Production method for induced pluripotent stem cells

Abstract

The present invention provides a production method for iPS cells that uses a cell processing device. The cell processing device has a closed system portion at which a sealed container for material supply and a centrifugation chamber for counterflow centrifugation are connected by a connection conduit. The production method involves performing the following steps in order while keeping the closed system portion closed: a step (s1) for bringing an initialization factor into contact with somatic cells while performing counterflow centrifugation in the centrifugation chamber; and a step (s2) for establishing iPS cells from the somatic cells.

Description

Method for producing induced pluripotent stem cells

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

[0002] In recent years, research into regenerative medicine using differentiated cells derived from induced pluripotent stem cells (hereinafter also referred to as 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 (Non-Patent Documents 1 and 2).

[0003] International Publication No. 2017 / 040548

[0004] 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

[0005] When iPS cell-derived cells are autologously transplanted into a patient for precision medicine (treatment tailored to an individual patient), it is preferable to collect as few somatic cells as possible from the patient in order to reduce the biological burden on the patient. Furthermore, from an economic perspective, it is preferable to reduce the cost of cell processing. To this end, it is preferable to make the configuration of cell processing equipment smaller and simpler, and to establish iPS cells with fewer processing steps.

[0006] Systems for automatically producing iPS cells from somatic cells in a closed system have been proposed (e.g., Patent Document 1). However, conventional systems are large-scale systems in which independent processing devices dedicated to each process are connected in series, and building and automatically operating such systems requires significant costs. Furthermore, such relatively large systems are typically not suitable for processing small amounts of somatic cells. Considering losses at the connections between the devices, a large amount of somatic cells and viral vectors must be input. Therefore, the present inventors have addressed the problem that a system for automatically producing iPS cells in a closed system with a smaller volume, lower cost, and higher efficiency, while reducing the burden on cell production workers, is yet to be provided, which is suitable for precision medicine.

[0007] An object of the present invention is to provide a new method for producing iPS cells that alleviates the above problems, requires a smaller amount of patient-derived somatic cells as a raw material, and can reduce the use of reagents (e.g., viral vectors, etc.) compared to conventional methods.

[0008] The main features of the present invention are as follows: [1] A method for producing induced pluripotent stem cells using a cell processing apparatus, wherein the cell processing apparatus has a closed system in which a sealed container for supplying materials and a rotatable sealed chamber configured for counterflow centrifugation are connected via a connecting pipeline, and the method comprises the steps of: (s1) contacting somatic cells with reprogramming factors while performing counterflow centrifugation in the sealed chamber while maintaining the closed nature of the closed system; and (s2) establishing induced pluripotent stem cells from the somatic cells, in this order. [2] The somatic cells are blood cells, and the method further comprises, prior to step (s1), a step (s0) of separating blood cells from whole blood in the sealed chamber while maintaining the closed nature of the closed system, wherein in step (s0), blood cells are separated from whole blood by elutriation achieved by counterflow centrifugation and the blood cells are left in the sealed chamber. [3] The method for producing induced pluripotent stem cells according to [1] or [2], wherein the central axis of the rotational motion of the sealed chamber passes outside the internal space of the sealed chamber. [4] The sealed chamber has an inlet port and an outlet port on the wall on the central side of the rotational motion, and is configured so that materials can be introduced and discharged through the inlet port and the outlet port when the sealed chamber is rotating or stationary, and a tube connected to the inlet port extends into the sealed chamber and has an open end in an area radially outward of the rotational motion so as to generate a counterflow during the rotational motion of the sealed chamber. [5] The method for producing induced pluripotent stem cells according to any one of [1] to [4], wherein the shape of the internal space defined by the inner wall surface surrounding the internal space of the sealed chamber is conical, with the base of the cone located on the central side of the rotational motion and the apex of the cone located radially outward of the rotational motion.[6] The method for producing induced pluripotent stem cells according to [2] above, wherein the step (s0) comprises: a step (s0-1) of removing red blood cells from whole blood by elutriation; and a step (s0-2) of removing granulocytes and monocytes from the whole blood from which the red blood cells have been removed by further elutriation after the step (s0-1), leaving lymphocytes in the sealed chamber. [7] The method for producing induced pluripotent stem cells according to [6], wherein in the step (s0-2), while keeping the counterflow flow rate Q [ml / min] constant, the relative centrifugal force f1 [G] acting on the contents in the sealed chamber is gradually reduced over a period of 20 to 100 seconds, thereby reducing the ratio f1 / Q of the relative centrifugal force f1 [G] to the flow rate Q from 50 to 70 to 20 to 35, and then, while keeping the relative centrifugal force f1 constant, the flow rate Q is gradually increased over a period of 30 to 100 seconds, thereby reducing the ratio f1 / Q from 20 to 35 to 10 to 20. [8] The method for producing induced pluripotent stem cells according to any one of [1] to [7] above, wherein in step (s1), the ratio f1 / Q of the relative centrifugal force f1 [G] acting on the contents in the sealed chamber to the counterflow flow rate Q [ml / min] is 50 to 400, and the time for which step (s1) is performed is 30 to 180 minutes. [9] The method for producing induced pluripotent stem cells according to [8] above, wherein the time for which step (s1) is performed is 120 minutes.

[10] The method for producing induced pluripotent stem cells according to any one of [1] to [9] above, wherein steps (s1) and (s2) are performed under suspension culture conditions.

[11] The method for producing induced pluripotent stem cells according to any one of [1] to

[10] above, further comprising, after step (s2), a step (s3) of expanding the induced pluripotent stem cells in the sealed chamber or in a sealed container for expansion culture aseptically connected to the closed system part while maintaining the closed nature of the closed system part.

[12] A method for producing differentiated cells, comprising the steps of the method for producing induced pluripotent stem cells according to any one of [1] to

[10] above, and further comprising, after step (s2), a step (s4) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closed nature of the closed system part, wherein (I) materials necessary for differentiation induction are supplied into the sealed chamber, and step (s4) is carried out in the sealed chamber, or (II) the induced pluripotent stem cells present in the sealed chamber are moved into a sealed container for differentiation induction that is aseptically connected to the closed system part, and materials necessary for differentiation induction are supplied into the sealed container for differentiation induction, and step (s4) is carried out in the sealed container for differentiation induction.

[13] The method for producing differentiated cells described in

[12] , further comprising, between steps (s2) and (s4), a step (s3) of expanding the artificial pluripotent stem cells in the sealed chamber or in the sealed container for expansion culture connected aseptically to the closed system part, while maintaining the closure of the closed system part.

[0009] The production method according to the present invention uses a cell processing device equipped with a rotatable sealed chamber capable of performing counterflow centrifugation, and carries out a step (s1) of contacting somatic cells with reprogramming factors while performing counterflow centrifugation in the sealed chamber. More specifically, in step (s1), somatic cells dispersed in a liquid are collected in a small region (a region radially outward from the center of rotation) within the sealed chamber by centrifugal force, and while maintaining this state, a fluid containing reprogramming factors is passed through the sealed chamber from the outer periphery of the rotation to the center as a counterflow (a flow toward the center of rotation against the centrifugal force). This causes the somatic cells collected in the small region to float as a cell bed, enabling the reprogramming factors to come into contact with the somatic cells with a high probability.

[0010] Hereinafter, the "rotatable sealed chamber configured for counterflow centrifugation" used in the present invention will be referred to as the "centrifugation chamber" or "chamber" for short. As exemplified by the centrifugation chamber provided in the CTS Rotea Counterflow Centrifugation System (Thermo Fisher Scientific) described below, centrifugation chambers for counterflow centrifugation have a smaller volume than other conventional commercially available sealed chambers for centrifugation. This allows for the appropriate processing of smaller amounts of somatic cells, enabling the required amount of iPS cells to be obtained. This allows for smaller amounts of somatic cells (e.g., whole blood) to be collected from patients, thereby reducing the biological burden on the patient.

[0011] As described above, in step (s1) of contacting somatic cells with reprogramming factors while performing counterflow centrifugation, centrifugal force causes the somatic cells to aggregate in a predetermined small area within the centrifugation chamber, and a fluid containing the reprogramming factors passes between the aggregated somatic cells as a counterflow. This increases the probability that the reprogramming factors will encounter and come into contact with the somatic cells. This reduces the use of expensive reagents such as viral vectors for introducing reprogramming factors into somatic cells, thereby reducing the cost of iPS cell production. Step (s1) of the production method according to the present invention can also be referred to as a spinoculation step.

[0012] Establishing iPS cells from somatic cells typically requires multi-stage, lengthy processing steps, resulting in high costs for numerous specialized devices and labor costs for operating them. In a preferred embodiment of the present invention, blood cells are used as the somatic cells serving as the source of iPS cells, and a step (s0) of separating the blood cells from whole blood in the centrifuge chamber is performed prior to the step (s1). That is, the step (s0) of separating blood cells from whole blood, the step (s1) of contacting the blood cells with reprogramming factors, and the step (s2) of establishing iPS cells are all performed in a single, closed-system cell processing device. This reduces cell contamination during production and enables the automation of the entire production process, from somatic cell isolation to iPS cell establishment, using a single cell processing device. This reduces the number of personnel required for iPS cell production at conventional cell culture processing facilities (CPCs). Furthermore, the use of a closed-system device eliminates the need to upgrade the facility to Grade A, thereby reducing the cost of iPS cell production.

[0013] FIG. 1 is a block diagram showing an example of the closed system configuration of a cell processing apparatus used in the manufacturing method of the present invention. The thick lines designated by the reference numerals 30B, 30C, 30D, 30E, 30F, 30G, and 30H indicate connecting pipes made of flexible tubing or the like. Connectors and other components are omitted from the illustration. The solid line designated by the reference numeral 30A indicates a connecting pipe similar to the connecting pipes designated by the reference numerals 30B to 30H, but is shown thinner than the other connecting pipes for easier identification. The connecting pipe 30A and the connecting pipes (30B, 30C, and 30D) intersect with each other (i.e., their internal flow paths do not intersect). FIG. 2 is a schematic diagram illustrating the principle of counterflow centrifugation in a sealed chamber of a cell processing apparatus used in the manufacturing method of the present invention, showing a cross-sectional view of the interior of the sealed chamber. FIG. 3 is a schematic diagram illustrating an example of counterflow centrifugation in the sealed chamber shown in FIG. 2, showing a cross-sectional view of the interior of the sealed chamber. Figure 4 is a photograph (advertising photograph issued by the manufacturer of the cell processing device) of a commercially available cell processing device preferably used in the manufacturing method of the present invention, illustrating a state in which a closed system component is attached to the mechanical component of the cell processing device, enabling cell processing. Figure 5 is a photograph (advertising photograph issued by the manufacturer of the cell processing device) showing a disposable unit component called a single-use kit that directly engages with the mechanical component of the closed system component of the cell processing device shown in Figure 4. Figure 6 is a photograph (published by the manufacturer of the cell processing device) showing only the mechanical component of the cell processing device shown in Figure 4 with the closed system component removed. Figure 7 is an actual photograph of the closed system component shown in Figure 1, showing various sealed containers connected to the single-use kit shown in Figure 5. Figure 8 is an actual photograph of a cell processing device with the closed system component shown in Figure 7 attached to the mechanical component. Figures 9(a) to 9(c) are photographs illustrating the process of hemolyzing and removing red blood cells from whole blood by performing counterflow centrifugation in a sealed chamber in an embodiment of the present invention. The photograph in FIG. 9 is an image captured at the moment when the rotating sealed chamber passes in front of the imaging device (the same applies to FIGS. 10, 11, and 12(a)).Figures 10(a) to 10(c) are photographs showing the process of performing counterflow centrifugation in a sealed chamber to further remove granulocytes and monocytes from whole blood from which red blood cells have been removed, leaving lymphocytes in the sealed chamber, in an example of the present invention. Figure 11 is a photograph showing the process of contacting white blood cells with a Sendai virus vector while performing counterflow centrifugation in a sealed chamber, in an example of the present invention. Figure 12 is a photograph showing the recovery of cells into which reprogramming factors have been introduced into a syringe through a filter that removes dead cells. Figure 13 is a micrograph of iPS cells after 3D culture in Example 1. The scale bar in the figure is 200 μm. Figure 14 is a graph showing the results of flow cytometry analysis of the expression of TRA-1-60, OCT3 / 4, and SSEA4 in the iPS cells obtained in Example 1. Figure 15 is a fluorescent micrograph showing the results of cell staining to confirm the expression of NANOG and OCT3 / 4 in the iPS cells obtained in Example 1. FIG. 16 is a graph showing the results of examining whether the number of white blood cells isolated in Example 2 differs depending on the donor. FIG. 17 is a graph showing the results of examining the effect of elutriation in Example 3. FIG. 18 is a graph showing the results of examining the time of SeV (Sendai virus) infection in Example 4. FIG. 19 is a graph showing the results of examining the ratio f1 / Q of relative centrifugal force f1 to flow rate Q in Example 5. FIG. 20 is a photograph and graph showing the results confirming that iPS cells produced in Example 6 can be induced to differentiate. FIG. 21 is a photograph and graph showing the results of evaluating cardiomyocytes obtained by inducing differentiation of iPS cells produced in Example 7. FIG. 22 is a photograph (left) and a fluorescence microscope photograph (right) of iPSC spheroids cultured 15 days after SeV infection.

[0014] A method for producing iPS cells according to the present invention is described in detail below. This method produces iPS cells using a cell processing apparatus. The cell processing apparatus has a closed system, which, as illustrated in FIG. 1 , comprises sealed containers (e.g., 21-26) for supplying materials and a centrifuge chamber 10 connected via connecting pipelines (e.g., 30A-30G, 33, 34, 35). FIG. 1 illustrates a preferred embodiment of the closed system, which includes not only the components necessary for steps (s1) and (s2) described below, but also the components necessary for step (s0), described below. The method involves operating the cell processing apparatus to perform step (s1) in the centrifuge chamber 10 while maintaining the closed nature of the closed system, followed by step (s2). Step (s2) may be performed in the centrifuge chamber 10 or in another container, as described below. Step (s1) is a step of contacting somatic cells with reprogramming factors while performing counterflow centrifugation. Step (s2) is a step of establishing iPS cells from the somatic cells contacted with the reprogramming factors (in one embodiment, a step of culturing the somatic cells contacted with the reprogramming factors to establish iPS cells). The operation of the cell processing device for carrying out steps (s1) and (s2) may be fully automatic, or may involve manual operation by an operator during each step or between steps.

[0015] By carrying out steps (s1) and (s2) using the cell processing device, the reprogramming factors are efficiently brought into contact with the somatic cells under the suspension conditions characteristic of counterflow centrifugation, and iPS cells are then established, thereby achieving the above-mentioned effects and alleviating or resolving the problems mentioned above.

[0016] Other processing steps may be added between steps (s1) and (s2). Furthermore, the following operations may be appropriately performed in steps (s1) to (s2): (i) circulating a cell suspension (hereinafter sometimes simply referred to as a suspension) containing the somatic cells to be processed or a medium (liquid) through a predetermined path including the centrifugation chamber in order to perform counterflow centrifugation favorably; (ii) transferring the somatic cells to be processed from the centrifugation chamber to a sealed container in the closed system portion or to an external device, and then returning them to the centrifugation chamber; (iii) conducting random testing of samples of the somatic cells in the middle of processing in accordance with additional processing such as quality control measurements. These operations may be performed automatically, manually, or a combination of these.

[0017] (Cell Processing Apparatus) The cell processing apparatus usable in the present invention has a closed system portion and a mechanical portion. These will be described in detail below. The mechanical portion drives the closed system portion while maintaining its closed nature, and cooperates with the closed system portion to sequentially perform steps (s1) to (s2). Such a cell processing apparatus may be equipped with a centrifugation chamber capable of performing counterflow centrifugation. The "CTS Rotea Counterflow Centrifugation System" manufactured by Thermo Fisher Scientific is particularly recommended (hereinafter, this cell processing apparatus will also be referred to as Rotea). Rotea is a commercially available product that boasts automatic "cell washing, concentration, and recovery" through its counterflow centrifugation function. The basic configuration and basic operation of Rotea are described in detail in the manufacturer's manual. The configuration and functions of Rotea are described in detail, for example, in International Publication WO 2018 / 204992 A1 and International Publication WO 2019-140491 A1. However, iPS cells are known to be vulnerable to physical stress, and even when manually established, there is considerable individual variability depending on the operator's handling. Therefore, the idea of ​​utilizing the counterflow centrifugation functions (washing, concentration, recovery) of Rotea, a cell processing device for CAR-T cells, for the establishment of iPS cells (particularly, the step (s1) of contacting somatic cells with reprogramming factors) had not previously been conceived, and the process for doing so had not previously been disclosed.

[0018] The following describes the manufacturing method in more detail, taking Rotea as an example of a cell processing device preferred for the present invention, and explaining its configuration in detail. However, the manufacturing method can also be implemented with other cell processing devices configured to perform counterflow centrifugation by modifying and operating the cell processing device in accordance with the following explanation.

[0019] (Configuration of Rotea) As illustrated in FIG. 4, Rotea has a closed system portion 300 and a mechanical portion 400 that operates the closed system portion 300 from the outside in order to process cells within the closed system portion 300. The closed system portion 300 is detachable from the mechanical portion 400 and is disposable. The mechanical portion 400 includes a peristaltic pump, pinch valves, a drive motor, various sensors, and a control unit including a control program configured to implement the manufacturing method. Each element of the mechanical portion 400 acts on the attached closed system portion 300 from the outside, thereby maintaining the closed nature of the closed system portion and sequentially implementing each step of the manufacturing method in accordance with commands from the control unit. FIG. 4 illustrates a conventional, general usage state in which the closed system portion 300 is attached to the mechanical portion 400.

[0020] FIG. 5 shows the central unit of the closed system portion. This unit portion is a disposable portion (hereinafter also referred to as a single-use kit) provided by the manufacturer. The single-use kit includes a centrifuge chamber 310 and is configured to be detachably engaged with the mechanism portion 400. Although not shown in FIG. 5, the connecting pipes 320a and 320b are connected together as shown in FIG. 4, and the connecting pipe 320a is the portion attached to the peristaltic pump. The connecting pipe 320b is connected to one port of the centrifuge chamber 310 corresponding to the connecting pipe 34 in FIG. 1, and the connecting pipe 330 is connected to the other port of the centrifuge chamber 310 corresponding to the connecting pipe 35 in FIG. 1. The connecting pipe 300P in FIG. 4 is added to the connecting pipes 300P1 and 300P2 of the single-use kit shown in FIG. 5, and the sealed container 200 in FIG. 4 is connected to form the closed system portion 300. The sealed container can be provided as needed for purposes such as storing various materials required for the manufacturing process, serving as a temporary passageway when circulating materials or suspensions within the closed system, storing waste liquids, recovering intermediate products, and recovering iPS cells.

[0021] Figure 6 is a photograph of the Rotea mechanism 400. The mechanism 400 shown in Figure 6 has its front door 430 open, allowing the single-use kit portion of the closed system to be attached to the front. The front of the mechanism 400 is equipped with a total of 10 pinch valve heads (400A-400G, 400H, 400J, 400K), a peristaltic pump 400M, a cylindrical recess 410 for the rotation of the centrifuge chamber, a chamber carrier 420 connected to the drive motor to rotate the centrifuge chamber, various sensors, and operation buttons. The various sealed containers included in the closed system are suspended from hanger posts 440. The door 430 also functions as a fixed base facing the pinch valve head (movable head) and constitutes a pinch valve that opens and closes the pinch points on each connecting line (soft tubing) in the single-use kit. Even when using a cell processing device other than Rotea, the present invention can be implemented by arranging the sealed container, connecting pipelines, pinch valves, peristaltic pumps, etc. in an equivalent manner to the Rotea exemplified in this specification and operating them in the same manner as the Rotea exemplified in this specification.

[0022] In the example shown in FIG. 1 , the sealed containers are used for the following purposes: sealed container 21 contains a liquid culture medium and is connected to connecting pipeline 30B. The liquid culture medium is also used as a liquid for washing the flow paths and the centrifugation chamber. sealed container 22 contains a liquid containing raw somatic cells and is connected to connecting pipeline 30C. In the example shown in FIG. 1 , sealed container 22 contains (diluted) whole blood in order to perform step (s0) of separating raw blood cells from whole blood before step (s1). sealed container 23 contains a hemolyzing agent (a buffer solution for lysing red blood cells (e.g., a buffer solution mainly composed of ammonium chloride, potassium bicarbonate, and EDTA or its salts)) and is connected to connecting pipeline 30D. sealed container 24 is an empty container connected to connecting pipelines 30A and 30E and is used as a temporary retention space for materials being processed, suspensions, etc., when circulating in the centrifugation chamber 10. In the clockwise direction in FIG. 1 , this circulation flow path is a circular flow path connecting the centrifugation chamber 10, connecting conduit 35, branch point 36, connecting conduit 30A, sealed container 24, connecting conduit 30E, junction 32, connecting conduit 33, connecting conduit 34 (which receives pressure from the peristaltic pump), and centrifugation chamber 10 in this order. In the example of FIG. 1 , syringes S1 and S2 are connected to connecting conduit 30A for obtaining test samples from the conduit. Sealed container 25 contains a liquid medium for flushing out the reprogramming factors and is connected to connecting conduit 30F. In the example of FIG. 1 , to prevent the reprogramming factors from remaining in the sealed container or connecting conduit (i.e., to utilize as much as possible), the reprogramming factors are injected into the connecting conduit from syringes S3 or S4 connected to connecting conduit 30F. The reprogramming factors injected from the syringes into connecting conduit 30F are swept away by the liquid medium flowing out of sealed container 25 and can flow into the centrifugation chamber 10. The sealed container 26 is a container for receiving various waste liquids generated in the processing steps, and is connected to a connecting pipe line 30G.

[0023] The connecting pipe 30H can be preferably used for output (recovery), etc. Although not shown in Fig. 1, a sealed container, a syringe, various external processing devices, etc. can be connected to the connecting pipe 30H as needed.

[0024] The structure of each sealed container is not particularly limited, and reference can be made to sealed containers conventionally used in Rotea. Preferred examples include flexible bags and syringes made of flexible films or flexible sheets. The flexible films are flexible enough to be deformed according to the amount of contents contained in the bags. The flexible films are suitable for preventing the O2 required for cell culture. ２ YaCO ２ The capacity of each sealed container varies depending on the application and is not particularly limited, but is about 20 to 200 ml when used to supply raw somatic cells, and about 200 to 3000 ml when used to supply liquid culture medium or to store waste liquid.

[0025] (Pinch Points) Each connecting pipe line (30A, 30B, 30C, 30D, 30E, 30F, 30G, 30H, 37, 38) has a pinch point (30A1, 30B1, 30C1, 30D1, 30E1, 30F1, 30G1, 30H1, 30J1, 30K1) made of soft tubing. As shown in FIG. 1, each pinch point is provided at a predetermined position in the single-use kit. When the single-use kit is attached to the mechanism portion 400 (FIG. 6), as described above, each pinch point (30A1 to 30H1, 30J1, 30K1) is positioned corresponding to the pinch valve heads (400A to 400H, 400J, 400K) of the mechanism portion 400, and is compressed / released in response to the operation of each pinch valve head, thereby opening or closing each connecting pipe line. In the following description of the opening and closing of each pinch point, the description of the opening and closing of the pinch valve corresponding to each pinch point will be omitted.

[0026] (Arrangement of Connecting Pipes) The connecting pipes (30B, 30C, 30D) extending from the sealed containers (21, 22, 23) enter the single-use kit, pass through pinch points (30B1, 30C1, 30D1), and are connected to a single bubble trap 31. The connecting pipe 38 extending from the lower side of the bubble trap 31 passes through pinch point 30K1 and then reaches junction 32. Meanwhile, the connecting pipes (30E, 30F) extending from the sealed containers (24, 25) enter the single-use kit, pass through pinch points (30E1, 30F1), and then reach junction 32. These connecting pipes (38, 30E, 30F) merge into one at junction 32 to form connecting pipe 33. The connecting pipe 33 branches into connecting pipe 30H for output. After passing through pinch point 30H1, connecting line 30H can be connected to various containers or devices as needed. Connecting line 33 also branches into connecting line 34, which is connected to a peristaltic pump. Connecting line 34 is connected to a first port of centrifuge chamber 10. Connecting line 35 is connected to a second port of centrifuge chamber 10, and connecting line 35 reaches branch point 36, where it branches into connecting lines 30A, 37, and 30G. After passing through pinch point 30A1, connecting line 30A exits the single-use kit and connects to sealed container 24. After passing through pinch point 30J1, connecting line 37 connects below bubble trap 31 and merges with the flow exiting bubble trap 31 to form connecting line 38. As described above, connecting line 38 passes through pinch point 30K1 and reaches merge point 32. The connecting pipe 30G passes through the pinch point 30G1 without joining with other connecting pipes (i.e., passing near the joining point 32), exits the single-use kit, and is connected to the sealed container 26.

[0027] The peristaltic pump 400M, shown by a dashed line in FIG. 1 , is a device belonging to the mechanical section 400. It is also called a tube pump or peristaltic pump. It uses a rotating roller head to sequentially press a connecting conduit (flexible tube) from the outside to move a fluid within the connecting conduit. In FIG. 1 , when the peristaltic pump 400M operates counterclockwise, material within the connecting conduit 34 flows in the direction of arrow a1. This flow direction generates a counterflow within the centrifuge chamber 10. Hereinafter, the flow direction of arrow a1 will also be referred to as the "forward direction." Conversely, when the peristaltic pump 400M operates clockwise in FIG. 1 , material within the connecting conduit flows in the direction of arrow a2. Hereinafter, the flow direction of arrow a2 will also be referred to as the "reverse direction." By controlling the opening and closing of any pinch valve and the rotation of the peristaltic pump, material can be moved in any direction within any selected connecting conduit.

[0028] The tubing members constituting the connecting pipeline are preferably made of a soft material (such as silicone or vinyl chloride) because the pinch valve and peristaltic pump are used. The connecting pipeline may also include tubing members made of a hard material at joints and other portions.

[0029] (Closed or airtight) The closed or airtight nature of the interior of the closed system part means not only a state in which the interior is kept airtight or liquidtight, but also a state in which the interior is isolated from the outside world to such an extent that microorganisms and viruses cannot enter from the outside world, i.e., to such an extent that the sterility of the interior is maintained. For example, a sealed container provided with a porous filter (e.g., pore size of about 0.2 μm or less, particularly about 0.1 to 0.2 μm) that does not allow bacteria or viruses to pass through but allows fluids (particularly gases) to pass through is airtight because outside air can pass through the porous filter and into the sealed container but bacteria and viruses cannot enter, and the sterility of the closed system part is maintained. Also, the wall of the container is a gas-permeable membrane that does not allow bacteria, viruses, etc. to pass through, and the container is airtight. ２ Gas molecules and CO ２A container that is permeable to gas molecules is also considered airtight. Therefore, a "closed system" does not only refer to an airtight or liquidtight system, but also to a system whose interior is isolated from the outside world to the extent that microorganisms and viruses cannot enter from the outside world.

[0030] (Centrifugal Chamber and Counterflow Centrifugation) As shown in Fig. 1, the centrifugation chamber 10 is an element included in the closed system portion, and has a rotatable pipe joint structure similar to a rotary joint, which allows it to rotate while maintaining the hermeticity of the closed system portion, even while the connecting pipes (34, 35) remain connected and even while liquids and the like are flowing in and out through the connecting pipes. In the example of Fig. 1, the chamber 10 is driven to rotate in the direction of arrow a3 about a central axis y1 perpendicular to the plane of the paper in Fig. 1.

[0031] 2 is a diagram illustrating counterflow centrifugation in a centrifugal chamber 10, showing a schematic cross section of the chamber 10 and the connecting pipes in a block diagram format. As shown in FIG. 2, the central axis (rotational axis) y1 of the rotational motion of the centrifugal chamber 10 passes outside the internal space of the chamber 10, and the chamber 10 is driven to rotate (revolve) around the rotational axis y1 as indicated by arrow a3. Thus, the internal space of the chamber 10 has an outer periphery side (a side radially outward from the rotational axis y1) and a rotational center side (a side closer to the rotational axis y1).

[0032] The centrifugation chamber 10 has two ports on the wall on the rotation center side, and these two ports function as an inlet port 12 and an outlet port 13 in the flow in the forward direction a1. The roles of these inlet and outlet ports are reversed in the flow in the reverse direction. In the example of Fig. 2, the inlet port 12 is connected to a connecting pipe 33, and the outlet port 13 is connected to a connecting pipe 34, so that liquids and materials can be introduced and discharged through the inlet port 12 and the outlet port 13 whether the chamber 10 is rotating or stationary.

[0033] The structure of the centrifuge chamber 10 is not particularly limited, and a counterflow inlet on the outer periphery of the chamber can be used to generate a counterflow during rotation of the chamber. In the Rotea chamber, as shown in Figure 2, a tube 14 connected to an inlet port 12 extends into the centrifuge chamber 10 and has an open end at the outer periphery. The flow f2 of liquid m1 flowing out of the open end of the tube 14 inside the centrifuge chamber is called the counterflow, and flows from the outer periphery to the center of rotation in the opposite direction to the relative centrifugal force f1. The antagonistic action between the relative centrifugal force f1 and the counterflow f2 achieves fractionation (elutriation) based on particle size and weight. Specifically, as illustrated in Figure 2, larger, heavier particles e1 collect at the outer periphery, while smaller, lighter particles e2 collect at the center of rotation, resulting in layered separation within the centrifuge chamber. Furthermore, even smaller particles (such as viral vectors used for contacting with reprogramming factors) do not remain in the centrifugation chamber, but flow as a counterflow together with the liquid m1.

[0034] 3A and 3B are diagrams showing an example of counterflow centrifugation. In the example of Fig. 3A, as shown in Fig. 3A, only one type of particle (e.g., somatic cells) e1 is dispersed in a liquid (e.g., liquid medium) m1 inside a centrifuge chamber 10. The operation of concentrating the dispersed particles e1 on the outer periphery by centrifugal force and counterflow as shown in Fig. 3B also corresponds to counterflow centrifugation in that it separates the particles into a particle layer e1a and a liquid layer m1a.

[0035] (Shape and volume of the internal space of the centrifugation chamber) The shape of the internal space of the centrifugation chamber is determined by the inner wall surface surrounding the internal space. A preferred basic shape of the internal space is a cone, with the bottom of the cone located toward the center of rotation and the apex of the cone located toward the outer periphery, as shown in Figure 2. The volume of the chamber is not particularly limited, but from the viewpoint of more efficiently processing a small amount of somatic cells, it is preferably about 20 to 10 ml, more preferably about 15 to 10 ml. In one embodiment, the volume of the centrifugation chamber of the Rotea used in the examples of the present invention is 10 ml.

[0036] (Ratio f1 / Q of relative centrifugal force f1 [G] to flow rate Q) In order to perform counterflow centrifugation more effectively and appropriately, the ratio f1 / Q between the relative centrifugal force f1 [G] corresponding to the rotation speed of the chamber and the flow rate Q of the counterflow f2 sent by the peristaltic pump is important. The specific value of the ratio f1 / Q will be described later.

[0037] (Controller) The controller (including a computer and computer program) included in the mechanism operates each pinch valve, peristaltic pump, and centrifugal chamber to automatically perform steps (s1) to (s2) of the manufacturing method in sequence. In a preferred embodiment, it also automatically performs step (s0) described below, automatically performing the entire processing steps (s0, s1, s2). The computer program executed by the controller's computer allows for preset parameters, such as the timing of opening and closing any pinch valves, the operation period of the peristaltic pump, the feed rate, the counterflow rate Q, the operation period and relative centrifugal force f1 of the centrifugal chamber, etc., to be entered or modified. By placing the necessary sealed containers in the closed system and executing the preset computer program, the steps of the novel manufacturing method of the present invention are automatically performed in sequence. The execution of the computer program (i.e., the operation of Rotea) can be controlled by inputting commands using switches, etc., to temporarily stop and restart the device, stop a process while continuing circulation, or transition to the next process. Therefore, manual processing (such as taking samples for quality control or injecting materials into the pipelines) can be added as desired during each process or between processes.

[0038] (Preparation for Operation of the Apparatus) In order to carry out each step of the production method using a cell processing apparatus equipped with a centrifugation chamber such as Rotea, preparations such as filling each flow path of the closed system (centrifugation chamber, connecting pipes, circulation flow path) with a liquid medium are required. Such preparations can be carried out appropriately according to the operation manual of the cell processing apparatus so that each step of the production method is carried out appropriately.

[0039] (Step (s1): Contacting somatic cells with reprogramming factors) First, as shown in FIG. 3( a), the somatic cells to be processed and a medium (such as a liquid culture medium) m1 are sent from a predetermined sealed container to a centrifugation chamber 10. Next, the medium (first suspension) in which these somatic cells are dispersed is circulated within a circulation channel (described below) including the chamber 10, while undergoing counterflow centrifugation. As a result, a cell layer e1a is formed within the chamber 10, as shown in FIG. 3( b). Next, while maintaining the cell layer e1a, a medium (second suspension) in which reprogramming factors are dispersed is supplied into the circulation channel. As a result, the reprogramming factors pass between the somatic cells densely packed in the cell layer e1a while circulating within the circulation channel, thereby increasing the probability of contact with the somatic cells. The iPS cells to be produced, the raw somatic cells and medium, the reprogramming factors, and other necessary materials will be described below.

[0040] (Ratio f1 / Q in Step (s1)) In the counterflow centrifugation of step (s1), the ratio f1 / Q of the relative centrifugal force f1 [G] acting on the somatic cells in the chamber to the counterflow flow rate Q [ml / min], and the time for performing step (s1), are important for the reprogramming factors to contact the somatic cells as efficiently as possible and for the reprogramming factors to contact as many somatic cells as possible. Although this ratio varies depending on the shape, capacity, temperature, fluid properties, particle shape, etc. of the centrifugation chamber, when Rotea is used at room temperature and a conventional liquid medium is used as the medium for the first and second suspensions, the ratio f1 / Q is approximately 50 to 400 [G·min / ml]. In this case, the relative centrifugal force f1 is preferably approximately 300 to 3,000 G, more preferably approximately 400 to 2,500 G, and the flow rate Q is preferably approximately 1 to 30 ml / min, more preferably approximately 1 to 10 ml / min. The combination of the relative centrifugal force f1 and the flow rate Q is selected so that the ratio f1 / Q is about 50 to 400 [G·min / ml]. The time for carrying out step (s1) under the above conditions, i.e., the time for contacting the reprogramming factors with as many somatic cells as possible, is not particularly limited as long as it is possible to achieve the establishment of desired iPS cells, but is preferably about 30 to 180 minutes, and more preferably about 120 minutes.

[0041] (Circulation Flow Path) To perform counterflow centrifugation, it is necessary to circulate a liquid or suspension at an appropriate ratio f1 / Q in a predetermined circulation flow path (including the centrifugation chamber 10). During this circulation, the necessary cell layer forms inside the chamber 10. A preferred circulation flow path that can be used in the closed system portion of the Rotea is, for example, a circular flow path connecting the outlet of the centrifugation chamber 10, connecting line 35, branch point 36, connecting line 37, pinch point 30J1, pinch point 30K1, connecting line 38, junction 32, connecting line 33, connecting line 34, and the inlet of the centrifugation chamber 10 in this order, as shown in Figure 1. This circulation flow path is preferably used when continuing circulation while performing counterflow centrifugation. It is also possible to configure the circulation flow path to pass through a sealed container 24 for circulation. 1 , the circulation flow path is a circular flow path that connects, in this order, the outlet of the centrifugation chamber 10, connecting pipe 35, branch point 36, connecting pipe 30A (including pinch point 30A1), sealed container 24, connecting pipe 30E (including pinch point 30E1), junction 32, connecting pipe 33, connecting pipe 34, and the inlet of the centrifugation chamber 10. This circulation flow path can be preferably used, for example, in a circulation process in which lymphocytes are separated from other blood cells by elutriation, sent to a bag (sealed container), allowed to temporarily reside in the bag, and then the lymphocytes are sent back to the centrifugation chamber.

[0042] (Step (s2): Step of establishing iPS cells) In this step, the somatic cells that were contacted with the reprogramming factors in step (s1) are cultured in a liquid medium to establish iPS cells. Step (s2) may be performed inside the centrifuge chamber 10, or may be performed while gently circulating the somatic cells that have been contacted with the reprogramming factors in an arbitrary circulation channel, or may be performed by transferring the somatic cells that have been contacted with the reprogramming factors to an arbitrary sealed container or external processing device. The iPS cells are not particularly limited as long as the desired iPS cells can be obtained, and may be in the form of, for example, two-dimensional colonies, spheroids, or the like.

[0043] In one embodiment, step (s2) may be carried out in a centrifuge chamber 10 configured to adjust the temperature to a temperature suitable for establishing iPS cells (in one embodiment, a temperature suitable for culturing somatic cells contacted with reprogramming factors to establish iPS cells) (e.g., 30°C to 40°C, preferably 37°C). Examples of configurations for adjusting the temperature inside the centrifuge chamber to a temperature suitable for establishing iPS cells include providing the chamber with a heater that heats the inside of the centrifuge chamber to an appropriate temperature, and feeding a liquid medium heated to an appropriate temperature into the centrifuge chamber. These configurations may be used alone or in combination.

[0044] Examples of heaters that heat the interior of the centrifuge chamber or the liquid medium to an appropriate temperature include electric heaters attached to the exterior of the centrifuge chamber or the sealed container, heating lamps installed at a distance from the object to be heated, hot air heaters that send hot air into the space surrounding the object to be heated, and air conditioners that adjust the temperature inside the case or room surrounding the entire Rotea to a temperature appropriate for the establishment of iPS cells. These configurations may be used alone or in any combination.

[0045] The temperature control for adjusting the temperature inside the centrifuge chamber to a temperature suitable for the establishment of iPS cells can be performed using a conventional control method such as feedback control, and a temperature sensor or controller can be used appropriately. In this embodiment, the rotation of the centrifuge chamber can be stopped and the chamber can be used as a stationary sealed container, and the counterflow mechanism can be used for exchanging the liquid medium.

[0046] In another embodiment, step (s2) may be performed by aseptically transferring the somatic cells contacted with the reprogramming factors from the centrifugation chamber to an appropriate sealed container or culture vessel (a sealed container equipped with the necessary ports to enable cell culture) within the closed system portion (i.e., by transferring the cells so as to maintain the closed nature of the closed system portion), and maintaining the sealed container or culture vessel at the appropriate temperature using a water bath or the like. Examples of the culture vessel include, but are not limited to, the disposable culture vessel included with the CellPet 3D-iPSC manufactured by JTEC Corporation, and the G-Rex (registered trademark) 10N-CS and G-Rex 100N-CS culture vessels manufactured by Wilson Wolf. When these culture vessels are connected to the closed system portion of the Rotea used in the production method via a connecting conduit so as to maintain the internal closed nature of the vessel, the culture vessel may be considered part of the closed system portion or an external element connected to the closed system portion.

[0047] In yet another embodiment, step (s2) may be performed in a sealed chamber or sealed container in another cell processing device. The sealed chamber or sealed container in such another cell processing device may be connected to the closed system part of the Rotea via a connecting pipe, or cells may be transferred aseptically using a transfer means such as a syringe. Examples of such other cell processing devices include CliniMACS Prodigy (registered trademark) manufactured by Miltenyi Biotec, Cocoon (registered trademark) manufactured by Lonza, and Xuri (registered trademark) manufactured by Cytiva. TM Examples include the Cell Expansion System.

[0048] The establishment of iPS 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 which step (s2) is performed is not particularly limited as long as iPS cells are established, but is typically, for example, 10 days or more, and preferably 14 days or more. There is also no particular upper limit, but it is typically 40 days or less, and preferably 30 days or less. However, while the concept of Quality By Test (QbT) requires confirmation of the establishment of iPS cells, the concept of Quality By Design (QbD) does not necessarily require confirmation of the establishment of iPS cells, as long as a process designed to produce differentiated cells is followed, for the purpose of obtaining differentiated cells for use in regenerative medicine from patient-derived somatic cells.

[0049] In the present invention, step (s1) is performed under suspension culture conditions. That is, by performing counterflow centrifugation, the reprogramming factors efficiently contact the somatic cells while the somatic cells remain suspended in the liquid medium. In one embodiment, step (s2) is also performed under suspension culture conditions. As used herein, "suspension culture" refers to culturing cells while maintaining a state in which the cells are suspended in the liquid medium (culture solution). More specifically, suspension culture refers to culturing under conditions that do not allow strong cell-substrate bonds to form between the cells and the cultureware, etc. (e.g., a sealed chamber, a sealed container, a circulation channel, an external processing device, etc.). Those skilled in the art can easily distinguish whether the cultured cells are in suspension culture or adherent culture by, for example, shaking the cultureware during microscopic observation. The suspension culture conditions in step (s2) may be achieved, for example, by using a scaffold material as described below, by counterflow centrifugation in a sealed chamber, by continuously flowing the cells through a circulation channel, by stirring the inside of a specified container or the like with a stirrer, a jet, a horizontally placed plate (e.g., up and down movement), or a stirring blade (e.g., rotational movement), or by periodically changing the tilt angle (e.g., a seesaw-like rocking movement). Furthermore, the suspension culture conditions may be achieved using an external processing device suitable for three-dimensional suspension culture. Other conditions for step (s2) are not particularly limited, but may be 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.

[0050] (Step (s0) of Separating Somatic Cells) In a preferred embodiment of the production method, a step (s0) of separating somatic cells that serve as a source of iPS cells from whole blood using the centrifugation chamber 10 is further added before the step (s1). Thus, in this embodiment, the somatic cells that serve as a source of iPS cells are blood cells. The whole blood, the blood cells to be separated, the medium, and other necessary materials will be described below. In the step (s0), blood cells necessary as a source of iPS cells are separated from various blood cells in the whole blood by elutriation achieved by counterflow centrifugation in the chamber 10. The separated blood cells are left in the centrifugation chamber and are subjected to the next step (s1). Here, in the step (s0), "the blood cells separated by elutriation are left in the centrifugation chamber" may mean either (i) or (ii) below. (i) The blood cells separated in the chamber are left as they are in the chamber without ever leaving the chamber. (ii) The blood cells separated in the chamber are first removed from the chamber, and then other unnecessary blood cells, etc. are removed from the chamber, after which the target blood cells are returned to the chamber, so that the target blood cells remain in the chamber.

[0051] In elutriation using counterflow centrifugation, the relative centrifugal force f1 and the counterflow flow rate Q are selected to have a predetermined ratio f1 / Q, and blood cells gather at specific locations according to their size and weight, as shown in Figure 2, thereby forming multiple cell layers. Of the multiple cell layers formed in the chamber, the cell layer closest to the center of rotation can be removed from the port closest to the center of rotation into a predetermined sealed container by, for example, increasing the flow rate Q while keeping the relative centrifugal force f1 constant, thereby decreasing the ratio f1 / Q.

[0052] Elutriation itself is a known separation technique, but in the present invention, the ratio f1 / Q of the relative centrifugal force f1 to the counterflow flow rate Q is appropriately selected to separate blood cells (raw material) from whole blood in a single centrifugation chamber, and then iPS cells are established by contacting the separated blood cells with reprogramming factors. This allows iPS cells to be produced from whole blood in a single, integrated process within a single centrifugation chamber, reducing production costs.

[0053] In one aspect, the blood cells to be separated from whole blood are white blood cells (particularly lymphocytes). In this aspect, step (s0) comprises two steps (step (s0-1): a hemolysis step; and step (s0-2): a step of removing granulocytes and monocytes). In the first step (s0-1), a hemolyzing agent is added to the whole blood, red blood cells are hemolyzed by the hemolyzing agent, and the lysed red blood cells are removed from the centrifugation chamber by elutriation. Here, removal of red blood cells refers not only to complete removal, but also to a treatment of reducing the number of red blood cells to a level that does not impair the establishment of iPS cells. In the second step (s0-2), granulocytes and monocytes are removed by elutriation from the whole blood from which the red blood cells have been removed, leaving lymphocytes in the centrifugation chamber. These lymphocytes are then subjected to the next step (s1) as a source of iPS cells. The term "removal of granulocytes and monocytes" used herein refers not only to complete removal, but also to treatment for reducing the number of granulocytes and monocytes to a degree that does not impede the establishment of iPS cells.

[0054] (Lymphocytes) In one aspect, lymphocytes may be selected as blood cells to be separated from whole blood, taking into consideration that lymphocytes are a suitable source of iPS cells and that lymphocytes are particles that are located on the port side and are easy to recover by elutriation.

[0055] (Ratio f1 / Q in Step (s0-1)) The ratio f1 / Q of the relative centrifugal force f1 to the counterflow flow rate Q in removing red blood cells varies depending on the shape, capacity, temperature, and fluid properties of the centrifugation chamber, but when Rotea is used at room temperature and a typical liquid medium and hemolyzing agent are used, the ratio f1 / Q is approximately 17 to 300 [G min / ml]. In this case, the relative centrifugal force f1 is preferably approximately 1000 to 3000 G, more preferably approximately 1500 to 2800 G, and the flow rate Q is preferably approximately 10 to 60 ml / min, more preferably approximately 15 to 50 ml / min. These combinations of relative centrifugal force f1 and flow rate Q are selected so that the ratio f1 / Q is approximately 17 to 300 [G min / ml]. Furthermore, in carrying out step (s0-1) under the above conditions, the hemolytic agent may be introduced into the flow path in small amounts, followed by repeatedly switching to a circulation circuit, and finally completely replacing the flow path with the hemolytic agent. In one embodiment, for example, the hemolytic agent is introduced stepwise in amounts of (1) 3 ml, (2) 4 ml, and (3) 5 ml, with the circulation time set to 25 to 35 seconds for each step, three times for each step, for a total of nine replacements, and then the amount of hemolytic agent delivered is set to 80 ml, and the liquid in the circulation flow path is completely replaced with the hemolytic agent.

[0056] (Ratio f1 / Q in Step (s0-2)) In removing granulocytes and monocytes by elutriation, first, the suspension from which red blood cells have been removed in Step (s0-1) is passed through the circulation flow path, and counterflow centrifugation is performed for 10 to 50 seconds with the ratio f1 / Q set to 50 to 70 (relative centrifugal force f1 of approximately 500 to 3000 G, counterflow flow rate Q of approximately 20 to 50 ml / min), to form a cell layer on the outer periphery of the centrifuge chamber. Granulocytes, monocytes, and lymphocytes are present within this cell layer. Next, while maintaining the flow rate Q constant, the relative centrifugal force f1 acting on the contents of the centrifuge chamber is gradually reduced over 20 to 100 seconds, reducing the ratio f1 / Q from 50 to 70 to 20 to 35. The relative centrifugal force f1 after the reduction is approximately 500 to 1000 G. The reduction in the ratio f1 / Q causes the cell layer to spread toward the center of rotation within the centrifuge chamber. Because lymphocytes are lighter than granulocytes and monocytes, they are thought to be distributed closer to the center of rotation in the cell layer than granulocytes and monocytes. The term "gradual decrease" used here does not necessarily mean a linear decrease, but may also include a gradual, curvilinear change, such as a smooth initial decrease and a smooth end at the end of the change. Next, while maintaining the relative centrifugal force f1 constant, the flow rate Q is gradually increased over 30 to 100 seconds, decreasing the ratio f1 / Q from 20 to 35 to 10 to 20. The flow rate Q after the increase is approximately 40 to 100 ml / min. The decrease in the ratio f1 / Q allows lymphocytes to flow out of the port on the center of rotation of the chamber. The effluent lymphocytes and medium (liquid culture medium) are collected in a sealed container 24 for circulation or the like. Here, "gradual increase" does not only mean a linear increase, but may also include a portion that changes gently in a curve, such as a smooth increase starting at the beginning of the change and a smooth end at the end of the change. Similarly, in the following description, a gradual change may not only mean a linear change, but also a portion that changes gently in a curve.

[0057] The iPS cells to be produced and the raw materials and ingredients required for the steps (s0) to (s2) will be explained below.

[0058] (Induced pluripotent stem cells) As used herein, "induced pluripotent stem cells (iPS cells)" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells through the introduction of reprogramming factors. Induced pluripotent stem cells can differentiate into tissues and cells with a variety of different morphologies and functions in the body, and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm).

[0059] In the present specification, iPS cells may be cells derived from a patient. Producing iPS cells from somatic cells derived from a patient and using them for clinical treatment can be an effective means of minimizing the risk of rejection.

[0060] The method for producing iPS cells of the present invention can be used for any currently available induced pluripotent stem cells.

[0061] 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.

[0062] In the present invention, "somatic cells" refer to original cells to be processed in the rotatable sealed chamber used in the present invention. As used herein, "somatic cells" refers to cells that constitute an animal, other than germ cells. Somatic cells are not particularly limited and include both mature, healthy and 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) or adherent 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, dental pulp cells, 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 (blood cell) cells (e.g., hematopoietic stem cells, peripheral blood mononuclear cells (PBMCs) (including T cells and non-T cells), leukocytes (e.g., lymphocytes), umbilical cord blood cells, etc.), epithelial cells, endothelial cells (e.g., vascular endothelial cells), muscle cells, etc.

[0063] In one embodiment, when blood cells (e.g., peripheral blood mononuclear cells) are used as somatic cells, the cells can be obtained by centrifuging whole blood (density gradient centrifugation, specific gravity centrifugation, etc.), separating the cells using a filter (leukocyte removal filter, etc.), or by using an antibody, a magnetic substance (magnetic beads, etc.), or a hydrophilic polysaccharide (Ficoll, ＴＭ etc.)

[0064] (Blood cells) As used herein, "blood cells" refers to all cells at various stages, from hematopoietic stem cells, through hematopoietic progenitor cells (including pluripotent hematopoietic progenitor cells and unipotent hematopoietic progenitor cells), to finally functional blood cells. Examples of blood cells include peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMNCs). As explained in step (s0) above, in the present invention, blood cells can be preferably used as a source of iPS cells.

[0065] In the present specification, the species from which the somatic cells are derived is not particularly limited, and the species from which the somatic cells are derived is preferably human.

[0066] (Whole Blood) As used herein, "whole blood" refers to blood collected from a subject such as a human, from which blood cells and the like have not been separated. Furthermore, as used herein, "whole blood" may include blood diluted by mixing with an appropriate buffer or the like, or blood to which additives such as blood coagulation inhibitors (e.g., heparin, EDTA, citric acid, etc.) or protease inhibitors have been added. The buffer used to dilute whole blood is not particularly limited as long as it does not cause hemolysis or other effects on blood cell components in the whole blood, and examples thereof include phosphate buffer solution (PBS), physiological saline, etc.

[0067] (Amount of Whole Blood Supplied) In the present invention, the amount of whole blood supplied to the rotatable sealed chamber is not particularly limited, but may be about 50 to 15 ml, preferably 40 to 20 ml, and more preferably 35 to 20 ml. The whole blood may be stored in a sealed container for supplying material in the state in which it is collected and then sent to the sealed chamber, or may be stored in a sealed container for supplying material in a state diluted with the above-mentioned buffer solution or the like and then sent to the sealed chamber.

[0068] 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.

[0069] As used herein, "processing" cells means subjecting cells to treatments such as culturing the cells, diluting a solution containing the cells, washing the cells, separating the target cells from a solution containing the cells, etc. It also means subjecting cells to chemical treatment, modification of biological properties, combination with non-cellular components, genetic engineering modification, etc. for the purpose of artificial proliferation / differentiation of the cells, establishment of a cell line, or activation of the cells.

[0070] (Liquid medium) The liquid medium delivered from the sealed container can be used not only for cell cultivation but also for various purposes in the production process, such as washing cells, diluting chemical solutions, etc. Examples of liquid media that can be used in the present invention include the following:

[0071] The liquid medium is not particularly limited, but examples thereof include StemFit (registered trademark) AK03 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) Basic03 medium, StemSpan™ (STEMCELL Technologies), and Essential8 medium (CTS TM Essential 8 TM Medium, Essential 8 TM Medium, Essential 8 TM Flex Medium, Essential 6 TM Examples of suitable media include StemFit® AK02 Medium (Thermo Fisher Scientific), StemFit® AK02 Medium (Ajinomoto Co., Inc.), 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. Other examples include, but are not limited to, MSCBM-CD, MSCGM-CD (both Lonza), and mixtures thereof.

[0072] If necessary, physiologically active substances and nutritional factors necessary for cell survival or proliferation can be added to the medium.

[0073] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the medium as needed.

[0074] In this specification, known serum can be used as the medium.

[0075] As used herein, the medium may or may not contain serum substitutes, as well as serum.

[0076] (Scaffold Material) In this specification, the term "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 adhesion culture or suspension culture using the scaffold material (in other words, it may be free in the medium), and examples include those containing or made of synthetic resin, and those made of flexible materials such as collagen. Microcarriers may also be used as scaffold materials. As an example, the scaffold material may contain atelocollagen. The scaffold material may be manufactured by a known method, or a commercially available product may be used. Examples of commercially available products include Cytodex-1 (manufactured by GE Healthcare) and Corning® Low Concentration Synthemax® II Microcarrier (manufactured by Corning).

[0077] The scaffold material may also contain or consist of an extracellular matrix, such as a basement membrane preparation (e.g., Matrigel (manufactured by Corning), Geltrex matrix (manufactured by Thermo Fisher Scientific), etc.), fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, vitronectin, etc., or a combination thereof. The exemplified extracellular matrices may be natural products, artificially synthesized by genetic engineering techniques, etc., fragments obtained by cleavage with restriction enzymes, etc., or synthetic proteins or synthetic peptides based on these biological substances.

[0078] The method of supplying the scaffold material to a sealed chamber, etc. is not particularly limited as long as it can appropriately process the desired cells, and the supply conditions (e.g., supply timing, volume, etc.) and type of scaffold material can be appropriately set by a person skilled in the art.

[0079] (Reprogramming Factors) 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. Any known combination of reprogramming factors may be used.

[0080] The reprogramming factor introduced into the somatic cell 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 cell may activate the cellular defense mechanism, so RNA for circumventing the defense mechanism may be introduced into the somatic cell.

[0081] 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).

[0082] Nucleic acids, 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.

[0083] (Other Substances to be Supplied) In addition to the above-mentioned materials, substances necessary for cell processing may be added as appropriate. The added materials can be stored in a sealed container or the like and connected to the sealed chamber, similar to the above-mentioned materials. Other materials to be added include, for example, liquids (e.g., saline, buffer solution, etc.), powders, additives, release agents, cryoprotectants, CO ２ Examples include:

[0084] A more detailed and preferred example of the preparation of materials and operating sequence for Rotea to sequentially carry out the above-mentioned steps (s0), (s1), and (s2) is shown in the following steps (1) to (14). The reference numerals for each part of the closed system and mechanism are shown in Figures 1 to 8. In the example of steps (1) to (14), the somatic cells used as the raw material for iPS cells are blood cells (particularly white blood cells, especially lymphocytes), and steps (2) to (7) exemplify preferred steps for separating white blood cells (particularly lymphocytes) from whole blood.

[0085] (1) Priming: When the closed system shown in Figure 1 is installed in the mechanism shown in Figure 6 and the mechanism is activated, all pinch valves are activated and all pinch points on the connecting lines are closed. Before whole blood is introduced into the centrifuge chamber, the circulation flow path is filled with liquid medium. In Figure 1, the circulation flow path is a circular flow path connecting pinch point 30K1, connecting lines (38, 33, 34), centrifuge chamber 10, connecting lines (35, 37), and pinch point 30J1 in this order. In the Rotea, if air is present in the centrifuge chamber, a sensor is activated and the rotation of chamber 10 is stopped. Therefore, as an initial operation, the circulation flow path and other connecting lines must be filled with liquid. To explain this filling step in more detail, first, the pinch points (30B1, 30G1, 30K1) are opened (i.e., the pinch valves closing these pinch points are opened), and the peristaltic pump 400M is operated to allow the liquid medium to flow from the sealed container 21 into the connecting conduit 30B at a predetermined flow rate (e.g., 100 ml / min). The liquid medium passes through the bubble trap 31 and the connecting flow paths (38, 33, 34) in order, filling the centrifuge chamber 10, passing through the connecting conduit 35, the branch point 36, and the connecting conduit 30G, and entering the sealed container 26 for waste liquid. This fills the flow path from the connecting conduit 30B to the connecting conduit 30G with the liquid medium. Similarly to this procedure, the pinch points 30C1, 30D1, and 30F1 are opened in order, filling the connecting conduits 30C, 30D, and 30F with the liquid in the sealed containers 22, 23, and 25, respectively. At this time, it is desirable to close pinch point 30B1 to prevent the hemolytic agent from entering the connecting pipe 30B, which is the liquid culture medium flow path, since this does not change the concentration of the liquid culture medium. In the Rotea, the peristaltic pump is always operating when the mechanical parts are operating, and liquid always flows through the flow path and centrifuge chamber with the pinch point open.

[0086] (2) Introducing the diluted whole blood from the sealed container 22 into the circulation flow path (step (s0)). The diluted whole blood contained in the sealed container 22 is introduced into the circulation flow path and the centrifugation chamber (FIG. 9(a)). While the diluted whole blood is circulated in the circulation flow path, counterflow centrifugation is performed in the centrifugation chamber 10. As a result, particles such as blood cells dispersed in the diluted whole blood gather in one location in the centrifugation chamber and remain as a cell layer, thereby concentrating the diluted whole blood in the centrifugation chamber (FIG. 9(b)). The diluted whole blood is obtained by diluting, for example, 35 ml of collected whole blood with a hemolytic agent, and the volumetric mixing ratio of the whole blood to the hemolytic agent is about 1:2 to 1:3.

[0087] (3) Gradually supplying a hemolytic agent to the circulation flow path to wash the whole blood. A hemolytic agent is further added to the diluted whole blood in the circulation flow path to hemolyze red blood cells and remove them from the diluted whole blood ( FIG. 9( c) ). Specifically, pinch point 30D1 on connecting line 30D extending from sealed container 23 is opened, and pinch point 30G1 on connecting line 30G extending from sealed container 26 for waste liquid is opened. A small amount of hemolytic agent is introduced from sealed container 23 into the circulation flow path to hemolyze red blood cells contained in the concentrated whole blood being counterflow centrifuged in the centrifuge chamber, and the liquid containing the hemolyzed red blood cells flowing through the circulation flow path is discharged into sealed container 26 for waste liquid. The pinch points 30D1 and 30G1 are closed to switch to the circulation flow path, and somatic cells that have flowed into the circulation flow path are collected in the centrifuge chamber during counterflow centrifugation (the outflow of somatic cells into the circulation flow path is caused by disturbance of the cell layer during counterflow centrifugation due to differences in liquid properties). Therefore, while gradually increasing the amount of hemolytic agent introduced, the introduction and drainage of the hemolytic agent and the switching to the circulation channel for counterflow centrifugation are alternately repeated. In this case, the conditions for the counterflow centrifugation, such as the relative centrifugal force and counterflow flow rate, are, for example, as follows: relative centrifugal force: 2000 G, flow rate: 30 ml / min, stepwise replacement of 3 ml, 4 ml, and 5 ml, circulation time: 25 to 35 seconds, number of treatments: 3 times each (i.e., the hemolytic agent is introduced into the circulation channel by first introducing 3 ml three times, then introducing 4 ml three times, and finally introducing 5 ml three times, with circulation for 25 to 35 seconds after each introduction, and the eluted somatic cells are recovered in the centrifugation chamber).

[0088] (4) Replacing the liquid in the circulation flow path with a hemolytic agent (liquid) and washing out somatic cells While draining the liquid in the circulation flow path into the sealed container 26, the hemolytic agent is introduced from the sealed container 23 into the circulation flow path, replacing the liquid in the circulation flow path with the hemolytic agent, completely hemolyzing the red blood cells in the whole blood, and removing the hemolyzed components from the suspension. In this case, the conditions of the relative centrifugal force of the counterflow centrifugation and the counterflow flow rate are, for example, as follows: Relative centrifugal force: 2000 G Flow rate: 30 ml / min Amount of hemolytic agent delivered: 80 ml

[0089] (5) Replacing the liquid (hemolytic agent) in the circulation flow path with liquid culture medium While draining the hemolytic agent in the circulation flow path into the sealed container 26, liquid culture medium is introduced from the sealed container 21 into the circulation flow path, and the liquid in the circulation flow path is replaced from the hemolytic agent to the liquid culture medium. This process of introducing small amounts of culture medium and switching to the circulation flow path is repeated. As described in step (3) above, differences in liquid properties disrupt the cell layer during counterflow centrifugation, causing somatic cells to flow out into the circulation flow path. Therefore, as in step (3) above, while gradually increasing the amount of liquid culture medium introduced, the introduction and drainage of the liquid culture medium and the switching to the circulation flow path for counterflow centrifugation are alternately repeated. In this case, the conditions for the relative centrifugal force and counterflow flow rate of the counterflow centrifugation are, for example, as follows: Relative centrifugal force: 2000 G Flow rate: 30 ml / min Stepwise replacement of 3 ml, 4 ml, and 5 ml Circulation time: 45 to 55 seconds Number of treatments: once each

[0090] (6) Sampling of somatic cells before elutriation Although not a required step, if sampling of somatic cells before elutriation is desired, it can be performed as follows. Close pinch point 30G1 of the drainage connection flow path, open pinch point 30A1 between the drainage connection flow path and the circulation bag 24, and pour 50 ml of liquid medium into the flow path to dilute the solution flowing in the flow path. Close pinch point 30B1 and open pinch point 30E1 to switch to a circulation flow path via the circulation bag 24. Sampling is performed using syringe S1.

[0091] (7) Removal of Granulocytes and Monocytes by Elutriation (Separation of Lymphocytes) First, a cell layer is formed in the centrifuge chamber by counterflow centrifugation at a relative centrifugal force of 2000 G and a flow rate of 30 mL / min for 30 seconds. After the cells before elutriation are sampled, the blood cells are returned from the sealed circulation container 24 to the centrifuge chamber. Next, while maintaining the flow rate at 30 mL / min, the relative centrifugal force is gradually reduced to 800 G over 50 seconds, spreading the cell layer toward the center of rotation in the centrifuge chamber (step (s0-1)). Because lymphocytes are lighter than granulocytes and monocytes, the elutriation achieved by counterflow centrifugation likely causes them to reside closer to the center of rotation than the granulocytes and monocytes ( FIG. 10( a)). Next, the direction of circulation of the liquid in the circulation channel is set to the forward direction, the pinch point 30A1 between the circulation sealed container 24 and the circulation channel is opened, and the flow rate is gradually increased to 50 ml / min over 60 seconds while maintaining the relative centrifugal force at 800 G, thereby discharging lymphocytes, which are theoretically thought to be contained in large amounts in the cell layer on the rotation center side in the centrifugation chamber, from the centrifugation chamber (FIG. 10(b)) and recovering them in the circulation sealed container 24 (step (s0-2)). Next, the direction of circulation of the liquid in the circulation channel is set to the forward direction, the relative centrifugal force is set to 2400 G, and the flow rate is set to 30 ml / min, and the cells remaining in the circulation channel are collected in the centrifugation chamber (FIG. 10(c)). Pinch point 400H before the syringe used to collect the liquid medium and cells removed by elutriation was opened, the liquid circulation direction in the circulation channel was reversed, and the liquid containing the cell layer that had accumulated on the outer periphery of the centrifuge chamber before elutriation was manually collected using syringe S4 at a relative centrifugal force of 2400 G, a flow rate of 30 mL / min, and a target volume of 40 mL (i.e., the control program was set to stop the inflow when the inflow volume reached 40 mL). The liquid circulation direction in the circulation channel was reversed at a relative centrifugal force of 10 G and 100 mL / min, and then pinch point 30E1 was opened. The remaining post-elutriation suspension was placed in sealed container 24, and the suspension was sampled using syringe S2 to verify the effectiveness of elutriation.The direction of circulation of the liquid in the circulation flow path is set to the forward direction, and the relative centrifugal force and flow rate are gradually changed to 2000 G and 30 ml / min over 60 seconds, respectively, to form a cell layer in the centrifugation chamber, and pinch point 30E1 is opened to move the post-elutriation suspension from the closed circulation container 24 into the centrifugation chamber, where the suspension is concentrated.

[0092] (8) Concentration The suspension is further concentrated in the centrifuge chamber by gradually increasing the relative centrifugal force to 1500 G and the flow rate to 5 ml / min for 60 seconds.

[0093] (9) Injection of reprogramming factors into the circulation flow path (step (s1)) Pinch point 30F1 of connecting line 30F to which syringe S3 (a syringe containing SeV-containing liquid) shown in FIG. 1 is connected is opened. As an example of releasing the pressure increase, pinch point 30C1 of connecting line 30C leading to sealed container 22 containing the raw material is opened. At a relative centrifugal force of 1500 G and a flow rate of 5 ml / min, the plunger of syringe S3 is manually pressed to inject SeV-containing liquid into the circulation flow path in small amounts. The increase in the amount of SeV-containing liquid due to the injection is released into sealed container 22. It is preferable that the length of the line into which the SeV-containing liquid is injected and the length of the line for releasing the increase in the amount of SeV-containing liquid are approximately the same. Therefore, in this example, sealed container 22 was used, but in principle, sealed container 26 for waste liquid may also be used.

[0094] (10) Contact of leukocytes (particularly lymphocytes) with reprogramming factors (step (s1)) Counterflow centrifugation was performed for 2 hours at a relative centrifugal force of 1500 G and a flow rate of 5 ml / min, while circulating the liquid medium containing the SeV vector ( FIG. 11 ). This caused the liquid medium containing the SeV vector to flow as a counterflow between the lymphocytes clustered at the outer periphery of the centrifugation chamber, thereby increasing the probability of contact between the lymphocytes and the SeV vector.

[0095] (10) Removal of reprogramming factors After the 2-hour treatment in (9) above, the liquid medium is injected into the centrifugation chamber at a relative centrifugal force of 1500 G and a flow rate of 5 ml / min to wash the leukocytes (particularly lymphocytes) and remove the SeV vector.

[0096] (11) Concentration (recovery) of leukocytes (particularly lymphocytes) that have come into contact with (infected by) SeV vectors. The circulation channel is closed, and the rotation speed is increased to circulate and concentrate the suspension (FIG. 12(a)). Then, the channel to the medium and 30H1 are opened, and the suspension (leukocytes (particularly lymphocytes) that have come into contact with SeV vectors) is recovered in a sealed container connected to channel 30H (FIGS. 12(b) and 12(d)). This process is repeated twice. When leukocytes (particularly lymphocytes) that have come into contact with SeV vectors are recovered in another sealed container, such as a CELLPET disposable culture vessel, it is desirable to pass them through a filter (e.g., Re-Strainer 200 μm pluriSelect) to trap and remove dead cells (FIG. 12(c)).

[0097] (12) Establishment of iPS cells (step (s2)) Cytokines are added to the collected cell suspension in accordance with the amount of the collected cell suspension, and the cells are cultured to establish iPS cells.

[0098] (13) Establishment of iPS cells The liquid medium in the sealed container is replaced or added.

[0099] (14) Establishment of iPS cells Colonies (in the case of adherent culture) or spheroids (in the case of suspension culture) are observed 14 to 21 days after the initiation of the cell culture.

[0100] (Step (s3) of expanding iPS cells) From the viewpoint of iPS cell yield, the production method may further include a step (s3) of expanding the iPS cells after the step (s2) of establishing iPS cells described above. The step (s3) may be carried out in a Rotea centrifuge chamber, or in a sealed container for expansion culture that is aseptically connected to the closed system portion so as to maintain the closed system. The step (s3) is carried out while maintaining the closed nature of the closed system portion, similar to the steps (s0) to (s2) described above.

[0101] The sealed containers for expansion culture include sealed containers connected to the connecting pipes (30A to 30H), sealed containers included in external expansion culture equipment (e.g., G-Rex (Wilson Wolf)), as well as tubes for air evacuation, tubes for medium exchange, and CO２ A sealed container equipped with a gas pipe can be used as appropriate. The connecting pipeline 30H shown in FIG. 1 is a pipeline suitable for output, and the sealed container connected to this connecting pipeline 30H, like the sealed containers (21-26) connected to the other connecting pipelines (30A-30G), is a sealed container that constitutes a closed system. Whether all sealed containers and devices connected to each connecting pipeline in a sterile manner to maintain a closed system belong to the closed system or to external devices can be determined as appropriate for convenient management. The iPS cells can be expanded by supplying a liquid medium suitable for expansion to elements for expansion (such as a centrifuge chamber or a sealed container for expansion).

[0102] The medium used in step (s3) can be appropriately selected by those skilled in the art from the liquid media described above. In one embodiment, when it is intended to expand the established iPS cells, a factor for maintaining undifferentiated states can be added. As used herein, the term "factor for maintaining undifferentiated states" refers to a substance that has the effect of suppressing the differentiation of induced pluripotent stem cells, and is not particularly limited as long as it is such a substance. Examples of factors for maintaining undifferentiated states commonly used by those skilled in the art include bFGF, FGF2, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, and TGFβ2. The factor for maintaining undifferentiated states used in the present invention is preferably isolated. "Isolated" means that the target components and factors other than cells have been removed, and the state is no longer that which exists in nature.

[0103] In step (s3), the number of times expansion culture is performed is not particularly limited as long as the desired number of iPS cells is obtained, but in a typical processing operation, it is preferably about 1 to 5 times, more preferably about 2 to 5 times. The period for performing step (s3) is not particularly limited as long as the desired number of iPS cells is obtained, but is typically, for example, 1 to 40 days, 3 to 20 days, or 5 to 10 days. If the Sendai virus vector used for reprogramming contains a GFP marker, it is desirable to perform expansion culture until the marker is lost.

[0104] (Quality Evaluation of iPS Cells) After step (s2) or step (s3) of the production method of the present invention, the quality of the obtained iPS cells may be evaluated by a known method.

[0105] (Method for producing differentiated cells) Next, a method for producing differentiated cells according to the present invention will be described. The method for producing differentiated cells comprises at least steps (s1) and (s2) in the method for producing iPS cells according to the present invention described above, and more preferably comprises at least steps (s0), (s1), and (s2), and further comprises a step (s4) of inducing differentiation of iPS cells after these steps. Step (s4), like steps (s1) and (s2), is carried out while maintaining the closed nature of the closed system.

[0106] Step (s4) is carried out, for example, as follows: (I) Materials necessary for differentiation induction are supplied into the centrifugation chamber, and iPS cells are induced to differentiate in the centrifugation chamber. (II) The iPS cells present in the centrifugation chamber are transferred to a sealed container for differentiation induction (not shown) connected to the closed system portion in a sterile manner so as to maintain the closed system, and materials necessary for differentiation induction are supplied into the sealed container for differentiation induction, and the iPS cells are induced to differentiate in the sealed container. The sealed container for supplying the materials necessary for differentiation induction and the sealed container for differentiation induction are not shown in Figures 1, 8, etc.

[0107] The sealed container for differentiation induction may be a sealed container connected to the connecting pipes (30A to 30H) similar to the sealed container for expansion culture, a sealed container included in an external device for differentiation induction, G-Rex (Wilson Wolf), a tube for air evacuation, a tube for medium exchange, and a CO ２ A suitable container may be used, such as a sealed container with a gas line.

[0108] The step (s4) of inducing differentiation of iPS cells may be performed after the establishment step (s2) in the method for producing iPS cells according to the present invention described above, without performing the step (s3) of expanding the iPS cells, or may be performed after the establishment step (s2) and the step (s3) of expanding the iPS cells.

[0109] After the step (s2) of establishing iPS cells and before the step (s4) of inducing differentiation of iPS cells, one or both of a cell sorting step and a gene transfer step may be further performed. The cell sorting step can be performed by connecting a known cell sorting device to the closed system portion. The gene transfer step may be performed, for example, on iPS cells derived from a patient with a genetic disease, by repairing the gene by genome editing or the like and then differentiating the cells into target cells with the intention of using the cells as a therapeutic agent for the disease. Electroporation, lipofection, or viral vectors can be used. For electroporation, an electroporator can be used. For methods using lipofection, liposomes, or viral vectors, Rotea can be used. The gene transfer method can be appropriately designed depending on the gene desired to be transferred.

[0110] (Differentiated cells) As used herein, "differentiated cells" refers to cells or organoids obtained by inducing differentiation of induced pluripotent stem cells. The cells obtained may be undifferentiated cells such as stem cells or progenitor cells, or may be terminally differentiated cells. As used herein, the term "differentiated cells" may be used to encompass both undifferentiated cells and terminally differentiated cells obtained by inducing differentiation of induced pluripotent stem cells. As used herein, "undifferentiated cells" refers to cells that have not reached terminal differentiation in the 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 ectodermal 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 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, skeletal muscle stem cells, adipose stem cells, kidney progenitor cells, and endodermal cells such as hepatic stem cells, liver progenitor cells, intestinal stem cells, and airway stem cells.

[0111] 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, hepatic mesothelial cells, bile duct epithelial cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, Kupffer cells, pit cells, vascular endothelial cells, blood cells, pancreatic duct epithelial cells, pancreatic duct cells, acinar centro-cells, acinar cells, islets of Langerhans, cardiac myocytes, 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, neurons, and glial cells. Examples of leukocytes include lymphocytes, granulocytes, and monocytes.

[0112] In one aspect, the cells or organoids (target cells or organoids) obtained by inducing differentiation of induced pluripotent stem cells are neural crest cells, neural progenitor cells, neurons, cerebral cortical organoids, hematopoietic progenitor cells, platelets, T cells, or cardiomyocytes.

[0113] (iPS cell differentiation inducer) In this specification, the term "differentiation inducer" refers to a substance that can induce differentiation from induced pluripotent stem cells into the differentiated cells or organoids described above. The differentiation inducer may be a known substance, or may be selected from those that are commonly used to induce differentiation of the desired differentiated cells or organoids. Specific examples include the substances described below.

[0114] The fluid used as a medium for containing the reprogramming factors, undifferentiated maintenance factors, and differentiation inducers is not particularly limited, but preferred examples include buffer solutions, culture media, and cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerin.

[0115] (Differentiation Induction Method) In the production method of the present invention, known methods can be used as the differentiation induction method for obtaining the target cells or organoids. For example, differentiation induction from pluripotent stem cells to 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), and then differentiated into neural crest cells by adhesion culture (suspension culture using a scaffold material) in a medium containing a TGFβ inhibitor and a GSK3β inhibitor.

[0116] Neural crest cells can also be used to produce cells such as mesenchymal stem cells, neural progenitor cells, neurons, glial cells, bone cells, chondrocytes, corneal cells, and melanocytes. 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 onto 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, 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.

[0117] 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.

[0118] 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 medium for inducing hematopoietic progenitor cells, 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.

[0119] 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).

[0120] Differentiation into monocytes can be induced by the method described in Di Cui., et al. Frontiers in Cell and Developmental Biology; vol 9, Article 656867 April 2021.

[0121] 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.

[0122] In the production method 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.

[0123] The manufacturing method of the present invention may include a step of recovering the obtained target cells or organoids. The recovered cells may be cryopreserved using a cell cryopreservation solution. The recovered cells may also be counted 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.

[0124] In the cell production method of the present invention, undifferentiated cells may be removed as appropriate. The method for removing undifferentiated cells is not particularly limited as long as it can remove cells other than cells produced by the cell production method of the present invention, 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.).

[0125] Quality testing may also be performed as appropriate to determine whether the cells, organoids, etc. obtained by the production method of the present invention are desirable. The test items for quality testing 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 testing devices suitable for each test item can be used.

[0126] In the examples shown below, the steps (s0) to (s4) of the production method according to the present invention described above were carried out using the following reagents, devices and materials, and the obtained iPS cells and differentiated cells were evaluated.

[0127] (Reagents) Whole blood: Blood from healthy volunteer donors (unprocessed, unfrozen, 1-2 days after collection) Hemolyzing agent: ACK lysing buffer Gibco TMProduct number (catalog number): A1049201 https: / / www.thermofisher.com / order / catalog / product / jp / ja / A1049201 DNase: Recombinant DNase I (RNase-free) Cat# 2270A Culture medium: StemFit AK03N (without C (containing growth factors). StemSpan is also acceptable) SeV: SRVTM iPSC-4 (Tokiwa Bio Co., Ltd. (a product with optimized gene expression levels for generating iPS cells from peripheral blood monocytes, peripheral blood mononuclear cells, and CD34-positive cells), containing six human-derived reprogramming genes: OCT3 / 4, KLF4, SOX2, c-MYC, NANOG, and LIN28, as well as EGFP and Puro resistance genes)

[0128] (Equipment, equipment) Cell processing device: Rotea Counterflow Centrifugation System (Thermo Fisher Scientific Inc.) Sealed container: Terumo Separation Bag Sterile splicer: TERUMO TSCD-II CELLPET: Disposable culture vessel JTEC Corp. CELLPET iPS / 3S / MA-2.1 (JTEC Corporation) Safety cabinet: phcbi Class II type A2 (PHC Corporation) Cell counter: celldrop FL (DeNovix) Incubator: thermo scientific FORMA STERI-CYCLE i160 (Thermo Fisher Scientific) CO2 incubator: MINIcell waken tech (Waken B Tech Co., Ltd.) Inverted microscope: ix83 p2zf (Evident Co., Ltd.) Flow cytometer: CytoFLEX (Beckman Coulter Inc.) Centrifuge: tomy lcx-100 (Tomy Seiko Co., Ltd.)

[0129] Example 11. Preparation (1) Mixing of whole blood and hemolytic agent In a safety cabinet, whole blood collected in a Terumo separation bag used as the sealed container 22 was aseptically mixed with the hemolytic agent and added with DNase at a volume of 10 units / ml or more so that the volumetric mixing ratio (whole blood:hemolytic agent) was 1:2 to 1:3, and the bag was left to stand at room temperature for 15 minutes. The hemolytic agent was added to lyse red blood cells and to reduce the viscosity of viscous blood.

[0130] (2) Preparation of the Closed System: Liquid medium and hemolytic agent were manually loaded into sealed containers 21 and 23, respectively, within a safety cabinet. SeV (2 tubes, 0.2 ml) was mixed with 1 ml of liquid medium within the safety cabinet and loaded into a syringe (S3). A sealed container 24 for circulation, sampling syringes (S1 and S2), a sealed container 26 for waste liquid, a harvesting syringe (S5 in Figure 7), and a syringe for recovering the liquid removed by elutriation (S5 in Figure 7) were connected to the respective flow paths. A sealed container 25 filled with liquid medium for introducing SeV liquid was connected to the end of the connecting conduit 30F of the syringe filled with SeV liquid (S3 in Figure 7). The syringe for recovering the liquid removed by elutriation (S4 in Figure 7) was connected closer to sealed container 25 than the syringe filled with SeV liquid (S3 in Figure 7). As shown in Figures 1 and 7, the prepared sealed containers (bags) and syringes were aseptically connected to the connecting lines (soft tubing) of the sterilized Rotea single-use kit using a sterile connector. The sealed container 24 for circulation has two ports, with connecting lines connected to the inlet and outlet of the liquid, leading to the flow path. The other sealed containers were connected to the single-use kit via connecting lines at only one port. All sealed containers were hung, and the tubing was checked for bends and clogs. The QR code (registered trademark) on the single-use kit was read, and the Rotea mechanism automatically closed all pinch points.

[0131] 2. Leukocyte Separation from Whole Blood The Rotea control program was run, and the following protocol was set up to obtain leukocytes by treating whole blood with a hemolytic agent and washing away plasma, platelets, and red blood cells. The protocol for the CTS Rotea system was designed in the Protocol Builder software to implement all of the processes described below.

[0132] (1) Priming First, the centrifugal chamber and the peristaltic pump were operated to fill all the flow paths of the closed system portion to be used with liquid at a relative centrifugal force of 10 G and a flow rate of 100 ml / min.

[0133] (2) Introducing whole blood into the centrifugal chamber Whole blood was sent from the suspended sealed container 22 into the centrifugal chamber 10. Rotation speed: 2400 G Flow rate: 35 mL / min Time: 90 seconds

[0134] (3) Washing with hemolytic agent A small amount of hemolytic agent was introduced from the sealed container 22 and then switched to the circulation flow path. This was done because the cell layer would be disturbed due to differences in liquid properties. The circulation flow path is a circular path that connects the outlet of the centrifuge chamber 10, connecting line 35, branch point 36, connecting line 37, pinch point 30J1, pinch point 30K1, connecting line 38, junction 32, connecting line 33, connecting line 34, and the inlet of the centrifuge chamber 10 in the closed system portion of Figure 1. Rotation speed: 2000G Flow rate: 30 mL / min Stepwise replacement of (1) 3 mL, (2) 4 mL, and (3) 5 mL Circulation time: 25-35 sec Number of treatments: 3 times each

[0135] (4) Complete replacement with hemolytic agent While the liquid in the circulation flow path was drained into the sealed container 26, the hemolytic agent was introduced from the sealed container 23 into the circulation flow path, and the liquid in the circulation flow path was replaced with the hemolytic agent, completely hemolyzing the red blood cells in the whole blood and removing the hemolyzed components from the suspension. Rotation speed: 2000 G Flow rate: 30 mL / min Amount of hemolytic agent delivered: 80 mL

[0136] (5) The liquid (hemolytic agent) in the circulation flow path is replaced with liquid culture medium. A small amount of liquid culture medium is introduced from the sealed container 21 into the centrifugation chamber and then repeatedly switched to the circulation flow path. This is to stabilize the cell layer that has been disturbed due to differences in liquid properties. Rotation speed: 2000G Flow rate: 30 mL / min Stepwise replacement of (1) 3 mL and (2) 5 mL Circulation time: 45-55 sec Number of treatments: 3 times each

[0137] (6) Sampling 50 ml of liquid medium was introduced from the sealed container 21 into the circulation flow path at a relative centrifugal force of 10 G and a flow rate of 100 ml / min for 50 seconds to dilute the suspension, and the pinch points (30A1, 30E1) between the sealed container 24 for circulation were opened to disperse the cells into the flow path including the sealed container 24 for circulation. The syringe S1 provided in the flow path between the sealed container 24 for circulation and the centrifugation chamber 10 was then manually pulled to sample the cells before elutriation.

[0138] (7) Elutriation Elutriation was performed as follows, except for the test group conducted without elutriation to verify the effect of elutriation. The relative centrifugal force of the centrifugation chamber was increased to 2000 G and the flow rate was 30 ml / min for 30 seconds to form a cell layer within the chamber. The cells were then returned from the closed circulation container 24 to the chamber. The relative centrifugal force was reduced to 800 G, and the cell layer was spread within the chamber at a flow rate of 30 ml / min for 50 seconds. Because lymphocytes are relatively lighter than larger cells such as granulocytes and monocytes, they are thought to reside closer to the center of rotation of the chamber. The fluid was circulated in the forward direction, and the valve between the closed circulation container 24 and the flow path was opened. The cell layer located closer to the center of rotation of the chamber was collected into the closed circulation container 24 at a relative centrifugal force of 800 G and a flow rate of 50 ml / min for 60 seconds.

[0139] The flow was circulated in the forward direction, and the relative centrifugal force was again increased to 2400 G. The cells remaining in the circulation channel were collected in the centrifuge chamber at a flow rate of 30 ml / min. Pinch point 30H1 before syringe S6, used for collecting the medium and cells removed by elutriation, was opened, and the flow was circulated in the reverse direction. The relative centrifugal force was 2400 G, the flow rate was 30 ml / min, and the liquid containing the cell layer collected in the centrifuge chamber (beyond the centrifuge chamber before elutriation) was manually withdrawn with the syringe at a target volume of 40 ml.

[0140] After flowing in the reverse direction at a relative centrifugal force of 10 G and a flow rate of 100 ml / min, pinch point 30A1 between the closed circulation container 24 was opened, and the remaining post-elutriation suspension was placed into the closed circulation container 24. The suspension was then sampled using syringe S4. A cell layer was formed by circulating in the forward direction at 2000 G and 30 ml / min for 60 seconds. Pinch point 30E1 between the closed circulation container 24 and the circulation path was then opened, and the post-elutriation suspension was transferred from the closed circulation container 24 to the centrifuge chamber. Spinoculation was then performed at 1500 G, 5 ml / min, and 60 seconds for concentration.

[0141] 3. SeV Vector Infection (Contact between Leukocytes (especially lymphocytes) and SeV Vector) A small amount of SeV-containing liquid was manually introduced into the circulation channel from syringe S3 at a relative centrifugal force of 1500 G and a flow rate of 5 ml / min. Lymphocytes were contacted with the SeV vector by spinoculation at 1500 G and 5 ml / min for 120 minutes. After contact, the SeV vector was removed by washing with medium at a relative centrifugal force of 1500 G and a flow rate of 5 ml / min.

[0142] 4. Harvesting: Lymphocytes infected with SeV vectors were concentrated for 25 seconds at a relative centrifugal force of 2400 G and a flow rate of 30 ml / min, and then transferred to a CELLPET disposable culture vessel (S5) connected to 30H to maintain the closure of the closed system.

[0143] 5. 3D Culture: iPS cells were expanded in the CELLPET disposable culture vessels in an incubator up to passage 8. The following six cytokines were added according to the volume of cell suspension collected in the CELLPET: IL-6: FUJIFILM Wako Pure Chemical; SCF: FUJIFILM Wako Pure Chemical; TPO: FUJIFILM Wako Pure Chemical; Flt3-L: FUJIFILM Wako Pure Chemical; IL-3: FUJIFILM Wako Pure Chemical; G-CSF: FUJIFILM Wako Pure Chemical. Half of the medium was replaced with StemFit AK03 medium every 2-3 days until day 14 of establishment. It was also confirmed that iPS cells could be established using a water bath or plate heater without using an incubator.

[0144] (1) Morphological Observation of iPS Cells The iPS cells established and 3D cultured in Example 1 were observed under a microscope. The iPS cells were as shown in the photograph in Figure 13 (scale bar, 200 µm) (230307_Rotea_lot1 p1 day 5, 23 days after SeV infection).

[0145] (2) Confirmation of GFP After harvesting, the cultured cell solution was set aside, and the portion for GFP evaluation was seeded onto a plate. FACS was performed 3 or 4 days after seeding. The results are shown in Tables 1 and 2 below. Tables 1 and 2 show that iPS cells were produced under various conditions using this production method, based on the observation of GFP fluorescence and the confirmation of undifferentiated markers.

[0146] (3) Number of spheroids after establishment The number of spheroids in each test group is as shown in Tables 1 and 2 below.

[0147]

[0148]

[0149] (4) Confirmation of Undifferentiated Cell Markers: The expression of iPS cell markers TRA-1-60, OCT3 / 4, and SSEA4 was examined by flow cytometry in iPS cells 53 days (4 / 24) after infection with the Rotea0302 2D p6 vector and 27 days (5 / 8) after infection with the Rotea0411 2D p2 vector. As shown in the graph in Figure 14, the expression of reprogramming markers was confirmed. Specifically, iPS cells were dispersed into single cells, washed once with D-PBS, and fixed with 4% paraformaldehyde (Nacalai Tesque) at room temperature for 15 minutes. Fixed cells were permeabilized with 0.5% Triton X-100 (Roche) and blocked with Blocking One (Nacalai Tesque). Cells were incubated with primary antibodies diluted in D-PBS containing 2% FBS for 30–60 minutes at room temperature. After washing twice with D-PBS containing 2% FBS, the cells were incubated with secondary antibodies diluted in D-PBS containing 2% FBS for 30–60 minutes at room temperature in the dark. The antibodies used were: anti-Oct-4A (Clone C30A3, CST), anti-SSEA-4 (Clone MC813-70, BD Biosciences), and anti-TRA-1-60 (Clone TRA-1-60, BD Biosciences). The stained cells were passed through a 45 μm cell strainer (BD Japan) and applied to a CytoFLEX (Beckman Coulter) column.

[0150] (4) Immunocytostaining: Cell staining confirmed that iPS cells produced with Rotea expressed the pluripotent cell markers NANOG and OCT3 / 4. Cells were used 67 days (5 / 8) after infection with the Rotea0302 2D p8 vector. The following reagents were used, and nuclei were stained with DAPI and observed under a fluorescent microscope. The results are shown in the photograph in Figure 15 (scale bar: 100 μm). In the actual image, NANOG is shown in green, OCT3 / 4 in magenta, and DAPI in blue.

[0151] BD Cytofix TMFixation Buffer: BD biosciences TritonX-100 solution: Roche 10x TBS: Nakalai Blocking One: Nakalai Rabbit monoclonal anti-Oct-4A: Cell Signaling Technology CF 640R Goat anti-rabbit IgG: BIOTIUM Mouse monoclonal anti-Nanog: Cell Signaling Technology Alexa 488 Plus Goat anti-mouse IgG: Thermo Fisher Scientific DAPI: Nakalai

[0152] Example 2: Differences in the number of leukocytes isolated using Rotea depending on the type of whole blood (donor) Lymphocytes were isolated using Rotea under the above-mentioned conditions from 20 ml (n=11) or 40 ml (n=5) of whole blood (different donors) as starting material. The isolated leukocytes (viable cells) were counted using a cell counter. The results are shown in the graphs in Figure 16 (left graph: viable cell count, right graph: viability).

[0153] Example 3: Verification of the Effect of Elutriation. Starting with 40 ml of whole blood from donor 1, leukocytes were isolated from the whole blood using Rotea under the isolation conditions described above. One sample was washed (elutriated) after isolation to increase the multiplicity of infection (MOI), and iPSCs were established from the other sample without elutriation. The number of recovered leukocytes was counted. The pre-isolation and post-isolation lymphocyte counts per ml of whole blood were as shown in Table 1 (230404) and the upper left graph in Figure 17 (dark bars before isolation, light bars after isolation; + indicates with elutriation, - indicates without elutriation). After isolation, the cultured portion of the cell solution was set aside, and the portion for GFP evaluation was plated. FACS was performed 3 or 4 days after plating. The number of GFP(+) cells per ml of whole blood was counted, and the difference in GFP(+) cell counts with and without elutriation was compared. The results are shown in the upper right graph in Figure 17. A similar experiment was conducted with Donor 2 using 35 ml. The results are shown in Test Group 230502 in Table 1 and the lower left and right graphs in the graph in Figure 17 (+ indicates with elutriation, - indicates without elutriation). As is clear from Table 1 and the graph in Figure 17, the effect of elutriation varied depending on the donor, but it was found that, depending on the blood used as raw material, elutriation can be effective in drastically reducing larger cells such as granulocytes and monocytes.

[0154] Example 4: Study of SeV Infection Time. SeV infection times of 30 and 120 minutes were used. Specifically, 28 ml of whole blood from donor 1 was used as the starting material. Leukocytes were separated from the whole blood using the CTS Rotea System under the above-mentioned separation conditions, including elutriation. The number of separated and recovered leukocytes was counted. The cell counts before and after separation are shown in the upper left graph in Figure 18. SeV infection was also performed using the CTS Rotea System in the same manner as the above SeV infection, except that the SeV infection time was 30 minutes. The number of GFP(+) cells per ml of whole blood was counted. The results are shown in the upper right graph in Figure 18 for test group 230421 in Table 1. A similar experiment was also performed with an SeV infection time of 120 minutes. Furthermore, a similar experiment was performed using 35 ml of whole blood from donor 2 as the starting material. The results are shown in test plot 230516 in Table 1 and the graphs at the bottom left and bottom right of the graph in FIG.

[0155] Example 5: Study on the ratio f1 / Q of relative centrifugal force f1 and flow rate Q A study was conducted in the same manner as in Example 3, except that the SeV infection time was kept the same and the ratio f1 / Q was changed to 80 or 300 (flow rate Q: 5 ml / min, relative centrifugal force f1: 400 G or 1500 G). The results were as shown in Table 1 for test plots 230421 and 230509 and in the graph in Figure 19.

[0156] Example 6: Differentiation into three germ layers It was confirmed that iPS cells produced using Rotea can be induced to differentiate into three germ layers. (1) Differentiation into three germ layers iPS cells produced using 230307 3D p12 in Table 1 were differentiated using STEMdiff TM Trilineage Differentiation Kit (STEMCELL Technologies) was used to differentiate into three germ layers according to the manufacturer's protocol. 3 × 10 cells were used for differentiation into ectoderm and mesoderm. 5 iPS cells were used, and 4 × 10 cells were used for endoderm differentiation. 5iPS cells were used. SB431542 (Nacalai Tesque) was added to the medium for ectodermal differentiation induction at a final concentration of 10 μM. The medium was changed daily, and induction culture was continued for 7 days for ectodermal differentiation and 5 days for mesodermal and endodermal differentiation.

[0157] (2) Flow cytometry identification of tri-germ-differentiated cells. Tri-germ-differentiated cells were dispersed into single cells, washed once with D-PBS, and fixed with 4% paraformaldehyde (Nacalai Tesque) for 15 minutes at room temperature. Fixed cells were permeabilized with 0.5% Triton X-100 (Roche) and blocked with Blocking One (Nacalai Tesque). Cells were incubated with primary antibodies diluted in D-PBS containing 2% FBS for 30-60 minutes at room temperature. After washing twice with D-PBS containing 2% FBS, cells were incubated with secondary antibodies diluted in D-PBS containing 2% FBS for 30-60 minutes at room temperature in the dark. The antibodies used were as follows: anti-Nestin (Clone 10C2, CST), anti-PAX6 (Clone D3A9V, CST), anti-NCAM (Clone 123C3, CST), anti-Brachyury (Clone D2Z3J, CST), anti-FOXA2 (Clone 7E6, Abcam), anti-SOX17 (Clone D1T8M, CST), and anti-Mouse IgG Alexa Fluor. TM The stained cells were stained with IgG CF® 640R (BIOTIUM Plus 555, Thermo Fisher Scientific) and anti-Rabbit IgG CF® 640R (BIOTIUM). After a brief wash with D-PBS containing 2% FBS, the stained cells were passed through a 45 μm cell strainer (BD Japan) and applied to an SA3800 cell analyzer (SONY). As shown in the photographs and graphs in Figure 20, the three germ layer markers PAX6, Nestin (ectoderm), NCAM, Brachyury (mesoderm), FOXA2, and SOX17 (endoderm) were confirmed.

[0158] Example 7: Differentiation into Cardiomyocytes and Evaluation (1) Differentiation into Cardiomyocytes Rotea0307 3D iPS cells at passage 12 were induced to differentiate into cardiomyocytes. The differentiation induction method was as described in S, F., K, M., T, T., C, O., T, H., K, C., M, N., I, T., A, O., M, N., et al. (2016). Enhanced engraftment, proliferation, and therapeutic potential in the heart using optimized human iPSC-derived cardiomyocytes. Scientific reports 6. 10.1038 / srep19111, with minor modifications. Specifically, the procedure is as follows. Undifferentiated iPSCs were detached and cultured using TrypLE. TM Single cells were dissociated by incubating with Select Enzyme for 5 minutes. The single cells were then resuspended in 10 μl / ml GlutaMax (Thermo Fisher Scientific), 50 μg / ml ascorbic acid (Sigma), 4 × 10 -4 StemPro supplemented with M monothioglycerol (SIGMA), 150 μg / ml transferrin (Roche), 10 μM Y-27632, 0.5% Matrigel (Corning), and 2 ng / ml human recombinant BMP4 (R&D Systems). TMThe cells were suspended in -34 SFM (Thermo Fisher Scientific), placed in a low-adhesion 6-well dish, and cultured for 24 hours to form embryoid bodies. On day 1, medium containing human recombinant activin A (R&D Systems), BMP4, and bFGF (R&D Systems) was added to the wells. The final concentrations were 6 ng / ml activin A, 10 ng / ml BMP4, and 5 ng / ml bFGF. On day 3, the medium was changed to StemPro medium supplemented with 10 ng / ml VEGF (R&D Systems), 5.4 nM SB431542, 0.06 μM Dorsomorphin (Sigma), and 1 μM Wnt inhibitor IWP-3 (Stemgent). TM On day 7, the medium was replaced with 10 μl / ml GlutaMax, 50 μg / ml ascorbic acid, 4 × 10 -4 The medium was replaced with StemPro™-34 medium supplemented with 100 μg / ml monothioglycerol, 150 μg / ml transferrin, and 5 ng / ml VEGF. To maintain iPS cell-derived cardiomyocytes, the medium was changed every 2–3 days. On the day of cell injection, embryoid bodies were dissociated using collagenase II (Worthington Biochemical Company) for 3–6 hours and TrypLE select for 30 minutes. As a result, cardiomyocytes were observed on day 15 of differentiation, as shown in the upper photograph of Figure 21 (40x magnification).

[0159] (2) Confirmation of cardiomyocytes by flow cytometry The obtained iPS cell-derived cardiomyocytes were dispersed into single cells on day 15 of differentiation and fixed in Fixation Buffer (BD Biosciences) for 10 minutes. The fixed cells were then washed with 1x Perm / Wash. TMThe cells were permeabilized with BD Biosciences and incubated with anti-cardiac toroponin T (Clone 13-11, BD Biosciences, USA) diluted in D-PBS containing 2% FBS for 30 minutes at room temperature in the dark. After a brief wash with D-PBS containing 2% FBS, the stained cells were passed through a 45 μm cell strainer (BD Japan) and applied to the CytoFLEX. The resulting differentiation efficiency was approximately 70%, as shown in the lower graph of Figure 21.

[0160] Example 8: iPS Cell Culture in a Centrifugal Chamber After SeV infection of cells in Rotea, the cells were not harvested but were still retained in the Rotea chamber (centrifugation chamber) when the Rotea was stopped. The in and out tubes connected to the chamber were cut using a sterile connector (TERUMO TSCD-II), and the ends of the tubes were clamped as a precaution. The chamber was fixed in a CO2 incubator with the tip facing downward, and cells were cultured at 37°C with 5% CO2 for 15 days. Syringes were connected to the front and back of the tubes to change the medium, and the liquid was exchanged without aspirating the cells from the end of the chamber. Specific culture conditions are as follows.

[0161] <Culture method> ・Culture medium used: StemFit AK03 ・Culture period: 15 days ・CO2 incubator: MINIcell waken tech (Waken B Tech Co., Ltd.) ・CO2 atmosphere in incubator: 5% ・Inverted microscope: ix83 p2zf (Evident Co., Ltd.) ・Medium changes were performed in the same manner as in Example 1. Half of the medium was changed with StemFit AK03 medium every 2-3 days until day 14 after establishment.

[0162] A photograph (left) of iPS cell spheroids and a fluorescent microscope photograph (right) of iPS cells cultured 15 days after SeV infection are shown in Figure 22. The reprogrammed GFP marker emitted fluorescence, demonstrating that the iPS cells could be maintained or proliferated. This demonstrates that iPS cells can be cultured in a Rotea chamber (centrifugation chamber).

[0163] The production method of the present invention can reduce the use of expensive reagents such as viral vectors for introducing reprogramming factors into somatic cells, thereby making it possible to reduce the production costs of iPS cells.

[0164] This application is based on patent application No. 2024-052336 filed in Japan (filing date: March 27, 2024), the contents of which are incorporated in their entirety herein.

[0165] 10 Sealed chamber for counterflow centrifugation 21-26 Sealed containers 30A-30H, 33-35, 37, 38 Connecting pipes 30A1-30H1 Pinch point 400M Peristaltic pump

Claims

1. A method for producing induced pluripotent stem cells using a cell processing device, wherein the cell processing device has a closed system portion in which a sealed container for supplying materials and a rotatable sealed chamber configured to perform counterflow centrifugation are connected via a connecting pipeline, and the method for producing induced pluripotent stem cells comprises the steps of: (s1) contacting somatic cells with reprogramming factors; and (s2) establishing induced pluripotent stem cells from the somatic cells while performing the counterflow centrifugation in the sealed chamber, while maintaining the closed nature of the closed system portion.

2. The method for producing induced pluripotent stem cells described in claim 1, wherein the somatic cells are blood cells, and the method further comprises, before step (s1), a step (s0) of separating blood cells from whole blood in the sealed chamber while maintaining the closed nature of the closed system portion, wherein in step (s0), blood cells are separated from whole blood by elutriation achieved by counterflow centrifugation, and the blood cells are left in the sealed chamber.

3. A method for producing artificial pluripotent stem cells as described in claim 1 or 2, wherein the central axis of the rotational movement of the sealed chamber passes outside the internal space of the sealed chamber.

4. The method for producing artificial pluripotent stem cells described in claim 3, wherein the sealed chamber has an inlet port and an outlet port on the wall on the center side of the rotational movement, and is configured so that materials can flow in and out through the inlet port and the outlet port when the sealed chamber is rotating or stationary, and a tube connected to the inlet port extends into the sealed chamber and has an open end in an area radially outward of the rotational movement so as to generate a counterflow when the sealed chamber is rotating.

5. A method for producing artificial pluripotent stem cells according to any one of claims 1 to 4, wherein the shape of the internal space defined by the inner wall surface surrounding the internal space of the sealed chamber is conical, the bottom of the cone being located towards the center of the rotational motion, and the apex of the cone being located radially outward of the rotational motion.

6. The method for producing induced pluripotent stem cells according to claim 2, wherein step (s0) comprises: a step (s0-1) of removing red blood cells from whole blood by elutriation; and a step (s0-2) after step (s0-1) of removing granulocytes and monocytes from the whole blood from which the red blood cells have been removed by further elutriation, leaving lymphocytes in the sealed chamber.

7. The method for producing induced pluripotent stem cells described in claim 6, wherein in step (s0-2), while keeping the counterflow flow rate Q [ml / min] constant, the relative centrifugal force f1 [G] acting on the contents in the sealed chamber is gradually reduced over 20 to 100 seconds to reduce the ratio f1 / Q of the relative centrifugal force f1 [G] to the flow rate Q from 50 to 70 to 20 to 35, and then, while keeping the relative centrifugal force f1 constant, the flow rate Q is gradually increased over 30 to 100 seconds to reduce the ratio f1 / Q from 20 to 35 to 10 to 20.

8. A method for producing artificial pluripotent stem cells according to any one of claims 1 to 7, wherein in step (s1), the ratio f1 / Q of the relative centrifugal force f1 [G] acting on the contents in the sealed chamber to the counterflow flow rate Q [ml / min] is 50 to 400, and the time for which step (s1) is carried out is 30 to 180 minutes.

9. The method for producing induced pluripotent stem cells described in claim 8, wherein the time for which step (s1) is carried out is 120 minutes.

10. A method for producing induced pluripotent stem cells described in any one of claims 1 to 9, wherein steps (s1) and (s2) are carried out under suspension culture conditions.

11. A method for producing induced pluripotent stem cells according to any one of claims 1 to 10, further comprising, after step (s2), a step (s3) of expanding the induced pluripotent stem cells in the sealed chamber or in a sealed container for expansion connected aseptically to the closed system part while maintaining the closed nature of the closed system part.

12. A method for producing differentiated cells, comprising the steps of the method for producing induced pluripotent stem cells according to any one of claims 1 to 10, and further comprising, after step (s2), a step (s4) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closed nature of the closed system portion, wherein (I) materials necessary for differentiation induction are supplied into the sealed chamber, and step (s4) is carried out in the sealed chamber, or (II) the induced pluripotent stem cells present in the sealed chamber are moved into a sealed container for differentiation induction that is aseptically connected to the closed system portion, and materials necessary for differentiation induction are supplied into the sealed container for differentiation induction, and step (s4) is carried out in the sealed container for differentiation induction.

13. The method for producing differentiated cells described in claim 12, further comprising a step (s3) between steps (s2) and (s4) of expanding and culturing the induced pluripotent stem cells in the sealed chamber or in the sealed container for expansion culture connected aseptically to the closed system part, while maintaining the closure of the closed system part.

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