confirmed, and the opening of the connected lock connector for the second input / output port of container A2 was closed using the connector of the fluid dispensing connector. The third input / output port of container A2 was also closed using the cap of the fluid dispensing connector.

[0194] The entire device was incubated for 14 days in a safety cabinet equipped with a hot plate at 37° C. This allowed iPS cells to be established.

[0195] To continuously supply liquid medium, a syringe containing liquid medium (StemFit AK03) can be connected to the third inlet / outlet port of container A3 via an extension tube (Seaman CTL1001S2) without closing the third inlet / outlet port of container A3, allowing for continuous supply of the liquid medium. Alternatively, to discharge waste liquid, a syringe or a waste liquid bag can be connected via an extension tube without closing the third inlet / outlet port of container A3. The waste liquid bag may be a hygroscopic waste liquid bag containing a hygroscopic material (e.g., a water-absorbent resin, more specifically, polyacrylic acid, sodium polyacrylate, polyacrylic acid copolymer, etc.) or an absorbent article containing such a material (e.g., an absorbent pad, absorbent sheet, etc.). In this example, liquid medium was continuously supplied (0.1 ml / h). A Coudec syringe pump (manufactured by Daiken Medical Co., Ltd., CSP-120) was used as the automatic liquid delivery pump, and a pump uniter stand (manufactured by Daiken Medical Co., Ltd., PUS-200S) and a pump uniter (manufactured by Daiken Medical Co., Ltd., PU3-200S) were used to fix the syringe pump.

[0196] (Step s4: Expansion of iPS cells) To terminate the 14-day incubation in step s3, the syringe pump was stopped. The syringe (50 mL, manufactured by Terumo Corporation) was removed, and the first inlet / outlet port, which was a gas port of container A3, was opened. Subsequently, the waste liquid syringe connected to the second inlet / outlet port of container A3 was pulled to release the positive pressure in container A3.

[0197] A lock connector (Terumo Corporation, TS-LC11), a three-way stopcock (Terumo Corporation, Terufusion), and a sampling syringe (Terumo Corporation, 2.5 ml) were used. The three-way stopcock was connected to the lock connector and then to the third inlet / outlet port of the first expansion culture vessel B1. The sampling syringe was connected to the side branch of the three-way stopcock.

[0198] First, as shown in Figure 24, the three-way stopcock connected to the third inlet / outlet port of container B1 was closed, and then the clip on the first inlet / outlet port of container A3 was closed. Next, the three-way stopcock (Terumo Corporation, Terufusion) connected to the third inlet / outlet port of container B1 was turned on in all directions, and all of the liquid medium contained in the syringe (Terumo Corporation, 50 mL) connected to the third inlet / outlet port of container A3 was pushed out. As a result, the contents of container A3 were transferred to container B1.

[0199] The sampling syringe connected to the three-way stopcock was pulled to sample the cell suspension. In this example, 1 / 10 (1.5 ml) of the total volume of the contents of Container A3 containing the established iPS cells was sampled. Then, as shown in Figure 25, the three-way stopcock was operated to turn the direction of the sampling syringe OFF.

[0200] Add 1 / 50 of the amount of Human GloLIVE TRA-1-60® Northern Lights to the sampled cell suspension. ＴＭ NL557-conjugated antibody was added, and after 30 minutes, the cells were washed twice with liquid medium (StemFit AK03). TRA-1-60 positive cells were detected using a fluorescence microscope.

[0201] The extension tube (Seaman, CTL1001S2) with a syringe (Terumo, 50 mL) attached to the third inlet / outlet port of container A3 was removed inside a safety cabinet. The third inlet / outlet port of container A3 was closed using the cap of the fluid dispensing connector. Next, the extension tube connected to the waste syringe connected to the second inlet / outlet port of container A3 was removed inside a safety cabinet. The second inlet / outlet port of container A3 was closed using the cap of the fluid dispensing connector. The closure of the gas port (first inlet / outlet port) of the used container A3 was confirmed.

[0202] Next, the three-way stopcock (Terumo Corporation, Terufusion) connected to the third inlet / outlet port of container B1 was removed inside a safety cabinet. A syringe (Terumo Corporation, 50 ml) containing liquid medium (StemFit AK03) was connected to the third inlet / outlet port of container B1 via an extension tube, and the liquid medium was continuously supplied. To discharge waste liquid, a syringe (Terumo Corporation, 50 ml) was connected to the third inlet / outlet port of container B1 via an extension tube. (A waste liquid bag may be connected, and the waste liquid bag may be a hygroscopic waste liquid bag containing a hygroscopic material (e.g., absorbent resin, more specifically, polyacrylic acid, sodium polyacrylate, polyacrylic acid copolymer, etc.) or an absorbent article containing such a material (e.g., absorbent pad, absorbent sheet, etc.).)

[0203] The entire apparatus was placed in a safety cabinet equipped with a hot plate at 37°C, and expansion culture was carried out for 7 days.

[0204] (Evaluation of iPS cells after expansion culture) A 10 ml sample was taken from 15 ml of the cell suspension after the expansion culture, and live staining was performed using Anti-TRA-1-60, Mouse-Mono (TRA-1-60), NL557, and GloLIVE (R&D). ＴＭ The number of colonies was visually confirmed using a fluorescence microscope together with GFP, a fluorescent protein from the iPSC-2 Vector. Figure 26 shows the number of TRA-1-60-positive + GFP-positive colonies (iPS cell colonies).

[0205] (Results) As shown in Figure 26, PBMCs (1.5 x 10 ７ The number of TRA-1-60 positive + GFP positive colonies that could be confirmed in the first expansion culture (P2) using 1000 cells (approximately 1000 cells) was one.

[0206] The number of iPS cell colonies contained in 10 ml of the 15 ml medium was 1 to 2. From this result, it was found that PBMCs (1.5 × 10 ７ The number of iPS cell colonies established using the culture medium (approximately 100 cells) is thought to be 1 to 2.

[0207] Example 2 (Production of iPS cells from commercially available PBMCs) In this example, iPS cells were obtained by carrying out steps s1 to s4 in the same manner as in Example 1 above, except that commercially available PBMCs were used (i.e., the whole blood centrifugation step was omitted).

[0208] (Step s1: Step of contacting somatic cells (PBMCs) with reprogramming factors) Commercially available PBMCs contained in a vial were collected with a syringe. Commercially available vectors, which were also filled in vials, were also collected with a syringe. In both steps, in the production of clinical cells, the vectors are provided sealed in a container equipped with a connector that can be connected aseptically.

[0209] Using a syringe (Terumo, 5 mL) and a needle (Nipro, NIPRO 18G x 1-1 / 2), SRV iPS-2 Vector (4.6 x 10 7 0.1 mL of PBS (10 CIU / mL) and the entire amount of commercially available PBMCs (Human PBMC 10M (PRECISION 93210-10M)) were sucked up. This was connected to a Luer lock connected to the third input / output port of container A1 and injected into container A1. This allowed the PBMCs to come into contact with the reprogramming factors.

[0210] The entire device including the container A1 was incubated for 2 hours in a safety cabinet equipped with a hot plate at 37°C, completing step s1.

[0211] Step s2 (reducing the concentration of reprogramming factors in container A2), step s3 (establishing iPS cells in container A3), and step s4 (expansion culture in container B1) were carried out under the same conditions and with the same procedures as in Example 1 above.

[0212] (Evaluation of iPS cells at the completion (establishment) of step s3) After 14 days of culture in step s3 (after establishment of iPS cells), a 1.5 ml sample of the cell suspension was obtained from container A3. The sample was subjected to live staining using Anti-TRA-1-60, Mouse-Mono (TRA-1-60), NL557, and GloLIVE (R&D). SRV ＴＭ The number of colonies was visually confirmed using a fluorescence microscope together with GFP, a fluorescent protein from the iPSC-2 Vector. Figure 27 shows the number of TRA-1-60-positive + GFP-positive colonies (iPS cell colonies).

[0213] (Results) As shown in Figure 27, commercially available PBMCs (1 x 10 ７ After establishment, one TRA-1-60-positive + GFP-positive colony was confirmed.

[0214] (Discussion) The number of iPS colonies contained in the above 1.5 ml sample was 1. From this result, it is believed that the number of iPS cell colonies established using commercially available PBMCs was 10.

[0215] (Evaluation of iPS cells after expansion culture) SRV TM After infection with 100 μL of iPSC-2 Vector (Tokiwa Bio), 50 ml of cell suspension was subcultured twice (2 x Day 7) after establishment on Day 14. A 10 ml sample was obtained from the cell suspension and subjected to live staining using Anti-TRA-1-60, Mouse-Mono (TRA-1-60), NL557, and GloLIVE (R&D). ＴＭ The number of colonies was visually confirmed using a fluorescence microscope together with GFP, a fluorescent protein from the iPSC-2 Vector. Figure 28 shows the number of TRA-1-60-positive + GFP-positive colonies.

[0216] (Results) As shown in Figure 28, PBMCs (1 x 107 In the second expansion culture (P3) using TRA-1-60+GFP-positive colonies, only one TRA-1-60-positive + GFP-positive colony was confirmed.

[0217] (Discussion) The number of iPS colonies contained in 10 ml of 15 ml of liquid medium was one. From this result, it was found that commercially available PBMC (1 × 10 ７ It is estimated that the number of iPS cell colonies established using the iPS cell culture medium is 1 to 2.

[0218] Example 3 (Production of iPS cells from human whole blood (1)) In this example, human whole blood was centrifuged to obtain PBMCs, and iPS cells were produced from the PBMCs in the same manner as in Example 1, except that a GREX 10M-CS (manufactured by Wilson Wolf Corporation) was used as the sealed container constituting the cell production device according to the present invention.

[0219] The GREX 10M-CS is a generally cylindrical sealed container having a container body and a lid (multi-port cap). The bottom of the container body is made of a gas-permeable membrane. The volume of the container is 100 ml. In this example, the containers were connected in series, and steps s1 to s4 were carried out.

[0220] The GREX 10M-CS multi-port cap is equipped with the following input / output ports: Sample Line Tubing (a MicroClave (registered trademark) Connector is attached to the tip of the Sample Line Tubing). Hereinafter, this will also be referred to as the sample port. Reduction Line Tubing: A weldable reduction line branches off from the reduction line tubing. Harvest Line Tubing: Hereinafter, this will also be referred to as the harvest port. Hereinafter, a weldable harvest line branches off from the harvest port. Gas input / output port connected to a Pall Versapor Vent Filter: Hereinafter, this will also be referred to as the gas port.

[0221] (Step s1: step of contacting PBMCs with reprogramming factors in container A1) PBMCs were obtained from human whole blood in the same manner as in Example 1. As shown in Figure 29, a BD Vacutainer CPT container GREX 10M-CS, a Luer lock access device, a syringe (manufactured by Terumo Corporation, 50 ml), an injection needle (manufactured by Terumo Corporation, 23G x 1), and a three-way stopcock R type (manufactured by Terufusion Corporation) were prepared to accommodate the PBMCs as a fraction after centrifugation.

[0222] A three-way stopcock R type and a luer lock access device were connected to the MicroClave Connector attached to the sample port of the container A1.

[0223] Commercially available vectors are filled in vials, so the vectors were extracted from the vials using a syringe. In the production of clinical cells, the reprogramming vectors are provided sealed in a container that can be connected via a sterile connector. Approximately 25 ml of the syringe was drawn into the syringe in advance. A needle was attached to the syringe, and the SRV iPS-2 Vector (4.6 x 10 ７ 0.1 ml (total volume) of 0.1 ml of 0.1 CIU / ml was taken.

[0224] The injection needle was removed from the syringe, and the syringe was connected to the side flow path of the R-type three-way stopcock. The three-way stopcock was turned off in the direction of the MicroClave Connector of container A1. The BD Vacutainer CPT, in which PBMCs had been centrifuged, was set in the Luer lock access device. The BD Vacutainer CPT was pushed into the Luer lock access device. By aspirating with the syringe connected to the side flow path of the R-type three-way stopcock, the contents of the BD Vacutainer CPT flowed into the syringe and were mixed with the vector that had been previously sealed in. The cock of the three-way stopcock was turned off in the direction of the Luer lock access device.

[0225] The syringe containing the serum and PBMCs was rotated 90 degrees to a vertical position, and the syringe was pushed in while the contents were held at the bottom of the syringe, causing the cell suspension to flow into the container A1. The BD Vacutainer CPT was removed from the Luer lock access device. ２ The mixture was incubated in an incubator for 2 hours.

[0226] (Step s2: Step of reducing the concentration of reprogramming factors in the liquid medium in container A2) Only the Reduction Line Tubing of container A1 was opened, and the gas port and harvest port were closed. A syringe containing 50 ml of liquid medium StemFit AK03 was connected to the Reduction Line Tubing of container A1, and 50 ml of liquid medium was injected. As a result, the contents of container A1 flowed into container A2 together with the injected 50 ml of liquid medium. The Reduction Line Tubing of container A1 was closed. CO was added until the cells (PBMCs) naturally settled in container A2. ２ The mixture was left to stand in an incubator for 30 minutes. At this stage, container A2 contained PBMCs in an amount 1 / 1 of the amount in the original vial and reprogramming vector diluted 500-fold relative to the amount in the original vial.

[0227] After 30 minutes, the device was ２ The container was removed from the incubator, and the supernatant was removed in a safety cabinet. The tube of the weldable reduction line of container A2 was opened, and an empty syringe (50 ml) for collecting waste liquid was connected. (A waste liquid bag may also be used, and the waste liquid bag may be a hygroscopic waste liquid bag containing a hygroscopic material (e.g., a water-absorbent resin, more specifically, polyacrylic acid, sodium polyacrylate, polyacrylic acid copolymer, etc.) or an absorbent article containing such a material (e.g., a water-absorbent pad, a water-absorbent sheet, etc.).

[0228] A syringe (50 ml) with the plunger pulled back was connected via the fluid dispensing connector to inject 50 ml of air into the opening of the gas port of container A2. The tube of the gas port of container A2 was released. Air was pushed into container A2 from the syringe connected to the gas port of container A2. As a result, the supernatant of the liquid medium in container A2 (the liquid present in the region up to 8 cm from the top end of the container body) was collected as waste liquid into a waste liquid collection syringe (50 ml) connected to the weldable reduction line of container A2.

[0229] The liquid medium containing PBMCs remained in container A2, approximately 2 cm above the bottom. The gas port tube and the weldable reduction line tube of container A2 were closed. At this stage, container A2 contained 1 / 1 the amount of PBMCs compared to the original vial and reprogramming vectors diluted 500-fold (relative to the original vial).

[0230] (Step s3: Establishment of iPS cells by culturing for 14 days) The Weldable Harvest Line of container A2 is connected to the MicroClave Connector of container A3. The MicroClave Connector can be connected only once even in a general environment (it cannot be reconnected). 10 ml of atelocollagen bead solution (KOKEN: MIC-00) contained in a syringe was injected from the harvest port of container A2 in a safety cabinet. A syringe containing 50 ml of StemFit AK03 liquid medium was connected to the Reduction Line Tubing of container A2, and 50 ml of the liquid medium was injected. As a result, the contents of container A2 flowed into container A3 together with the injected 50 ml liquid medium. Thereafter, the Reduction Line Tubing of container A2 was closed. The device was then placed in a CO ２ The cells were placed in an incubator and cultured for 14 days.

[0231] In this example, on day 7 of the 14-day culture, additional medium was added to container A3 to bring the volume up to 100 ml. At this stage, container A3 contained PBMCs in a volume 1 / 1 of the original vial and reprogramming vectors diluted 3000-fold compared to the original vial.

[0232] The various connections made by the operator in the safety cabinet were made in advance in the actual production. Liquid medium was injected from a syringe via an extension tube (Seaman CTL1001S2). To achieve this, a Coudec Syringe Pump CSP-120 (Daiken Medical Co., Ltd. CSP-120) was used as the syringe pump, as shown in Figure 32. A Pump Uniter Stand (Daiken Medical Co., Ltd.: PUS-200S) and a Pump Uniter (Daiken Medical Co., Ltd.: PU3-200S) were used to secure the syringe pump.

[0233] (Operation to inject additional liquid medium on day 7 of 14-day culture) The clip on the tubing connected to the Reduction Line Tubing of Container A3 was released, and a syringe (Terumo, 50 ml) containing 50 mL of liquid medium (StemFit AK03) was connected. The Weldable Harvest Line of Container A2 was closed with a clip. As shown in Figure 33, the liquid medium was poured from the syringe into Container A3. The clip on the Reduction Line Tubing tubing was closed.

[0234] (Step s4: Expansion of iPS cells) A syringe (JMS, 100 ml) was connected to the Weldable Reduction Line of container A3 (a waste liquid bag can also be used, and the waste liquid bag may be a hygroscopic waste liquid bag containing a hygroscopic material (e.g., a water-absorbent resin, more specifically, polyacrylic acid, sodium polyacrylate, polyacrylic acid copolymer, etc.) or an absorbent article containing such a material (e.g., an absorbent pad, a water-absorbent sheet, etc.)). A fluid dispensing connector was connected to the gas port of container A3, and an air-filled syringe (manufactured by Terumo Corporation, 50 ml) was connected. The clip of the Weldable Harvest Line of container A2 connected to the MicroClave Connector of container A3 was closed. Air was sent from the syringe, and the supernatant medium in container A3 was discharged into the waste liquid syringe. This operation was repeated twice, and the syringe connected to the fluid dispensing connector was detached and refilled with air. The entire supernatant in container A3 was discharged into a waste syringe. The gas port of container A3 was then clipped shut to prevent backflow. The weldable reduction line of container A3 was also clipped shut to prevent backflow of waste liquid.

[0235] As shown in Figure 34, the Weldable Harvest Line of container A3 was connected to the MicroClave Connector of container B1 via a three-way stopcock (Terufusion TERUMO). A syringe (Terumo, 2.5 ml) for sample acquisition was connected to the three-way stopcock. The MicroClave Connector can be connected only once in a general environment (it cannot be reconnected).

[0236] It is possible to transfer the approximately 20 ml of medium remaining in container A3 directly to container B1. However, because the liquid containing 10 ml of atelocollagen beads may clog the flow path, a cell suspension prepared by first adding 50 ml of liquid medium (StemFit AK03) was transferred from container A3 to container B1. To allow the liquid medium in container A3 to flow in, the clip on the gas port of container B1 was released. 50 ml of liquid medium was poured into container A3 through the harvesting port. After the liquid medium was poured in, the clip on the harvesting port of container A3 was closed. The MicroClave Connector of container B1 was turned on in all directions. A fluid dispensing connector was connected to the gas port of container A3, and an air-filled syringe (Terumo, 50 ml) was connected. Air was introduced into container A3 through the gas port using the syringe.

[0237] After air was introduced into container A3, the clip on the gas port was closed. After transferring the contents from container A3 to container B1, the cell suspension was collected using a sample syringe. After collection, the three-way stopcock was operated to turn the direction of the sample syringe OFF. The Weldable Harvest Line of container A3 was then closed with a clip.

[0238] Add 1 / 50 of the amount of Human GloLIVE TRA-1-60® Northern Lights to the cell suspension obtained from the sample. ＴＭ NL557-conjugated antibody was added, and after 30 minutes, the cells were washed twice with liquid medium (StemFit AK03). TRA-1-60 positive cells were detected using a fluorescence microscope.

[0239] CO without removing the piping of the equipment ２ The cells were placed in an incubator and cultured for 7 days.

[0240] (Evaluation of iPS cells after expansion culture) A 10 ml sample was obtained from 50 ml of the cell suspension after the above expansion culture (single subculture (1x Day 7)). Live staining was performed using Anti-TRA-1-60, Mouse-Mono (TRA-1-60), NL557, and GloLIVE (R&D). SRV ＴＭ The number of colonies was visually confirmed using a fluorescence microscope together with GFP, a fluorescent protein from the iPSC-2 Vector. Figure 35 shows the number of TRA-1-60-positive + GFP-positive colonies (iPS cell colonies).

[0241] (Results) As shown in Figure 35, PBMCs (1.5 x 10 ７ The number of TRA-1-60 positive + GFP positive colonies confirmed in the first expansion culture (P2) using 1000 cells (approximately 1000 cells) was one.

[0242] (Discussion) The number of iPS colonies contained in 10 ml of the 50 ml medium was one. From this result, it was found that PBMCs (1.5 × 10 ７ The Ronnie number for establishing iPS cells using the 500 cells (approximately 1000 cells) is thought to be 5.

[0243] (Production of iPS cells from human whole blood (2)) Based on the results of Production (1) in Example 3 above, an embodiment was also carried out in which the amount of reprogramming factor was increased by 5 times. In Production (2), the amount of vector contacted with PBMCs in Step s1: a step of contacting PBMCs with reprogramming factors in Container A1 was increased by 5 times (SRV iPS-2 Vector (4.6 × 10 ７ After completion of step s3: establishment of iPS cells by 14-day culture, 0.5 ml of 0.1 ml of ... ７ Immediately after establishment (Day 14) using approximately 1000 cells, the number of TRA-1-60-positive + GFP-positive colonies that could be confirmed was 3. This indicates that the reprogramming efficiency can be further increased by changing the implementation conditions.

[0244] Example 4 (Production of iPS cells from commercially available PBMCs) In this example, iPS cells were obtained by carrying out steps s1 to s4 in the same manner as in Example 3 above, except that commercially available PBMCs were used (i.e., the whole blood centrifugation step was omitted).

[0245] (Evaluation of iPS cells after expansion culture) SRV ＴＭ After infection with 100 μl of iPSC-2 Vector (Tokiwa Bio), 50 mL of cell suspension was subcultured twice (2x Day 7) after establishment on Day 14. A 10 mL sample was obtained from the cell suspension and subjected to live staining using Anti-TRA-1-60, Mouse-Mono (TRA-1-60), NL557, and GloLIVE (R&D). ＴＭ The number of colonies was visually confirmed using a fluorescence microscope together with GFP, a fluorescent protein from the iPSC-2 Vector. Figure 36 shows the number of TRA-1-60-positive + GFP-positive colonies (iPS cell colonies).

[0246] (Results) As shown in Figure 36, PBMCs (1 x 10 ７ In the second expansion culture (P3) using TRA-1-60+GFP-positive colonies, only one TRA-1-60-positive + GFP-positive colony was confirmed.

[0247] (Discussion) The number of iPS colonies contained in 10 ml of 50 ml of liquid medium was one. From this result, it was found that commercially available PBMC (1 × 10 ７ It is considered that the number of iPS cell colonies established using the iPS cell line was 5.

[0248] Example 5 (Production and evaluation of cardiomyocytes from iPS cells (step s5 of inducing differentiation of iPS cells)) In this example, the cell production device of the present invention was used to carry out the method for producing differentiated cells of the present invention, and cardiomyocytes were actually produced by inducing differentiation of iPS cells. In a preferred embodiment of the method for producing differentiated cells of the present invention, iPS cells obtained by the production method of the present invention are used, but in this example, the method for producing differentiated cells of the present invention (step s5 above) was carried out using iPS cells provided by the Kyoto University iPS Cell Research Foundation (a research strain of human clinical iPS cells (15M66)).

[0249] In this example, step s5 of inducing differentiation of iPS cells is further divided into three steps (s5-1, s5-2, s5-3), and each of these steps corresponds one-to-one to three sealed containers (C1, C2, C3) as follows. Each step was performed sequentially in each container while transferring the contents from one container to another. Step s5-1: Inducing differentiation of iPS cells in container C1 to obtain cardiac mesoderm via mesoderm. Step s5-2: Transferring cardiac mesoderm to container C2 and inducing differentiation of the cardiac mesoderm in container C2 to obtain cardiac progenitor cells. Step s5-3: Transferring cardiac progenitor cells to container C3 and inducing differentiation of the cardiac progenitor cells in container C3 to obtain cardiomyocytes.

[0250] In this example, the three sealed containers used were GREX 10M-CS, the same containers used in Examples 3 and 4. The outline of the cell manufacturing apparatus for carrying out steps s5-1, s5-2, and s5-3 is as shown in Figure 12, and corresponds to the case where q = 3 in the configuration of the same figure. The external appearance of the sealed container GREX 10M-CS is as shown in Figure 29.

[0251] (Step s5-1 (Induction of Differentiation from iPS Cells to Cardiac Mesoderm)) First, an iPS cell line (15M66, 1 × 10 6 The iPS cells, an atelocollagen bead solution (KOKEN:MIC-00), and 10 mL of liquid medium StemFit AK03 (Ajinomoto) (containing 10 μM Y-27632 (Fujifilm Wako)) were poured into the container C1. Next, 20 mL of medium A from the PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) was poured into the container C1. The medium A was continuously supplied at a flow rate of 0.5 mL / h, and the container was cultured for two days while the medium was being drained. This induced differentiation of the iPS cells into mesoderm and cardiac mesoderm.

[0252] (Operation to transfer the contents of container C1 to container C2) First, air was pumped into container C1, and the supernatant in container C1 was pushed into the waste syringe (other inlet / outlet ports were closed as appropriate so that the supernatant would be pushed out by the pumping of air into the container). As a result, the settled cardiac mesoderm and atelocollagen beads, along with a small amount of liquid medium containing them, remained at the bottom of container C1. Next, the connections of the inlet / outlet ports were switched, and air was injected into container C1 using the syringe, pushing the settled portion (cardiac mesoderm, atelocollagen beads, and a small amount of liquid medium) remaining at the bottom into container C2, completing the transfer of the settled portion.

[0253] (Step s5-2 (Induction of Differentiation from Cardiac Mesoderm to Cardiac Progenitor Cells)) First, 20 ml of medium B from the PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) was poured into the container C2. Next, the medium B was continuously supplied at a flow rate of 0.5 ml / h, and the cells were cultured for 2 days while the medium in the container was being discharged. This resulted in induction of differentiation of cardiac mesoderm, and cardiac progenitor cells were obtained.

[0254] (Operation of transferring the contents of container C2 to container C3) Similar to the operation of transferring the contents of container C1 to container C2, air was pumped into container C2, and the supernatant in container C2 was pushed into a waste syringe. As a result, the settled cardiac progenitor cells and atelocollagen beads, along with a small amount of liquid medium containing them, remained at the bottom of container C2. Next, the connection of the input / output ports was switched, and air was injected into container C2 using a syringe. The settled portion (cardiac progenitor cells, atelocollagen beads, and a small amount of liquid medium) remaining at the bottom was pushed into container C3, completing the transfer of the settled portion.

[0255] (Step s5-3) Induction of differentiation from cardiac progenitor cells to cardiomyocytes First, 20 ml of C medium from the PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) was poured into the container C3. Next, the C medium was continuously supplied at a flow rate of 0.5 ml / h, and the cells were cultured for 10 days while the medium in the container was being discharged. This resulted in induction of differentiation of cardiac progenitor cells, and cardiomyocytes were obtained.

[0256] (Cardiomyocyte marker-positive cells) On day 14, the contents of the container were sampled and immunostained for troponin T (TNNT2), a marker protein for cardiomyocytes, according to standard methods (using an anti-troponin antibody: BD Pharmingen Alexa Fluor 647 Anti-Cardiac Troponin T (BD 565744)). The results of double staining with Hoechst staining (nuclear staining) are shown in Figure 37. As shown in Figure 37, many cells (i.e., cardiomyocytes) with troponin T signals around the Hoechst signals (nuclei) were observed in the cell clusters on the atelocollagen. This demonstrates that the production method of the present invention efficiently induced differentiation of iPS cells into cardiomyocytes.

[0257] (Induction of Cardiomyocyte and Cardiac Progenitor Cell Marker Expression) On Day 14, the contents of the vessel (step s5-3) were sampled and treated with collagenase to dissolve the atelocollagen beads. mRNA was extracted from the resulting cell pellet using the SuperPrep II Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd.: SCQ-401), and cDNA was synthesized. Real-time PCR analysis was performed using the StepOnePlus system (Life Technologies, Carlsbad, CA, USA) and Luna Universal qPCR Master Mix (New England Biolabs Inc., Ipswich, MA, USA) to analyze the expression levels of T brachyury (mesodermal marker), NKX2.5 (cardiomyocyte marker: cardiac-specific transcription factor), KDR, and ISL1 (cardiac progenitor cell marker). An iPS cell line (15M66) cultured on atelocollagen without differentiation induction was used as a control. The results are shown in Figure 38.

[0258] As shown in Figure 38, expression of the mesoderm marker (T brachyury) was not detected in either the control or differentiation-induced cells. In contrast, expression of cardiomyocyte markers (NKX2.5 and TNNT2) was very high (approximately 8-10 times higher than the control) in the differentiation-induced cells, and expression of cardiac progenitor cell markers (KDR and ISL1) was also high (approximately 4-6 times higher than the control). This suggests that this method successfully induced differentiation of iPS cells into cardiac progenitor cells and then into cardiac myocytes. These results demonstrate that cell differentiation can be induced using the production method and production device of the present invention.

[0259] Example 6 (Production and Evaluation of Pancreatic Progenitor Cells from iPS Cells (Step s5 of Inducing Differentiation of iPS Cells)) In this Example, the cell production device of the present invention was used to carry out the method for producing differentiated cells of the present invention, and pancreatic progenitor cells were actually produced by inducing differentiation of iPS cells. In a preferred embodiment of the method for producing differentiated cells of the present invention, iPS cells obtained by the production method of the present invention are used, but in this Example, the method for producing differentiated cells of the present invention (step s5 above) was carried out using iPS cells provided by the Kyoto University iPS Cell Research Foundation (a research strain of human clinical iPS cells (15M66)).

[0260] In this example, step s5 of inducing differentiation of iPS cells was further divided into three steps (s5-1, s5-2, s5-3), and each of these steps was assigned to one of three sealed containers (C1, C2, C3) as described below. Each step was carried out sequentially in each container while transferring the contents from one container to another. Step s5-1: Inducing differentiation of iPS cells in container C1 to obtain a primitive gut tube via definitive endoderm. Step s5-2: Transferring the primitive gut tube to container C2 and inducing differentiation of the primitive gut tube in container C2 to obtain posterior foregut endoderm. Step s5-3: Transferring the posterior foregut endoderm to container C3 and inducing differentiation of the posterior foregut endoderm in container C3 to obtain pancreatic progenitor cells.

[0261] In this example, the three sealed containers used were GREX 10M-CS, the same containers used in Examples 3 and 4. The outline of the cell manufacturing apparatus for carrying out steps s5-1, s5-2, and s5-3 is as shown in Figure 12, and corresponds to the case where q = 4 in the configuration of the same figure. The external appearance of the sealed container GREX 10M-CS is as shown in Figure 29.

[0262] (Step s5-1 (Induction of Differentiation of iPS Cells to Definitive Endoderm)) First, an iPS cell line (15M66, 1 × 10 6The iPS cells, atelocollagen bead solution (KOKEN:MIC-00), and 10 mL of liquid medium StemFit AK03 (Ajinomoto) (containing 10 μM Y-27632 (Fujifilm Wako)) were injected into the vessel C1. The procedure thereafter followed the recommended protocol for the TEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies) (https: / / cdn.stemcell.com / media / files / pis / DX20464-PIS_1_4_0.pdf). Next, 10 mL of Medium 1A from the STEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies) was injected into the vessel C1. From Day 1, Medium 1B was continuously supplied at a flow rate of 0.5 mL / h, and the cells were cultured until Day 5. This induced differentiation of the iPS cells, resulting in the production of definitive endoderm.

[0263] (Operation to transfer the contents of container C1 to container C2) First, air was pumped into container C1, and the supernatant in container C1 was pushed into a waste syringe (other input / output ports were closed as appropriate so that the supernatant would be pushed out by pumping air into the container). As a result, the settled definitive endoderm and atelocollagen beads, along with a small amount of liquid medium containing them, remained at the bottom of container C1. Next, the connections of the input / output ports were switched, and air was injected into container C1 with a syringe, pushing the settled portion (definitive endoderm, atelocollagen beads, and a small amount of liquid medium) that remained at the bottom into container C2, completing the transfer of the settled portion.

[0264] (Step s5-2 (Induction of Differentiation from Definitive Endoderm to Primitive Gut)) First, 10 ml of Medium 2A from the STEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies) was poured into the container C2. From Day 7, Medium 2B was continuously supplied at a flow rate of 0.5 ml / h, and the cells were cultured until Day 9. This induced differentiation of the definitive endoderm, resulting in the production of a primitive gut.

[0265] (Operation of transferring the contents of container C2 to container C3) Similar to the operation of transferring the contents of container C1 to container C2, air was pumped into container C2, and the supernatant in container C2 was pushed into a waste syringe. As a result, the settled primitive intestine and atelocollagen beads, along with a small amount of liquid medium containing them, remained at the bottom of container C2. Next, the connection of the inlet / outlet ports was switched, and air was injected into container C2 with a syringe, pushing the settled portion (primitive intestine, atelocollagen beads, and a small amount of liquid medium) remaining at the bottom into container C3, completing the transfer of the settled portion.

[0266] (Step s5-3) Differentiation induction from the primitive gut to posterior foregut endoderm First, 10 ml of Medium 3 from the STEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies) was poured into the container C3. From Day 10, Medium 3 was continuously supplied at a flow rate of 0.5 ml / h, and the cells were cultured until Day 13. This induced differentiation of the primitive gut to obtain posterior foregut endoderm.

[0267] (Operation of transferring the contents of container C3 to container C4) Similar to the operation of transferring the contents of container C1 to container C2, air was pumped into container C3, and the supernatant in container C3 was pushed into a waste syringe. As a result, the settled posterior foregut endoderm and atelocollagen beads, along with a small amount of liquid medium containing them, remained at the bottom of container C3. Next, the connection of the inlet / outlet ports was switched, and air was injected into container C3 using a syringe. The settled portion (posterior foregut endoderm, atelocollagen beads, and a small amount of liquid medium) remaining at the bottom was pushed into container C4, completing the transfer of the settled portion.

[0268] (Step s5-4) Differentiation induction from posterior foregut endoderm to pancreatic progenitor cells First, 10 ml of Medium 4 from the STEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies) was injected into the container C3. From Day 10, Medium 4 was continuously supplied at a flow rate of 0.5 ml / h, and the cells were cultured until Day 19. This induced differentiation of posterior foregut endoderm, and pancreatic progenitor cells were obtained.

[0269] (Pancreatic progenitor cell marker-positive cells) On Day 19, the contents of the container (step s5-4) were sampled and immunostained for pancreatic progenitor cell marker proteins PDX-1 (pancreatic progenitor cell marker) and NKX6.1 (pancreatic progenitor cell marker) according to standard methods. The results of double staining using an anti-PDX-1 antibody (Human / Mouse PDX-1 / IPF1 Alexa Fluor 647 MAb (Clone 267712) (R&D Systems, Inc. IC2419R-100UG)) and Hoechst staining (nuclear staining) are shown in Figure 39 (Pancreatic progenitor cells PDX-1). The results of double staining using an anti-NKX6.1 antibody (BD Pharmingen Alexa Fluor 647 Mouse Anti-NKX6.1 (BD 563338)) and Hoechst staining (nuclear staining) are shown in Figure 40 (Pancreatic progenitor cells NKX6.1). As shown in Figure 39 (pancreatic progenitor cells PDX-1) and Figure 40 (pancreatic progenitor cells NKX6.1), numerous cells (i.e., pancreatic progenitor cells) bearing PDX-1 and NKX6.1 signals coexisting with Hoechst signals were observed in the cell clusters on atelocollagen. This demonstrates that the production method of the present invention efficiently induces differentiation of iPS cells into pancreatic progenitor cells.

[0270] (Induction of pancreatic progenitor cell marker expression) On Day 19, the contents of the container (step s5-4) were sampled and treated with collagenase to dissolve the atelocollagen beads. mRNA was extracted from the resulting cell pellet using SuperPrep II Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd.: SCQ-401), and cDNA was synthesized. The StepOnePlus system (Life Technologies, Carlsbad, CA, USA) and TaqMan TM Real-time PCR analysis was performed using Fast Advanced Master Mix (Life Technologies, Carlsbad, CA, USA) to analyze the expression levels of SOX17 (endodermal marker), HNF1b and FoxA2 (primitive gut markers), and SOX9, PDX-1, and NKX6.1 (pancreatic progenitor cell markers). An iPS cell line (15M66) cultured on atelocollagen without differentiation induction was used as a control. The results are shown in Figure 41 (Gene expression in pancreatic progenitor cells).

[0271] As shown in Figure 41 (gene expression in pancreatic progenitor cells), the expression of the endoderm marker (SOX17) in cells after differentiation induction was significantly increased by approximately 1.5-fold compared to the control. The expression of pancreatic progenitor cell markers (SOX9, PDX-1, and NKX6.1) in cells after differentiation induction was significantly increased by approximately 1.3-1.4-fold compared to the control. The expression of the primitive gut marker (FoxA2) was significantly increased by approximately 1.4-fold compared to the control. These results suggest that this method successfully induced differentiation of iPS cells into pancreatic progenitor cells via the primitive gut. These results demonstrate that cell differentiation can be induced using the production method and production device of the present invention.

[0272] The method for producing iPS cells, the method for producing differentiated cells, and the cell production apparatus of the present invention enable iPS cells and differentiated cells to be produced more cheaply and easily than before, and also enable automated production. From a clinical perspective, the present invention is particularly suitable for applications such as establishing iPS cells from the somatic cells of a patient requiring transplantation therapy, producing various differentiated cells from the iPS cells, and transplanting them into the patient (autotransplantation).

[0273] This application is based on International Application (PCT / JP) 2023 / 006753 (filing date: February 24, 2023), the contents of which are incorporated in their entirety herein.

[0274] A1 to An (n≧3) Sealed container J1 to J(n-1) Connecting pipe F1 to F(n-1) Feeding mechanism G1 to Gn Material supply source

Claims

1. A method for producing induced pluripotent stem cells, comprising: The method comprises: sequentially moving cells in one direction from a first sealed container (A1) to an nth sealed container (An) by a feed mechanism among n (n≧3) sealed containers connected in series via a connecting pipeline; and sequentially carrying out a production process of induced pluripotent stem cells in each sealed container; Each of the sealed containers (A1 to An) has one or more openable / closable inlet / outlet ports, The connecting pipe is configured to be switchable between a communicating state and a non-communicating state, the feed mechanism is a mechanism for moving the contents from each sealed container to the next sealed container through the connecting pipe line switched to a communicating state, The process for producing the induced pluripotent stem cells comprises: A step (s1) of contacting a reprogramming factor with a somatic cell in a liquid medium in a first sealed container (A1); A step (s2) of reducing the concentration of the reprogramming factor in the liquid medium in the second sealed container (A2) to the (n-1)th sealed container (A(n-1)); and (s3) culturing the somatic cells in a liquid medium in the n-th closed container (An) to establish induced pluripotent stem cells. A method for producing the induced pluripotent stem cells.

2. After the step (s3), the method further includes a step (s4) of expanding the induced pluripotent stem cells p times (p≧1), p sealed containers (B1 to Bp) corresponding to the number of times of expansion culture are connected in series to the n-th sealed container (An) via a connecting pipeline; The artificial pluripotent stem cells are sequentially moved in one direction from the sealed container (An) to the sealed container (Bp) by the respective feeding mechanisms, and one expansion culture is carried out in each of the sealed containers (B1 to Bp), thereby carrying out a total of p expansion cultures; Each of the sealed containers (B1 to Bp) has one or more openable / closable inlet / outlet ports, The connecting pipe is configured to be switchable between a communicating state and a non-communicating state, The feed mechanism is a mechanism for moving the contents from each sealed container to the next sealed container through the connecting pipe line switched to the communicating state. A method for producing the induced pluripotent stem cell according to claim 1.

3. The sealed container is A sealed container having a container body made of a flexible material; A sealed container having a container body made of a hard material; A sealed container having a container body made of a composite material of a flexible material and a hard material A sealed container selected from the group consisting of: A method for producing the induced pluripotent stem cell according to claim 1.

4. The feed mechanism is A mechanism for pushing and transferring the contents of the source sealed container to the next sealed container by adding fluid to the source sealed container; A mechanism for pushing and transferring the contents of the source sealed container to the next sealed container by reducing the volume of the source sealed container; a mechanism for transferring the contents of the source sealed container to the next sealed container by a pump device provided in the connecting pipeline; a mechanism for transferring the contents of the source sealed container to the next sealed container by applying suction force from the next sealed container to the source sealed container; A mechanism for transferring the contents of the source sealed container to the next sealed container by utilizing gravity; and A mechanism for attaching cells to magnetic microcarriers and then applying an external magnetic force to the microcarriers to move the microcarriers and the cells attached to them in the original sealed container to the next sealed container. A mechanism selected from the group consisting of: or a combination of two or more mechanisms selected from the group; A method for producing the induced pluripotent stem cell according to claim 1.

5. A method for producing differentiated cells, comprising: A step of producing induced pluripotent stem cells by the production method according to any one of claims 1 to 4; and a step (s5) of inducing differentiation of the produced induced pluripotent stem cells, a sealed container (C1) for carrying out the step (s5) is further connected to the rear end of the sealed container (X1) among the sealed containers used in the method for producing induced pluripotent stem cells via a connecting pipeline; The sealed container (C1) has one or more openable / closable inlet / outlet ports, and a material necessary for differentiation induction in step (s5) is supplied to the inside of the sealed container (C1) through the inlet / outlet ports; The artificial pluripotent stem cells are transferred from the sealed container (X1) to the sealed container (C1) by a transfer mechanism, and the step (s5) is carried out in the sealed container (C1); The connecting pipe is configured to be switchable between a communicating state and a non-communicating state, The feed mechanism is a mechanism for moving the contents from the sealed container (X1) to the sealed container (C1) through the connecting pipe line switched to a communicating state. The method for producing the differentiated cells.

6. The method further comprises a step (s6) of removing undifferentiated cells after the differentiation induction step, a sealed container (D1) for carrying out the step (s6) is further connected via a connecting pipeline to the rear end of the sealed container (X2) among the sealed containers used in the differentiation induction step; The sealed container (D1) has one or more openable / closable inlet / outlet ports, The differentiated cells are transferred from the sealed container (X2) to the sealed container (D1) by a transfer mechanism, and the step (s6) is carried out in the sealed container (D1); The connecting pipe is configured to be switchable between a communicating state and a non-communicating state, The feed mechanism is a mechanism for moving the contents from the sealed container (X2) to the sealed container (D1) through the connecting pipe line switched to a communicating state. The method for producing differentiated cells according to claim 5 .

7. A cell manufacturing device comprising: A sealed container (A1) for carrying out a step (s1) of contacting a reprogramming factor with a somatic cell in a liquid medium; A sealed container (A2) for carrying out a step (s2) of reducing the concentration of the reprogramming factor in the liquid medium; and a sealed container (A3) for carrying out a step (s3) of culturing the somatic cells in the liquid medium to establish induced pluripotent stem cells; Each of the sealed containers (A1) to (A3) has one or more openable / closable inlet / outlet ports, The sealed containers (A1) to (A3) are connected in series in the order of the steps via a connecting pipe line that can be switched between a communicating state and a non-communicating state, or are capable of being connected in series in the order of the steps, and The cell manufacturing device comprises: a feed mechanism for moving the content of the sealed container (A1) to the sealed container (A2) through the connecting pipe line switched to a communicating state, a feed mechanism for moving the content of the sealed container (A2) to the sealed container (A3) through the connecting pipe line switched to a communicating state; The cell manufacturing device.

8. The method further includes p sealed containers (B1 to Bp) for carrying out a step (s4) of expanding the induced pluripotent stem cells in a liquid medium p times (p≧1), Each of the sealed containers (B1 to Bp) has one or more openable / closable inlet / outlet ports, (i) When the number of times of expansion culture, p, is 1, The p number of sealed containers is one sealed container (B1), the sealed container (B1) is connected to or can be connected to the sealed container (A3) via a connecting pipe that can be switched between a communicating state and a non-communicating state; the cell manufacturing apparatus has a transfer mechanism that transfers the content of the sealed container (A3) to the sealed container (B1) through the connecting pipeline that has been switched to a communicating state; (ii) When the number of times of the expansion culture, p, is 2 or more, The p number of sealed containers is two or more sealed containers (B1 to Bp), the sealed container (B1) is connected to or can be connected to the sealed container (A3) via a connecting pipe that can be switched between a communicating state and a non-communicating state; the sealed containers (B1) to (Bp) are connected in series or are connectable in the order of the step (s4) via connecting pipes that can be switched between a communicating state and a non-communicating state; The cell manufacturing apparatus has a feed mechanism that moves the contents of the sealed container (A3) to the sealed containers (B1) to (Bp) in order through the connecting pipeline that is switched to a communicating state. The cell manufacturing device according to claim 7.

9. The method further includes q (q≧1) sealed containers (C1) to (Cq) for carrying out the step (s5) of inducing differentiation of the induced pluripotent stem cells, Each of the sealed containers (C1) to (Cq) has one or more openable / closable inlet / outlet ports, (i) When the q number of sealed containers is one sealed container (C1), the sealed container (C1) is connected or connectable to the sealed container (A3) or to the last sealed container (Bp) of the sealed containers (B1) to (Bp) via a connecting pipe that can be switched between a communicating state and a non-communicating state; the cell manufacturing apparatus has a transfer mechanism that transfers the contents of the sealed container (A3) or the sealed container (Bp) to the sealed container (C1) through the connecting pipeline that has been switched to a communicating state; (ii) When the q number of sealed containers is two or more sealed containers (C1) to (Cq), the sealed containers (C1) to (Cq) are connected in series or are connectable in the order of the step (s5) via a connecting pipe that can be switched between a communicating state and a non-communicating state, and the sealed container (C1) is connected or is connectable to the sealed container (A3) or to the last sealed container (Bp) among the sealed containers (B1) to (Bp) via a connecting pipe that can be switched between a communicating state and a non-communicating state, The cell manufacturing apparatus has a feed mechanism that moves the contents of the sealed container (A3) or the last sealed container (Bp) to the sealed containers (C1) to (Cq) in sequence through the connecting pipeline that has been switched to a communicating state. The cell manufacturing device according to claim 7.

10. The method further includes a sealed container (D1) for carrying out a step (s6) of removing undifferentiated cells from the content of the last sealed container (Cq) among the sealed containers (C1) to (Cq), The sealed container (D1) has one or more openable / closable inlet / outlet ports, the sealed container (D1) is connected to or can be connected to the rearmost sealed container (Cq) via a connecting pipe that can be switched between a communicating state and a non-communicating state; The cell manufacturing device comprises: A feed mechanism is provided for moving the contents of the rearmost sealed container (Cq) to the sealed container (D1) through the connecting pipe line switched to a communicating state. The cell manufacturing device according to claim 9.

11. The sealed container is A sealed container having a container body made of a flexible material; A sealed container having a container body made of a hard material; A sealed container having a container body made of a composite material of a flexible material and a hard material A sealed container selected from the group consisting of: The cell manufacturing device according to claim 7.

12. The feed mechanism is A mechanism for pushing and transferring the contents of the source sealed container to the next sealed container by adding fluid to the source sealed container; A mechanism for pushing and transferring the contents of the source sealed container to the next sealed container by reducing the volume of the source sealed container; a mechanism for transferring the contents of the source sealed container to the next sealed container by a pump device provided in the connecting pipeline; a mechanism for transferring the contents of the source sealed container to the next sealed container by applying suction force from the next sealed container to the source sealed container; A mechanism for transferring the contents of the source sealed container to the next sealed container by utilizing gravity; and A mechanism for attaching cells to magnetic microcarriers and then applying an external magnetic force to the microcarriers to move the microcarriers and the cells attached to them in the original sealed container to the next sealed container. A mechanism selected from the group consisting of: or a combination of two or more mechanisms selected from the group; The cell manufacturing device according to claim 7.

13. Further comprising a substrate for arranging all of the sealed containers; The sealed containers are disposed on the substrate, and each sealed container is fixed to the substrate; The sealed containers are connected or connectable in the order of the steps via the connecting pipes that can be switched between a communicating state and a non-communicating state. The cell manufacturing device according to claim 7.

14. The substrate can be folded in two around a folding center line, (i) in one region (e1) of two regions (e1) and (e2) on the substrate surface separated by the folding center line, a predetermined number of the sealed containers are arranged in order in one direction (d1) along the folding center line; (ii) in the other region (e2) of the two regions (e1) and (e2) of the substrate surface separated by the folding center line, the remaining sealed containers among the sealed containers are arranged in order along the folding center line in a direction (d2) opposite to the direction (d1); (iii) the rearmost sealed container among the sealed containers in the one region (e1) and the frontmost sealed container among the sealed containers in the other region (e2) are connected or in a connectable state by the connecting pipe line that can be switched between a communicating state and a non-communicating state; The cell manufacturing device according to claim 13.

15. The device further comprises two overlapping flexible sheets, the two flexible sheets are bonded to each other while leaving the regions that become all of the above-mentioned sealed containers, the region that becomes one or more inlet / outlet ports, and the region that becomes the connecting pipeline as non-bonded regions so that these regions are formed at predetermined positions between the two flexible sheets; a pressing actuator for opening and closing the connection pipe line is provided on the outer surfaces of the two flexible sheets; the pressing actuator operates to take a pressing position in which the region that will become the connecting pipeline is pressed from the outside of the flexible sheet to bring it into a non-communicating state, and a non-pressing position in which the region that will become the connecting pipeline is not pressed to bring it into a communicating state, and by operation of the pressing actuator, the region that will become the connecting pipeline functions as a connecting pipeline that can be switched between a communicating state and a non-communicating state; The region that will become one or more inlet / outlet ports extends from the region that will become each sealed container to the outer periphery of the two flexible sheets to form an open end, and the open end is provided with a structure for an openable / closable inlet / outlet port. The cell manufacturing device according to claim 7.

16. the outer peripheral shape of the two flexible sheets that are overlapped and joined to each other is a shape that can be folded in two around a folding center line; (i) In one of the two regions (e3) and (e4) separated by the folding center line, A predetermined number of regions that become sealed containers among the sealed containers are formed so as to be arranged in sequence in one direction (d3) along the folding center line, from an outer periphery of each of the regions that will become the predetermined number of sealed containers that is located farther from the folding center line, a region that will become the one or more inlet / outlet ports extends in a direction away from the folding center line and extends to an outer periphery of the two flexible sheets to form an open end, a region that becomes the connecting pipeline connecting the regions that become the sealed containers is formed between the regions that become the predetermined number of sealed containers, (ii) In the other region (e4) of the two regions (e3) and (e4) separated by the folding center line, The remaining sealed containers are arranged in order along the folding center line in a direction (d4) opposite to the one direction (d3), a region that becomes the one or more inlet / outlet ports extends from an outer periphery of each of the outer peripheries of the remaining region that becomes the sealed container, the outer periphery being located farther from the folding center line, in a direction away from the folding center line, and extends to an outer periphery of the two flexible sheets to form an open end; Between the remaining regions that will become the sealed containers, a region that will become a connecting pipe that connects the regions that will become the sealed containers is formed, (iii) the rearmost sealed container among the sealed containers in the one region (e3) and the frontmost sealed container among the sealed containers in the other region (e4) are connected by a region that becomes a connecting pipeline that crosses the folding center line; The cell manufacturing device according to claim 15.

17. A cell manufacturing method using the cell manufacturing device according to any one of claims 7 to 16, A step (s1) of contacting somatic cells with reprogramming factors in a liquid medium in the sealed container (A1); After completion of the step (s1), a step (s2) is performed in which the content of the sealed container (A1) is transferred into the sealed container (A2) through the connecting pipe line switched to a communicating state, and a concentration of the reprogramming factor in the liquid medium is reduced in the sealed container (A2); After completion of the step (s2), a step (s3) is performed in which the content of the sealed container (A2) is transferred into the sealed container (A3) through the connecting pipe line that has been switched to a communicating state, and induced pluripotent stem cells are established in the liquid medium in the sealed container (A3). The cell production method as described above, comprising at least the steps of: