Method for manufacturing induced pluripotent stem cells

Abstract

To provide a novel method for producing iPS cells that requires less patient-derived somatic cells as raw material and reduces the use of reagents (e.g., viral vectors) compared to conventional methods. [Solution] The present invention provides a method for producing iPS cells using a cell processing device. The cell processing device has a closed system portion in which a sealed container for supplying materials and a centrifugal chamber configured to perform counterflow centrifugal separation are connected via a connecting conduit. The production method involves performing a step (s1) of contacting somatic cells with reprogramming factors while performing counterflow centrifugal separation in the centrifugal chamber, while maintaining the closed nature of the closed system portion.

Description

[Technical Field]

[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 the said production method. [Background technology]

[0002] In recent years, research into regenerative medicine using differentiated cells derived from induced pluripotent stem cells (iPS cells) has been actively conducted. In particular, treatment methods that involve establishing iPS cells from a patient's somatic cells (e.g., peripheral blood mononuclear cells) and then transplanting various differentiated cells and organoids induced from these iPS cells into the patient (autologous transplantation) are attracting attention as treatment methods that can reduce the risk of rejection (Non-patent documents 1 and 2). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2017 / 040548 [Non-patent literature]

[0004] [Non-Patent Document 1] Shinsuke Yoshida., et al., CLINICAL AND TRANSLATIONAL RESOURCE AND TECHNOLOGY INSIGHTS VOLUME 4, ISSUE 1, P51-66.E10, JANUARY 13, 2023 [Non-Patent Document 2] Madrid, M., et al., Current Protocols,1, e88. doi: 10.1002 / cpzl.88 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] For precision medicine (treatment tailored to individual patients), when autologously transplanting iPS cell-derived cells into a patient, it is preferable to minimize the amount of somatic cells collected from the patient in order to reduce the biological burden on the patient. Furthermore, from an economic standpoint, it is preferable to reduce the cost of cell processing, and to achieve this, it is preferable to make the cell processing equipment smaller and simpler, and to enable the establishment of iPS cells with fewer processing steps.

[0006] To date, systems for automatically producing iPS cells from somatic cells in a closed system have been proposed (for example, Patent Document 1). However, conventional systems are large-scale, sequentially connected independent processing devices for each process, and constructing and automatically operating such a system requires considerable expense. Furthermore, such relatively large systems are usually not suitable for processing small amounts of somatic cells, and considering losses that occur at the connection points between each device, it is necessary to input a large amount of somatic cells or viral vectors. Therefore, the inventors have addressed the problem that a system suitable for precision medicine, which is smaller in volume, lower in cost, more efficient, and reduces the burden on cell manufacturing operators while automatically producing iPS cells in a closed system, has not yet been provided.

[0007] The object of the present invention is to mitigate the aforementioned problems and provide a novel method for producing iPS cells that requires less patient-derived somatic cells as raw materials and reduces the use of reagents (e.g., viral vectors) compared to conventional methods. [Means for solving the problem]

[0008] The main components of this invention are as follows: [1] A method for producing induced pluripotent stem cells using a cell processing device, The cell processing apparatus has a closed system section in which a sealed container for supplying material and a rotatable sealed chamber configured for counterflow centrifugation are connected via a connecting conduit. This manufacturing method maintains the closure of the closed system portion, The process (s1) involves bringing somatic cells into contact with the reprogramming factor while performing the counterflow centrifugation in the sealed chamber, The process of establishing induced pluripotent stem cells from the aforementioned somatic cells (s2) A method for producing induced pluripotent stem cells, performed in this order. [2] The somatic cells are blood cells, Prior to step (s1), the process further includes step (s0) of separating blood cells from whole blood within the sealed chamber while maintaining the closedness of the closed system portion, wherein in step (s0), blood cells are separated from whole blood by ertriation achieved by counterflow centrifugation, and the blood cells remain within the sealed chamber. A method for producing induced pluripotent stem cells as described in [1] above. [3] The method for producing induced pluripotent stem cells according to [1] or [2], wherein the central axis of rotation of the sealed chamber passes outside the internal space of the sealed chamber. [4] The sealed chamber is provided with an inlet port and an outlet port on the wall on the side of the center of rotation, and is configured so that material flows in and out through the inlet port and the outlet port when the sealed chamber is rotating or stationary, To generate counterflow during the rotational motion of the sealed chamber, the tube connected to the inlet port extends into the sealed chamber and has an open end in a region radially outward from the rotational motion. The method for producing induced pluripotent stem cells as described in [3] above. [5] The shape of the internal space, as determined by the inner wall surface surrounding the internal space of the sealed chamber, is conical. The base of the cone is located on the side of the center of the rotational motion, and the apex of the cone is located radially outward from the rotational motion. A method for producing induced pluripotent stem cells according to any one of the above [1] to [4]. [6] The above step (s0) is A step (s0-1) of removing red blood cells from whole blood by erythrocytapheresis, and After the step (s0-1), a step (s0-2) of removing granulocytes and monocytes from the whole blood from which the red blood cells have been removed by further erythrocytapheresis and leaving lymphocytes in the closed chamber The method for producing induced pluripotent stem cells according to [2], comprising: [7] In the step (s0-2), While keeping the counterflow rate Q [ml / min] constant, the relative centrifugal force f1 [G] acting on the contents in the closed chamber is gradually decreased over 20 to 100 seconds, and the ratio f1 / Q of the relative centrifugal force f1 [G] to the flow rate Q is decreased 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, and the ratio f1 / Q is decreased from 20 to 35 to 10 to 20. The method for producing induced pluripotent stem cells according to [6]. [8] In the step (s1), the ratio f1 / Q of the relative centrifugal force f1 [G] acting on the contents in the closed chamber to the counterflow rate Q [ml / min] is 50 to 400, The time for which the step (s1) is carried out is 30 minutes to 180 minutes. The method for producing induced pluripotent stem cells according to any one of [1] to [7]. [9] The method for producing induced pluripotent stem cells according to [8], wherein the time for which the step (s1) is carried out is 120 minutes.

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

[11] After the step (s2), a step (s3) of expanding the induced pluripotent stem cells is further included in the closed chamber while maintaining the closure of the closed system portion or in a closed container for expansion culture aseptically connected to the closed system portion. The method for producing induced pluripotent stem cells according to any one of [1] to

[10] .

[12] A method for producing differentiated cells, comprising: The manufacturing method is having the steps in the method for manufacturing induced pluripotent stem cells according to any one of [1] to

[10] , and after the step (s2), further having a step (s4) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closure of the closed system portion, (I) Materials necessary for differentiation induction are supplied into the sealed chamber, and the 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 aseptically connected to the closed system portion, materials necessary for differentiation induction are supplied into the sealed container for differentiation induction, and the step (s4) is carried out in the sealed container for differentiation induction, The method for manufacturing the differentiated cells.

[13] The method for manufacturing differentiated cells according to

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

Advantages of the Invention

[0009] In the manufacturing method according to the present invention, a cell processing apparatus having a rotatable sealed chamber capable of performing counterflow centrifugation is used, and in the sealed chamber, a step (s1) of bringing an initialization factor into contact with somatic cells while performing counterflow centrifugation is carried out. More specifically, in the step (s1), somatic cells dispersed in a liquid are collected by centrifugal force in a small region (a region away from the center of the rotational movement in the radial outward direction) in the sealed chamber, and while maintaining that state, a fluid containing an initialization factor is passed as a counterflow (a flow in a direction toward the center of rotation against the centrifugal force) from the outer peripheral side to the center side of the rotational movement in the sealed chamber. Thereby, the somatic cells collected in the small region float as a cell bed, and it becomes possible for the initialization factor to contact the somatic cells with a high probability.

[0010] Hereinafter, the "rotatable sealed chamber configured to perform counterflow centrifugation" used in the present invention will also be abbreviated as "centrifugation chamber" or "chamber." As exemplified by the centrifugation chamber provided in the CTS Rotea Counterflow Centrifugation System (Thermo Fisher Scientific) described later, a centrifugation chamber that performs counterflow centrifugation has a smaller volume than other conventional commercially available sealed chambers capable of centrifugation. Therefore, a smaller amount of somatic cells can be processed appropriately, and the required amount of iPS cells can be obtained. Thus, the amount of somatic cells (e.g., whole blood) collected from a patient can be reduced, thereby reducing the biological burden on the patient.

[0011] As described above, in the step (s1) in which somatic cells are brought into contact with reprogramming factors while performing counterflow centrifugation, somatic cells gather in a predetermined small area within the centrifugation chamber due to centrifugal force, and a fluid containing the reprogramming factors passes between the gathered somatic cells as a counterflow. This increases the probability that the reprogramming factors will encounter and come into contact with the somatic cells. Therefore, the use of expensive reagents such as viral vectors for introducing reprogramming factors into somatic cells can be reduced, and consequently, the manufacturing cost of iPS cells can be reduced. Step (s1) of the manufacturing method according to the present invention can also be called a spinoculation step.

[0012] Establishing iPS cells from somatic cells typically requires a multi-stage and lengthy processing process, resulting in high costs for numerous specialized devices and associated labor. In a preferred embodiment of the present invention, blood cells are used as the somatic cells that serve as the raw material for iPS cells, and a step (s0) of separating the blood cells from whole blood in the centrifugal chamber is performed prior to the aforementioned step (s1). That is, the steps of separating blood cells from whole blood (s0), contacting the blood cells with reprogramming factors (s1), and establishing iPS cells (s2) are all carried out consistently within a single closed-system cell processing apparatus. This reduces cell contamination during manufacturing and enables the automation of a consistent manufacturing process from somatic cell separation to iPS cell establishment using a single cell processing apparatus. Therefore, it becomes possible to reduce the personnel required for iPS cell production in conventional cell culture processing facilities (CPCs), and because it is a closed-system apparatus, it is not necessary to raise the facility's grade to Grade A, resulting in a reduction in the cost of producing iPS cells. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a block diagram showing an example of the configuration of the closed system portion of a cell processing apparatus used in the manufacturing method of the present invention. The thick lines indicated by reference numerals 30B, 30C, 30D, 30E, 30F, 30G, and 30H indicate connecting conduits made of flexible tubing or the like. Connectors and the like are not shown. The solid line indicated by reference numeral 30A indicates a connecting conduit similar to the connecting conduits indicated by reference numerals 30B to 30H, but is shown as a thinner solid line than the other connecting conduits for easier identification. Connecting conduit 30A and the connecting conduits (30B, 30C, 30D) pass each other (i.e., the internal flow paths of each do not intersect with each other). [Figure 2] Figure 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, and is a cross-sectional view showing the inside of the sealed chamber. [Figure 3]Figure 3 is a schematic diagram showing an example of the counterflow centrifugal separation state in the sealed chamber shown in Figure 2, and is a cross-sectional view showing the inside of the sealed chamber. [Figure 4] Figure 4 is a photographic illustration of a commercially available cell processing apparatus preferably used in the manufacturing method of the present invention (a photographic illustration for advertising issued by the manufacturer of the cell processing apparatus), illustrating a state in which a closed system is attached to the mechanism of the cell processing apparatus and cells can be processed. [Figure 5] Figure 5 is a photographic diagram (an advertising photograph issued by the manufacturer of the cell processing device) showing a disposable unit called a single-use kit that directly engages with the mechanical part of the closed system in the cell processing device shown in Figure 4. [Figure 6] Figure 6 is a photographic diagram (issued by the manufacturer of the cell processing device) showing only the mechanical part of the cell processing device shown in Figure 4, with the closed system removed. [Figure 7] Figure 7 is an actual photograph of the closed system shown in Figure 1, illustrating the state in which various sealed containers are connected to the single-use kit shown in Figure 5. [Figure 8] Figure 8 is an actual photograph of a cell processing apparatus in which the closed system portion shown in Figure 7 is attached to the mechanical part. [Figure 9] Figures 9(a) to 9(c) are photographic diagrams showing the process of performing counterflow centrifugation in a sealed chamber to lyse and remove red blood cells from whole blood in an embodiment of the present invention. The photograph in Figure 9 captures the moment when the rotating sealed chamber passes in front of the imaging device (the same applies to Figures 10, 11, and 12(a)). [Figure 10] Figures 10(a) to (c) are photographic diagrams showing the process in which, in an embodiment of the present invention, counterflow centrifugation is performed in a sealed chamber to remove red blood cells from whole blood, and then granulocytes and monocytes are further removed, leaving lymphocytes in the sealed chamber. [Figure 11]Figure 11 is a photographic diagram showing the process of contacting leukocytes with a Sendai virus vector while performing counterflow centrifugation in a sealed chamber in an embodiment of the present invention. [Figure 12] Figure 12 is a photograph showing how cells into which reprogramming factors have been introduced are collected into a syringe via a filter that removes dead cells. [Figure 13] Figure 13 is a micrograph of iPS cells after 3D culture in Example 1. In the figure, the scale bar represents 200 μm. [Figure 14] Figure 14 is a graph showing the results of flow cytometry analysis of TRA-1-60, OCT3 / 4, and SSEA4 expression in the iPS cells obtained in Example 1. [Figure 15] Figure 15 is a fluorescence micrograph showing the results of cell staining confirming that the iPS cells obtained in Example 1 express NANOG and OCT3 / 4. [Figure 16] Figure 16 is a graph showing the results of an investigation into whether the number of white blood cells isolated in Example 2 differed depending on the donor. [Figure 17] Figure 17 is a graph showing the results of verifying the effect of ertriation in Example 3. [Figure 18] Figure 18 is a graph showing the results of the examination of SeV (Sendai virus) infection time in Example 4. [Figure 19] Figure 19 is a graph showing the results of the study on the ratio f1 / Q of relative centrifugal force f1 to flow rate Q in Example 5. [Figure 20] Figure 20 shows photographs and graphs confirming that the iPS cells produced in Example 6 can be differentiated. [Figure 21] Figure 21 shows photographs and graphs illustrating the evaluation results of cardiomyocytes obtained by differentiating iPS cells produced in Example 7. [Figure 22] Figure 22 shows a photograph (left) and a fluorescence micrograph (right) of a cultured iPSC spheroid 15 days after SeV infection. [Modes for carrying out the invention]

[0014] The method for producing iPS cells according to the present invention will be described in detail below. The manufacturing method is a method for producing iPS cells using a cell processing device. The cell processing device has a closed system section, which, as illustrated in Figure 1, has a configuration in which a sealed container for supplying materials (21-26, etc.) and a centrifugation chamber 10 are connected via connecting conduits (30A-30G, 33, 34, 35, etc.). Figure 1 illustrates a preferred embodiment of the closed system section, and the closed system section in the figure includes not only the configuration necessary for the following steps (s1) and (s2), but also the configuration necessary for step (s0), which will be described later. The manufacturing method involves operating the cell processing device to maintain the closedness of the closed system section, carrying out step (s1) in the centrifugation chamber 10, and then carrying out step (s2). Step (s2) may be carried out in the centrifugation chamber 10, as will be described later, or it may be carried out in another container, etc. Step (s1) is a step of bringing reprogramming factors into contact with somatic cells while performing counterflow centrifugation. Step (s2) is a step of establishing iPS cells from somatic cells that have come into contact with a reprogramming factor (in one embodiment, a step of culturing somatic cells that have come into contact with a reprogramming factor and establishing iPS cells). The operation of the cell processing apparatus for carrying out steps (s1) and (s2) may not be entirely automatic, but may also involve manual operation by an operator during or between each step.

[0015] By performing steps (s1) and (s2) using the aforementioned cell processing apparatus, reprogramming factors efficiently come into contact with somatic cells under the suspension conditions unique to counterflow centrifugation, and subsequently, iPS cells are established. This yields the effects described above, and the problems mentioned in the above-mentioned challenges are mitigated or resolved.

[0016] Other processing steps may be added between steps (s1) and (s2). Furthermore, the following operations may be performed as appropriate in steps (s1) and (s2). (i) In order to preferably perform counterflow centrifugation, the cell suspension (hereinafter sometimes simply referred to as the suspension) or medium (liquid) containing the somatic cells to be processed is circulated through a predetermined path including the centrifugation chamber. (ii) The somatic cells to be processed are transferred from the centrifugation chamber to a sealed container or external equipment within a closed system, and then returned to the centrifugation chamber. (iii) Taking samples of somatic cells during processing and performing tests, etc., in accordance with additional processing such as quality control measurements. These operations may be performed automatically, manually, or in combination.

[0017] (Cell processing equipment) The cell processing apparatus usable in this invention comprises a closed system section and a mechanical section. Details of these will be described later. The mechanical section drives the closed system section while maintaining its closed nature, and cooperates with the closed system section to sequentially carry out steps (s1) to (s2). As such a cell processing apparatus, a cell processing apparatus equipped with a centrifugation chamber capable of performing counterflow centrifugation is available, and in particular, the "CTS Rotea Counterflow Centrifugation System" manufactured by Thermo Fisher Scientific is preferred and recommended (hereinafter this cell processing apparatus will also be referred to as Rotea). Rotea is a commercially available product that boasts the automatic "washing, concentration, and recovery of cells" through its counterflow centrifugation function. The basic configuration and operation of Rotea itself are described in detail in the manufacturer's manual, etc. Furthermore, the configuration and function of Rotea are described in detail in international publications WO 2018 / 204992 A1 and WO 2019-140491 A1, for example. However, iPS cells are known to be vulnerable to physical stress, and even in manual establishment, there are significant individual differences depending on how the operator handles them. Therefore, the idea of ​​using the counterflow centrifugation function (washing, concentration, and recovery) of the Rotea cell processing device for CAR-T cells for iPS cell establishment (especially the step of contacting somatic cells with reprogramming factors (s1)) was unprecedented, and no such process had been demonstrated before.

[0018] The manufacturing method will be described in more detail below, with Rotea being cited as an example of a cell processing apparatus preferred for the present invention, while describing its configuration in detail. However, the manufacturing method can also be carried out with other cell processing apparatuses configured to perform counterflow centrifugation by modifying and operating the apparatus in accordance with the following description.

[0019] (Rotea's composition) As illustrated in Figure 4, Rotea has a closed system portion 300 and a mechanism 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 mechanism portion 400 and is disposable. The mechanism portion 400 is equipped with a peristaltic pump, a pinch valve, a drive motor, various sensors, etc., and includes a control unit that contains a control program set to carry out the manufacturing method. Each element of the mechanism portion 400 acts on the attached closed system portion 300 from the outside, thereby maintaining the closedness of the closed system portion and sequentially carrying out each step of the manufacturing method in accordance with the commands of the control unit. Figure 4 illustrates a conventional general usage state in which the closed system portion 300 is attached to the mechanism portion 400.

[0020] Figure 5 shows the central unit of the closed system. This unit is a disposable part provided by the manufacturer (hereinafter also referred to as a single-use kit). The single-use kit includes a centrifugal chamber 310 and is configured to engage detachably with the mechanism 400. Although not shown in Figure 5, the connecting conduits 320a and 320b are connected together as shown in Figure 4, with connecting conduit 320a being the part attached to the peristaltic pump. Connecting conduit 320b is connected to one port of the centrifugal chamber 310, corresponding to connecting conduit 34 in Figure 1, and connecting conduit 330 is connected to the other port of the centrifugal chamber 310, corresponding to connecting conduit 35 in Figure 1. Connecting conduit 300P in Figure 4 is added to the connecting conduits 300P1 and 300P2 of the single-use kit shown in Figure 5, and the sealed container 200 in Figure 4 is connected, thereby forming the closed system 300. The sealed container can be provided as needed for purposes such as containing each material required in the manufacturing process, a temporary passage for circulating materials or suspensions within a closed system, containing waste liquid, recovering intermediate products, or recovering iPS cells.

[0021] Figure 6 is a photographic diagram showing the mechanism 400 of Rotea. In Figure 6, the mechanism 400 has its front door 430 open, allowing the single-use kit portion of the closed system to be mounted on the front. The front of the mechanism 400 is arranged with a total of 10 pinch valve heads (400A~400G, 400H, 400J, 400K), a peristaltic pump 400M, a cylindrical recess 410 which is the space for the centrifugal separation chamber to rotate, a chamber carrier 420 connected to a drive motor to rotate the centrifugal separation chamber, various sensors, and operation buttons. Various sealed containers included in the closed system are suspended from hanger posts 440. The door 430 also functions as a fixed base opposite the pinch valve heads (movable heads) and constitutes a pinch valve that opens and closes the pinch points on each connecting conduit (flexible tube) in the single-use kit. Even when using a cell processing apparatus other than Rotea, the present invention can be carried out by arranging the sealed container, connecting conduits, pinch valves, peristaltic pumps, etc., in an equivalent manner to the Rotea exemplified herein, and operating them in the same manner as the exemplified Rotea.

[0022] In the example shown in Figure 1, each sealed container is used for the following purposes: The sealed container 21 contains a liquid culture medium and is connected to the connecting conduit 30B. The liquid culture medium is also used as a liquid for cleaning each channel and the centrifuge chamber. The sealed container 22 contains a liquid containing somatic cells as the raw material and is connected to the connecting conduit 30C. In the example in Figure 1, the sealed container 22 contains (diluted) whole blood so that the step (s0) of separating the raw material blood cells from whole blood is performed before step (s1). The sealed container 23 contains a hemolytic agent (a buffer solution for lysing red blood cells (for example, a buffer solution mainly composed of ammonium chloride, potassium bicarbonate, and EDTA or its salts)) and is connected to the connecting conduit 30D. The sealed container 24 is an empty container connected to connecting conduits 30A and 30E, and is used as a temporary storage space when circulating materials or suspensions being processed in the centrifugal chamber 10. In the clockwise flow direction shown in Figure 1, this circulation path is an annular flow path that sequentially connects the centrifugal chamber 10, connecting conduit 35, branching point 36, connecting conduit 30A, sealed container 24, connecting conduit 30E, confluence point 32, connecting conduit 33, connecting conduit 34 (which receives pressure from the peristaltic pump), and centrifugal chamber 10. In the example shown in Figure 1, syringes S1 and S2 are connected to connecting conduit 30A for obtaining samples for inspection from within the conduit. The sealed container 25 contains a liquid culture medium for flushing the initialization factor and is connected to the connecting conduit 30F. In the example in Figure 1, to prevent the initialization factor from remaining in the sealed container or connecting conduit (i.e., to utilize it more completely), the initialization factor is injected into the connecting conduit 30F from a syringe S3 or S4 connected to the connecting conduit 30F. The initialization factor injected into the connecting conduit 30F from the syringe is flushed out by the liquid culture medium flowing out of the sealed container 25 and can flow into the centrifugation chamber 10. The sealed container 26 is a container for receiving various waste liquids generated during the processing steps, and is connected to the connecting pipeline 30G.

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

[0024] The configuration of each sealed container is not particularly limited, and conventional sealed containers used in Rotea can be referenced. Preferred examples include flexible bags or syringes made of flexible film or flexible sheet. The flexible film is a film that is flexible enough to deform according to the amount of contents in the bag. The flexible film may also be gas permeable, allowing O2 and CO2 necessary for cell culture to pass through. The capacity of each sealed container varies depending on the application and is not particularly limited, but for applications supplying somatic cells as raw materials, it is about 20 to 200 ml, and for applications supplying liquid culture media or containing waste liquid, it is about 200 to 3000 ml.

[0025] (Pinch point) Each connecting conduit (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 flexible tubing. Each pinch point is located in a predetermined position in the single-use kit, as shown in Figure 1. When the single-use kit is mounted on the mechanism part 400 (Figure 6), as described above, each pinch point (30A1~30H1, 30J1, 30K1) is positioned corresponding to the pinch valve heads (400A~400H, 400J, 400K) of the mechanism part 400, and is compressed / released in accordance with the operation of each pinch valve head, thereby opening and closing each connecting conduit. In the following explanation of opening and closing each pinch point, the explanation of opening and closing the pinch valve corresponding to each pinch point will be omitted.

[0026] (Layout and configuration of connecting conduits) The connecting conduits (30B, 30C, 30D) extending from the sealed containers (21, 22, 23) respectively enter the single-use kit, pass through pinch points (30B1, 30C1, 30D1), and are then connected to a single bubble trap 31. The connecting conduit 38 extending from the bottom of the bubble trap 31 passes through pinch point 30K1 and then reaches confluence point 32. Meanwhile, the connecting conduits (30E, 30F) extending from the sealed containers (24, 25) respectively enter the single-use kit and pass through pinch points (30E1, 30F1) respectively before reaching confluence point 32. These connecting conduits (38, 30E, 30F) merge into one at confluence point 32 to form connecting conduit 33. The connecting conduit 33 branches off to an output connecting conduit 30H. After passing through pinch point 30H1, the connecting conduit 30H can be connected to various containers and equipment as needed. The connecting conduit 33 also branches off to a connecting conduit 34 which is set on a peristaltic pump. The connecting conduit 34 is connected to the first port of the centrifugal separation chamber 10. A connecting conduit 35 is connected to the second port of the centrifugal separation chamber 10, and the connecting conduit 35 reaches a branching point 36, where it branches off to connecting conduits 30A, 37, and 30G. The connecting conduit 30A, after passing through pinch point 30A1, exits the single-use kit and connects to the sealed container 24. After passing through pinch point 30J1, connecting pipe 37 is connected below the bubble trap 31, where it merges with the flow coming out of the bubble trap 31, becoming connecting pipe 38. As described above, connecting pipe 38 passes through pinch point 30K1 and then reaches confluence point 32. The connecting conduit 30G passes through pinch point 30G1 without merging with other connecting conduits (i.e., passing near junction point 32), then exits the single-use kit and connects to the sealed container 26.

[0027] The peristaltic pump 400M, shown by the dashed line in Figure 1, is a device belonging to the mechanism part 400. Also called a tube pump or peristaltic pump, it moves the fluid inside the connecting pipe (flexible tube) by sequentially pressing it from the outside with a rotating roller head. In Figure 1, when the peristaltic pump 400M operates counterclockwise, the material in the connecting pipe 34 flows in the direction of arrow a1. This flow direction is the direction that generates a counterflow in the centrifugal chamber 10. Hereafter, this flow direction of arrow a1 will also be called the "forward direction". Conversely, in Figure 1, when the peristaltic pump 400M operates clockwise, the material in the connecting pipe flows in the direction of arrow a2. Hereafter, this flow direction of arrow a2 will also be called 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 pipe.

[0028] Given the use of the pinch valve and peristaltic pump described above, the pipe members constituting the connecting pipeline are preferably tubes made of soft materials (such as silicone or polyvinyl chloride). The connecting pipeline may also include pipe members made of hard materials in parts such as joints.

[0029] (Closed or airtight) The closed or airtight nature of a closed system refers not only to a state in which the interior is kept airtight or liquid-tight, but also to a state in which the interior is isolated from the outside world to the extent that microorganisms and viruses cannot enter from the outside, that is, to the extent that sterility is maintained inside. For example, a sealed container equipped with a porous filter (for example, with a pore size of about 0.2 μm or less, especially about 0.1 to 0.2 μm) that prevents bacteria and viruses from passing through but allows fluids (especially gases) to pass through, allows outside air to pass through the porous filter and into the sealed container, but prevents bacteria and viruses from entering, thus maintaining sterility within the closed system and thus possessing airtightness. Similarly, a container whose walls are made of a gas-permeable membrane, preventing bacteria and viruses from passing through but allowing O2 gas molecules and CO2 gas molecules to pass through, also possesses airtightness. Therefore, "closed system" similarly refers not only to a system that is closed in an airtight or liquid-tight manner, but also to a system in which the interior is isolated from the outside world to the extent that microorganisms and viruses cannot enter from the outside.

[0030] (Centrifugal chamber and counterflow centrifugation) As shown in Figure 1, the centrifugal separation chamber 10 is an element included in the closed system and is equipped with a rotatable pipe joint structure similar to a rotary joint, thereby allowing rotation while maintaining the airtightness of the closed system, even with the connecting pipes (34, 35) connected and even while liquids flow in and out through the connecting pipes. In the example in Figure 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 Figure 1.

[0031] Figure 2 is a diagram illustrating the counterflow centrifugal separation process in a centrifugal chamber 10, schematically showing a cross-section of the chamber 10 and illustrating the connecting pipelines in block diagram form. As shown in Figure 2, the central axis of rotation (rotation axis) y1 of the centrifugal chamber 10 passes outside the internal space of the chamber 10, and as indicated by arrow a3, the chamber 10 is driven to rotate (circumferentially) around the rotation axis y1. Therefore, the internal space of the chamber 10 has an outer circumference side (the side radially away from the rotation axis y1) and a rotation center side (the side closer to the rotation axis y1).

[0032] The centrifugal separation chamber 10 has two ports on the wall on the side of the center of rotation, which function as an inlet port 12 and an outlet port 13 in the forward flow a1. In the reverse flow, these inlet and outlet ports have opposite functions. In the example in Figure 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 or materials can flow in and out through the inlet port 12 and the outlet port 13 whether the chamber 10 is rotating or stationary.

[0033] The structure of the centrifugal separation chamber 10 is not particularly limited, and a chamber with an inlet for counterflow on its outer circumference can be used to generate counterflow during the rotational motion of the chamber. In Rotea's chamber, as shown in Figure 2, a tube 14 connected to the inlet port 12 extends into the interior of the centrifugal separation chamber 10 and has an open end on the outer circumference. The flow f2 of liquid m1 flowing out from the open end of the tube 14 inside the centrifugal separation chamber is called counterflow, and it flows inside the chamber from the outer circumference to the center of rotation in the opposite direction to the relative centrifugal force f1. The antagonistic effect of the relative centrifugal force f1 and the counterflow f2 achieves fractionation (elutriation) according to the size and weight of the particles. That is, as illustrated in Figure 2, in the centrifugal separation chamber, larger and heavier particles e1 gather on the outer circumference, and smaller and lighter particles e2 gather on the center of rotation, separating into layers. Furthermore, even smaller particles (such as viral vectors used for contact with initialization factors) do not remain in the centrifugation chamber but flow along with liquid m1 as a counterflow.

[0034] Figure 3 shows an example of counterflow centrifugation. In the example in Figure 3, as shown in Figure 3(a), only one type of particle (e.g., somatic cell) e1 is dispersed in the liquid (e.g., liquid culture medium) m1 inside the centrifugation chamber 10. The operation of gathering these dispersed particles e1 to the outer periphery using centrifugal force and counterflow, as shown in Figure 3(b), also corresponds to counterflow centrifugation, since it separates the particle layer e1a and the liquid layer m1a.

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

[0036] (Ratio of relative centrifugal force f1 [G] to flow rate Q: f1 / Q) 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 delivered by the peristaltic pump is important. The specific value of the ratio f1 / Q will be described later.

[0037] (Control Unit) The control unit (including a computer and computer program) included in the mechanism operates each pinch valve, peristaltic pump, and centrifugal chamber, and automatically executes each step (s1) to (s2) of the manufacturing method in order. In a preferred embodiment, it also automatically executes step (s0), which will be described later, thereby automatically executing a consistent processing process (s0, s1, s2). The computer program executed by the control unit's computer allows for the preset setting of parameters such as the opening and closing timing of any pinch valve, the operating period of the peristaltic pump, the feed rate, the counterflow rate Q, the operating period of the centrifugal chamber, and the relative centrifugal force f1, through the input and modification of parameters. By setting the necessary sealed containers within the closed system and executing the pre-configured computer program, the steps of the novel manufacturing method according to the present invention are automatically and sequentially carried out. The execution of the computer program (i.e., the operation of Rotea) allows for arbitrary stopping and restarting of the device, stopping a process while continuing circulation, and transitioning to the next process, all through the input of commands via switches, etc. Therefore, manual processes (such as taking samples for quality control or injecting materials into pipelines) can be arbitrarily added during or between each process.

[0038] (Preparation for device operation) In order to carry out each step of the manufacturing method using a cell processing apparatus equipped with a centrifugal chamber such as Rotea, preliminary preparations are necessary, such as filling each channel of the closed system (centrifugal chamber, each connecting conduit, and circulation channel) with liquid culture medium. Such preliminary preparations can be carried out appropriately in accordance with the operation manual of the cell processing apparatus so that each step of the manufacturing method can be carried out as desired.

[0039] (Step (s1): The process of exposing somatic cells to reprogramming factors) First, as shown in Figure 3(a), the somatic cells to be processed and the medium (such as a liquid culture medium) m1 are transferred from a designated sealed container to the centrifuge chamber 10. Next, the medium in which these somatic cells are dispersed (the first suspension) is circulated in a circulation channel, which includes the chamber 10 and will be described later, while counterflow centrifugation is performed. As a result, a cell layer e1a is formed in the chamber 10, as shown in Figure 3(b). Next, while maintaining the cell layer e1a, a medium (second suspension) in which the reprogramming factors are dispersed is supplied into the circulating channel. As a result, the reprogramming factors circulate through the circulating channel and pass between the somatic cells densely packed in the cell layer e1a, thereby coming into contact with the somatic cells with a higher probability. The iPS cells to be manufactured, the somatic cells and medium used as raw materials, the reprogramming factors, and other necessary materials will be described later.

[0040] (Ratio f1 / Q in process (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 rate Q [ml / min], and the duration of step (s1) are important for the reprogramming factors to come into contact with somatic cells more efficiently and for the reprogramming factors to come into contact with as many somatic cells as possible. Although it varies depending on the shape, volume, and temperature of the centrifuge chamber, as well as the properties of the fluid and the shape of the particles, when Rotea is used at room temperature and ordinary liquid culture media are 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 3000 G, more preferably approximately 400 to 2500 G, and the flow rate Q is preferably approximately 1 to 30 ml / min, more preferably approximately 1 to 10 ml / min. These combinations of relative centrifugal force f1 and flow rate Q are selected so that the ratio f1 / Q is approximately 50 to 400 [G·min / ml]. Furthermore, the time required to carry out step (s1) under the above conditions, that is, the time required to bring the reprogramming factor into contact with as many somatic cells as possible, is not particularly limited as long as the establishment of the desired iPS cells can be achieved, but is preferably 30 to 180 minutes, and more preferably 120 minutes.

[0041] (Circulation channel) To perform counterflow centrifugation, it is necessary to circulate a liquid or suspension in a predetermined circulation channel (including the centrifugation chamber 10) at an appropriate ratio f1 / Q. During this circulation, the necessary cell layer is formed inside the chamber 10. A preferred circulation channel usable in the closed system portion of Rotea is, for example, a ring-shaped channel in Figure 1 that connects the outlet of the centrifugal chamber 10, connecting conduit 35, branching point 36, connecting conduit 37, pinch point 30J1, pinch point 30K1, connecting conduit 38, confluence point 32, connecting conduit 33, connecting conduit 34, and the inlet of the centrifugal chamber 10 in this order. This circulation channel is preferably usable when circulation is to be continued while performing counterflow centrifugal separation. It is also possible to configure the circulation channel to pass through a sealed container 24 for circulation. In this case, the circulation channel is a ring-shaped channel in Figure 1 that connects the outlet of the centrifugal chamber 10, connecting conduit 35, branching point 36, connecting conduit 30A (including pinch point 30A1), sealed container 24, connecting conduit 30E (including pinch point 30E1), confluence point 32, connecting conduit 33, connecting conduit 34, and the inlet of the centrifugal chamber 10 in this order. This circulation channel is preferably used, for example, when elutriation separates lymphocytes from other blood cells, sends them to a bag (sealed container), allows them to temporarily reside in the bag, and then returns the lymphocytes to the centrifugal chamber.

[0042] (Step (s2): Step to establish iPS cells) In this step, iPS cells are established by culturing somatic cells that have come into contact with the reprogramming factor in step (s1) above in a liquid culture medium. Step (s2) may be carried out inside the centrifugation chamber 10, or while slowly circulating the somatic cells that have come into contact with the reprogramming factor in any circulation channel, or by transferring the somatic cells that have come into contact with the reprogramming factor to any sealed container or external processing device. The iPS cells are not particularly limited as long as the desired iPS cells can be obtained, but they may be in the form of, for example, two-dimensional colonies, spheroids, etc.

[0043] In one embodiment, step (s2) may be carried out in a centrifugation chamber 10 configured to adjust to a temperature suitable for establishing iPS cells (in one embodiment, a temperature suitable for culturing somatic cells that have come into contact with reprogramming factors to establish iPS cells) (for example, 30°C to 40°C, preferably 37°C). Examples of configurations for adjusting the temperature inside the centrifugation chamber to a temperature suitable for establishing iPS cells include, for example, a configuration in which a heater is attached to the chamber to heat the inside of the centrifugation chamber to an appropriate temperature, or a configuration in which a liquid culture medium heated to an appropriate temperature is sent into the centrifugation chamber. These configurations may be used individually or in combination.

[0044] Examples of heaters used to heat the centrifugation chamber and the liquid culture medium to an appropriate temperature include electric heaters attached to the outside of the centrifugation chamber or sealed container, heating lamps located away from the object to be heated, hot air heaters that supply hot air to the space surrounding the object to be heated, and air conditioning systems that adjust the temperature inside the case surrounding the entire Rotea or the room to a temperature suitable for establishing iPS cells. These components may be used individually or in any combination.

[0045] Temperature control to adjust the temperature inside the centrifugation chamber to a temperature suitable for establishing iPS cells can utilize conventionally known control methods such as feedback control, and temperature sensors and controllers can be used as appropriate. In this embodiment, the rotation of the centrifugation chamber may be stopped and used as a stationary sealed container, and the configuration for counterflow may be used for liquid culture medium exchange.

[0046] In other embodiments, step (s2) may be carried out by aseptically transferring somatic cells that have come into contact with the reprogramming factor from the centrifugation chamber to a suitable sealed container or culture vessel (a sealed container equipped with the necessary ports to enable cell culture) within the closed system (i.e., moving them in a way that maintains the closedness of the closed system), and maintaining these sealed containers or culture vessels at the appropriate temperature using a water bath or the like. Examples of the culture vessels are not particularly limited, but include the disposable culture vessels attached to CellPet 3D-iPSC manufactured by J-TEC Corporation, and the G-Rex® 10N-CS and G-Rex 100N-CS culture vessels manufactured by Wilson Wolf. When these culture vessels are connected to the closed system of the Rotea used in the manufacturing method via connecting conduits in a way that maintains internal closure, the culture vessels may be considered as part of the closed system, or as external elements connected to the closed system.

[0047] In yet another embodiment, step (s2) may be carried out in a sealed chamber or sealed container in another cell processing apparatus. Such a sealed chamber or sealed container in another cell processing apparatus may be connected to the closed system portion of Rotea via a connecting conduit, or cells may be aseptically transferred using a transfer means such as a syringe. Examples of such other cell processing apparatuses include CliniMACS Prodigy® from Miltenyi Biotec, Cocoon® from Lonza Corporation, and Xuri from Cytiva. TM Examples include Cell Expansion Systems.

[0048] The establishment of iPS cells can be appropriately confirmed by the expression of reprogramming factors introduced by known methods (e.g., Oct3 / 4, SOX2, Nanog, TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase, etc.). The duration of this step (s2) is not particularly limited as long as iPS cells are established, but typically it is, for example, 10 days or more, and 14 days or more is preferred. There is no particular upper limit, but typically it is 40 days or less, and 30 days or less is preferred. However, if the concept of Quality By Test (QbT) is followed, it is necessary to confirm the establishment of iPS cells, but if the concept of Quality By Design (QbD) is followed, it is not necessarily necessary to confirm the establishment of iPS cells as long as the process is designed to produce iPS cells and differentiated cells for the purpose of obtaining differentiated cells for use in regenerative medicine from patient-derived somatic cells.

[0049] In this invention, step (s1) is carried out under suspension culture conditions. That is, by performing counterflow centrifugation, the somatic cells remain suspended in the liquid culture medium, and the reprogramming factors efficiently come into contact with the somatic cells. In one embodiment, step (s2) is also carried out under suspension culture conditions. In this specification, "suspension culture" means culturing cells while maintaining a state in which they are suspended in a liquid medium (culture medium). More specifically, suspension culture means culturing under conditions that do not allow strong cell-substrate binding to be formed between cells and culture equipment, etc. (e.g., sealed chamber, sealed container, circulation channel, external processing device, etc.). It is easy for a person skilled in the art to determine whether the cultured cells are in a suspension culture state or adherent culture state, for example, by shaking the culture equipment during microscopic observation. The suspension culture conditions in step (s2) may be achieved, for example, by using scaffolding materials as described later, by counterflow centrifugation in a sealed chamber to keep the cells flowing through the circulation channel, by stirring the contents of a predetermined container with a stirrer, jet, horizontally placed plate (e.g., vertical movement), or stirring blade (e.g., rotational movement), or by periodically changing the tilt angle (e.g., a seesaw-like oscillating motion). Alternatively, the suspension culture conditions may be achieved using an external processing device suitable for three-dimensional suspension culture. Other conditions in step (s2) are not particularly limited, but are approximately 30-40°C, preferably approximately 37°C, and culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of preferably approximately 2-5%. For example, temperature control in each container can refer to conventionally known temperature control methods, such as room temperature control, provision of heaters to each container, or temperature control of the material supplied to the container.

[0050] (Step to separate somatic cells (s0)) In a preferred embodiment of the manufacturing method, a further step (s0) is added before step (s1) in which somatic cells to be used as raw materials for iPS cells are separated from whole blood using the centrifugal chamber 10. Therefore, in this embodiment, the somatic cells to be used as raw materials for iPS cells are blood cells. Whole blood, blood cells to be separated, medium, and other necessary materials will be described later. In step (s0), blood cells necessary as raw materials for iPS cells are separated from various blood cells in whole blood by eltriation achieved by counterflow centrifugation in the chamber 10. The separated blood cells are left in the centrifugation chamber and subjected to the next step (s1). Here, in step (s0), "[the blood cells separated by eltriation are left in the centrifugation chamber]" may mean either (i) or (ii) below. (i) The blood cells separated in the chamber remain inside the chamber without ever leaving it. (ii) The blood cells separated in the chamber are removed from the chamber, and then other unwanted blood cells 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 by counterflow centrifugation, the relative centrifugal force f1 and the counterflow rate Q are selected to have a predetermined ratio f1 / Q, and as shown in Figure 2, blood cells gather at specific locations according to their size and weight, thereby forming multiple cell layers. Of the multiple cell layers formed in the chamber, the cell layer on the rotation center side can be removed from the port on the rotation center side 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] While elutriation itself is a known classification technique, in this invention, the ratio f1 / Q of the relative centrifugal force f1 to the counterflow rate Q is appropriately selected to separate blood cells (raw material) from whole blood in a single centrifuge chamber, and then iPS cells are established by contacting the separated blood cells with reprogramming factors. This allows for the production of iPS cells from whole blood in a single, integrated process within a single centrifuge chamber, thereby reducing manufacturing costs.

[0053] In one embodiment, the blood cells to be separated from whole blood are white blood cells (particularly lymphocytes). In this embodiment, step (s0) comprises two steps (step (s0-1); hemolysis step, step (s0-2); step to remove granulocytes and monocytes). In the first step (s0-1), a hemolytic agent is added to whole blood, and the red blood cells are lysed by the hemolytic agent. The lysed red blood cells are then removed from the centrifugation chamber by elutriation. The removal of red blood cells here means not only complete removal but also a process that reduces the number of red blood cells to a degree that does not hinder the establishment of iPS cells. In the second step (s0-2), granulocytes and monocytes are removed from the whole blood from which the red blood cells have been removed by elutriation, leaving lymphocytes in the centrifugation chamber, which are then used as raw materials for iPS cells in the next step (s1). The removal of granulocytes and monocytes here means not only complete removal but also a process that reduces the number of granulocytes and monocytes to a degree that does not hinder the establishment of iPS cells.

[0054] (Lymphocytes) In one embodiment, lymphocytes may be selected as the blood cells to be separated from whole blood, taking into consideration that lymphocytes are suitable raw materials for iPS cells and that they are particles that are easily located on the port side and recovered by elutriation.

[0055] (Ratio f1 / Q in process (s0-1)) In red blood cell removal, the ratio f1 / Q of the relative centrifugal force f1 to the counterflow rate Q varies depending on the shape, volume, temperature, and properties of the fluid in the centrifuge chamber. However, when using Rotea at room temperature and a normal liquid culture medium and hemolytic agent, the ratio f1 / Q is approximately 17-300 [G·min / ml]. In this case, the relative centrifugal force f1 is preferably around 1000-3000 G, more preferably around 1500-2800 G, and the flow rate Q is preferably around 10-60 ml / min, more preferably around 15-50 ml / min. These combinations of relative centrifugal force f1 and flow rate Q are selected so that the ratio f1 / Q is approximately 17-300 [G·min / ml]. Furthermore, in carrying out the process (s0-1) under the above conditions, the hemolytic agent may be introduced into the flow path in small amounts, and the process may be repeated by switching to the circulation circuit, and finally the flow path may be completely replaced with the hemolytic agent. In one embodiment, for example, (1) 3 ml, (2) 4 ml, and (3) 5 ml may be introduced in stages, with a circulation time of 25 to 35 seconds for each, and after replacing the flow path 3 times for a total of 9 times, the amount of hemolytic agent delivered may be set to 80 ml to completely replace the liquid in the circulation flow path with the hemolytic agent.

[0056] (Ratio f1 / Q in process (s0-2)) In the removal of granulocytes and monocytes by elutriation, the suspension from which red blood cells have been removed in the above step (s0-1) is first flowed through the circulation channel, and counterflow centrifugation is performed for 10 to 50 seconds with a ratio f1 / Q of 50 to 70 (relative centrifugal force f1 of approximately 500 to 3000 G, counterflow flow rate Q of approximately 20 to 50 ml / min), forming a cell layer on the outer periphery of the centrifugation chamber. Granulocytes, monocytes, and lymphocytes are present in this cell layer. Next, while maintaining a constant flow rate Q, the relative centrifugal force f1 acting on the contents in the centrifugation chamber is gradually reduced over 20 to 100 seconds, decreasing 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 decrease in the ratio f1 / Q causes the cell layer to spread towards the center of rotation in the centrifugation chamber. At this time, since lymphocytes are lighter than granulocytes and monocytes, they are thought to be distributed more towards the center of rotation than granulocytes and monocytes within the cell layer. The term "gradually decreasing" here does not only mean a linear decrease, but may also include a portion of the change that is smoothly curvilinear, such as a smooth beginning to the change and a smooth ending to the change. Next, while maintaining a constant relative centrifugal force f1, the flow rate Q is gradually increased over 30 to 100 seconds, reducing the ratio f1 / Q from 20 to 35 to 10 to 20. The increased flow rate Q is approximately 40 to 100 ml / min. The decrease in the ratio f1 / Q allows lymphocytes to flow out from the port on the rotation center side of the chamber. The flowed-out lymphocytes and medium (liquid culture medium) are collected in a sealed container 24 for circulation. Here, "gradual increase" does not only mean a linear increase, but may also include a portion of the change that is smoothly curvilinear, such as starting with a smooth increase and ending with a smooth increase. Similarly, in the following explanation, a gradual change may include not only a linear change but also a portion of a smooth, curvilinear change.

[0057] The iPS cells to be manufactured, and the raw materials and ingredients required for the aforementioned steps (s0) to (s2) are described.

[0058] (induced pluripotent stem cells) In this specification, "induced pluripotent stem cell (iPS cell)" refers to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing reprogramming factors. Induced pluripotent stem cells have the ability to differentiate into various tissues and cells with different forms and functions in the living body, and to differentiate into any lineage of the three germ layers (endoderm, mesoderm, and ectoderm).

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

[0060] The method for producing iPS cells according to 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 patients with hereditary diseases. Cells differentiated from pluripotent stem cells derived from patients with hereditary diseases can serve as disease models that reflect the pathogenesis of the disease, and are therefore suitable for screening for therapeutic or preventive drugs for the disease. Alternatively, by repairing the genes of pluripotent stem cells derived from patients with hereditary diseases using genome editing with a CRISPR-Cas system or the like, and then differentiating them into target cells, it is possible to use these cells as therapeutic agents for the disease.

[0062] In this invention, “somatic cells” refers to the original cells to be processed in the rotatable sealed chamber used in this invention. In this specification, “somatic cells” means cells other than germ cells among the cells that make up an animal. Somatic cells are not particularly limited and include both mature, healthy or diseased somatic cells, and also include primary cultured cells, passaged cells, and established cell lines. Specifically, somatic cells may be, for example, suspension cells (e.g., blood cells) or adherent cells, but suspension cells are preferred. Examples of somatic cells used in the manufacturing method of the present invention include, but are not limited to, fibroblasts from the skin, skin cells, visual cells, brain cells, hair cells, oral mucosa cells, dental pulp cells, lung cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells, kidney cells, neural stem cells, mesenchymal stem cells derived from wisdom teeth, tissue stem cells, tissue progenitor cells, blood (hematopoietic) 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, these cells are separated by centrifugation of whole blood (density gradient centrifugation, specific gravity centrifugation, etc.), filtration (leukocyte removal filter, etc.), and removal of antibodies, magnetic materials (magnetic beads, etc.), or hydrophilic polysaccharides (Ficoll TM It is obtained by separation using (etc.).

[0064] (blood cells) In this specification, "blood cells" refers to all cells from hematopoietic stem cells through hematopoietic progenitor cells (including pluripotent hematopoietic progenitor cells and unipotent hematopoietic progenitor cells) to ultimately functional blood cells. Examples of blood cells include peripheral blood mononuclear cells (PBMCs) and umbilical cord blood mononuclear cells (CBMNCs). As described in step (s0) above, blood cells are preferably used as raw materials for iPS cells in the present invention.

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

[0066] (Whole blood) In this specification, "whole blood" means blood itself collected from a subject such as a human, in which blood cells and other components have not been separated. In this specification, "whole blood" may also include whole blood diluted by mixing with an appropriate buffer solution, or whole blood to which additives such as blood coagulation inhibitors (e.g., heparin, EDTA, citrate, etc.) or protease inhibitors have been added. The buffer solution used to dilute the whole blood is not particularly limited as long as it does not affect the blood cell components in the whole blood in a way such as hemolysis, but examples include phosphate buffer (PBS) and physiological saline.

[0067] (Whole blood supply) In the present invention, the amount of whole blood supplied to the rotatable sealed chamber is not particularly limited, but is approximately 50 to 15 ml, preferably 40 to 20 ml, and more preferably 35 to 20 ml. The whole blood may be placed in a sealed container for material supply in its collected state and sent to the sealed chamber, or it may be diluted with a buffer solution as described above and then placed in a sealed container for material supply and sent to the sealed chamber.

[0068] In this specification, unless otherwise specified, "cell" includes "cell population." A cell population may consist of one type of cell or two or more types of cells.

[0069] In this specification, "processing" cells means subjecting them to treatments such as cell culture, dilution of cell-containing solutions, cell washing, and separation of target cells from cell-containing solutions. It also means subjecting cells to treatments such as artificial proliferation and differentiation, cell line formation, cell activation, modification of biological properties, combination with non-cellular components, or genetic engineering modifications.

[0070] (Liquid culture medium) Liquid culture media delivered from sealed containers can be used not only for cell culture but also for various purposes in the manufacturing process, such as cell washing and dilution of drug solutions. Examples of liquid culture media that can be used in this invention include the following:

[0071] The liquid culture medium is not particularly limited, but examples include StemFit® AK03 medium (Ajinomoto Co., Inc.), StemFit® 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 include Thermo Fisher Scientific Medium, StemFit® AK02 medium (Ajinomoto Co., Inc.), Gibco CTS® KnockOut SR XenoFree Medium, 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 from Lonza), and mixtures thereof.

[0072] The culture medium can be supplemented with physiologically active substances and nutritional factors necessary for cell survival or proliferation, as needed.

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

[0074] In this specification, known serums may be used as the culture medium.

[0075] In this specification, the culture medium may or may not contain serum substitutes, as well as serum.

[0076] (Scaffolding materials) In this specification, "scaffold material" means a material or substrate that functions as a scaffold for cells in cell culture. Scaffold materials are not particularly limited as long as they can be used in adherent culture or suspension culture using scaffold materials (in other words, they may be free in the culture medium), but examples include those containing or made of synthetic resins, or those made of flexible materials such as collagen. Microcarriers may also be used as scaffold materials. Furthermore, as an example, scaffold materials may contain atelocollagen. Scaffold materials may be manufactured by known methods, or commercially available products may be used. Examples of commercially available products include Cytodex-1 (manufactured by GE Healthcare) and Corning® low-concentration Synthemax® II microcarriers (manufactured by Corning).

[0077] Furthermore, the scaffold material may contain or consist of an extracellular matrix, such as a basement membrane preparation (e.g., Matrigel (Corning), Geltrex matrix (Thermo Fisher Scientific), etc.), fibronectin, laminin or its fragments, entactin, collagen, gelatin, vitronectin, etc., or a combination thereof. The extracellular matrix as exemplified may be a natural product, an artificially synthesized product using genetic engineering technology, or a fragment cleaved with restriction enzymes, or a synthetic protein or synthetic peptide based on these biologically derived substances.

[0078] The method of supplying scaffolding materials to a sealed chamber or the like is not particularly limited as long as the desired cells can be processed appropriately, and the supply conditions (e.g., timing of supply, volume, etc.) and types of scaffolding materials can be appropriately set by those skilled in the art.

[0079] (initialization factor) In this specification, examples of "initialization 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, or Glis1. These initialization factors may be used individually or in combination. Any known combination of initialization factors can be used.

[0080] The reprogramming factor introduced into somatic cells may be in the form of a protein, a nucleic acid (RNA or DNA) encoding the protein, or an expression vector containing the nucleic acid. When the reprogramming factor is introduced in the form of RNA, the immunogenic RNA introduced into the cell may activate the cell's defense mechanism; therefore, RNA that evades this defense mechanism may be introduced into the somatic cell.

[0081] Examples of expression vectors include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, as well as plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors (piggyBac, piggyBat, TolII).

[0082] The introduction of nucleic acids, expression vectors containing said nucleic acids, or proteins (e.g., reprogramming factors) into cells can be carried out by various known methods. Examples of such methods include 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 materials mentioned above, any substances necessary for cell processing may be added as appropriate. The added materials can be placed in a sealed container or the like and connected to a sealed chamber, similar to the materials mentioned above. Other materials that may be added include, for example, liquids (e.g., physiological saline, buffer solution, etc.), powders, additives, release agents, cryoprotection solutions, and CO2.

[0084] A more preferred and detailed example of the preparation of materials for Rotea and the sequence of operations for carrying out the above steps (s0), (s1), and (s2) in order is shown in the following steps (1) to (14). The symbols 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 that are the raw material for iPS cells are blood cells (in particular leukocytes, especially lymphocytes), and steps (2) to (7) illustrate preferred steps for separating leukocytes (in particular lymphocytes) from whole blood.

[0085] (1) Priming When the closing system shown in Figure 1 is set into the mechanism shown in Figure 6 and the mechanism is activated, all pinch valves are activated first, closing all pinch points on the connecting pipeline. Before introducing whole blood into the centrifuge chamber, the circulation channel is filled with liquid culture medium. In Figure 1, the circulation channel is a ring-shaped channel connecting pinch point 30K1, connecting lines (38, 33, 34), centrifuge chamber 10, connecting lines (35, 37), and pinch point 30J1 in that order. In Rotea, if there is air in the centrifuge chamber, a sensor activates and the rotation of the chamber 10 stops. Therefore, as an initial operation, it is necessary to fill the circulation channel and other connecting lines with liquid. To explain this filling step in more detail, first, the pinch points (30B1, 30G1, 30K1) are opened (i.e., the pinch valves that close these pinch points are opened), and the peristaltic pump 400M is activated to allow the liquid culture medium to flow from the sealed container 21 into the connecting line 30B at a predetermined flow rate (e.g., 100 ml / min). The liquid culture medium passes through the bubble trap 31 and connecting channels (38, 33, 34) in sequence, filling the centrifugal chamber 10, and then through connecting conduit 35, branching point 36, and connecting conduit 30G to enter the sealed container 26 for waste liquid. This fills the aforementioned channels from connecting conduit 30B to connecting conduit 30G with the liquid culture medium. Similarly, the pinch points 30C1, 30D1, and 30F1 are opened in sequence to fill 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 hemolytic agent from entering connecting conduit 30B, which is the liquid culture medium channel, so that the concentration of the liquid culture medium does not change. In Rotea, the peristaltic pump is always operating when the mechanical parts are running, and liquid is always flowing through the channels with open pinch points and the centrifugal chamber.

[0086] (2) Diluted whole blood is transferred from the sealed container 22 into the circulation channel (step (s0)) Diluted whole blood contained in a sealed container 22 is introduced into the circulation channel and the centrifugation chamber (Figure 9(a)). While the diluted whole blood is circulated in the circulation channel, 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 place in the centrifugation chamber and remain as a cell layer, thus concentrating the diluted whole blood in the centrifugation chamber (Figure 9(b)). Diluted whole blood is, for example, 35 ml of collected whole blood diluted with a hemolytic agent, and the volume mixing ratio of whole blood to hemolytic agent is approximately whole blood:hemolytic agent = 1:2 to 1:3.

[0087] (3) A hemolytic agent is gradually supplied to the circulating pathway to wash the whole blood. A hemolytic agent is further added to the diluted whole blood in the circulating channel to lyse red blood cells and remove them from the diluted whole blood (Figure 9(c)). Specifically, a pinch point 30D1 on the connecting conduit 30D extending from the sealed container 23 is opened, and a pinch point 30G1 on the connecting conduit 30G extending from the sealed container 26 for waste liquid is opened, and a small amount of hemolytic agent is introduced from the sealed container 23 into the circulating channel to lyse the red blood cells contained in the concentrated whole blood during counterflow centrifugation in the centrifugation chamber, and the liquid containing the hemolyzed red blood cells flowing through the circulating channel is discharged into the sealed container 26 for waste liquid. The aforementioned pinch points 30D1 and 30G1 are closed to switch to the circulating channel, and somatic cells that have flowed into the circulating channel are recovered into the centrifugation chamber during counterflow centrifugation (the outflow of somatic cells into the circulating channel is caused by the disturbance of the cell layer during counterflow centrifugation due to the difference in liquid properties). Therefore, the amount of hemolytic agent introduced is gradually increased, and the introduction and draining of the hemolytic agent and counterflow centrifugation by switching to a circulation channel are repeated alternately. In this case, the conditions such as the relative centrifugal force and counterflow flow rate of the counterflow centrifugation are as follows, for example. Relative centrifugal force: 2000G Flow rate: 30ml / min Replace in stages, 3ml, 4ml, and 5ml at a time. Circulation time: 25~35 seconds Number of treatments: 3 times each (i.e., to introduce the hemolytic agent into the circulating channel, first 3 ml is introduced three times, then 4 ml is introduced three times, and finally 5 ml is introduced three times. After each introduction, circulation is performed for 25-35 seconds, and the flushed somatic cells are collected in the centrifuge chamber.)

[0088] (4) Replace the fluid in the circulating channel with a hemolytic agent (liquid) and wash the somatic cells. While draining the liquid in the circulating channel into the sealed container 26, a hemolytic agent is introduced from the sealed container 23 into the circulating channel, replacing the liquid in the circulating channel with the hemolytic agent, completely lysing the red blood cells in the whole blood, and removing the hemolytic components from the suspension. In this case, the conditions such as the relative centrifugal force and counterflow rate of the counterflow centrifugation are as follows, for example. Relative centrifugal force: 2000G Flow rate: 30ml / min Volume of hemolytic agent delivered: 80 ml

[0089] (5) Replace the liquid (hemolytic agent) in the circulation channel with liquid culture medium. While draining the hemolytic agent from the circulation channel into the sealed container 26, liquid culture medium is introduced from the sealed container 21 into the circulation channel, replacing the liquid in the circulation channel from the hemolytic agent to the liquid culture medium. A small amount of culture medium is introduced, and then the system is switched to the circulation channel, and this process is repeated. As described in step (3) above, the difference in liquid properties disrupts the cell layer during counterflow centrifugation, causing somatic cells to flow into the circulation channel. Therefore, as in step (3) above, the amount of liquid culture medium introduced is gradually increased, and the introduction and draining of the liquid culture medium and counterflow centrifugation with the system switched to the circulation channel are repeated alternately. In this case, the conditions such as the relative centrifugal force and counterflow flow rate of the counterflow centrifugation are, for example, as follows. Relative centrifugal force: 2000G Flow rate: 30ml / min Replace in stages, 3ml, 4ml, and 5ml at a time. Circulation time: 45~55 seconds Number of processing attempts: 1 each

[0090] (6) Sampling of somatic cells before elutriation Although not a mandatory step, if you wish to sample somatic cells before elutriation, you can do so as follows: Close pinch point 30G1 of the drainage connection channel, open pinch point 30A1 between it and the circulation bag 24, and add 50 ml of liquid culture medium to the channel to dilute the solution flowing through it. Close pinch point 30B1, open pinch point 30E1, and switch to the circulation channel via the circulation bag 24. Sampling is performed using syringe S1.

[0091] (7) Removal of granulocytes and monocytes by elutriation (separation of lymphocytes) First, the relative centrifugal force of the counterflow centrifuge is set to 2000G, and a cell layer is formed in the centrifuge chamber at a flow rate of 30 ml / min for 30 seconds. After sampling the cells before elutriation, the blood cells are returned from the sealed circulation container 24 to the centrifuge chamber. Next, while maintaining a flow rate of 30 ml / min, the relative centrifugal force is gradually reduced to 800 G over 50 seconds, spreading the cell layer towards the center of rotation in the centrifugation chamber (step (s0-1)). At this time, since lymphocytes are relatively lighter than granulocytes and monocytes, they are thought to be located closer to the center of rotation than granulocytes and monocytes due to the ertriation achieved by counterflow centrifugation (Figure 10(a)). Next, the direction of liquid circulation in the circulation channel is set to the positive direction, the pinch point 30A1 between the sealed circulation container 24 and the circulation channel is opened, and the flow rate is gradually increased to 50 ml / min over 60 seconds while maintaining a relative centrifugal force of 800 G. This theoretically discharges lymphocytes, which are thought to be abundant in the cell layer on the rotational side of the centrifugal chamber, from the centrifugal chamber (Figure 10(b)) and collects them in the sealed circulation container 24 (step (s0-2)). Next, with the liquid circulation direction in the circulation channel set to the positive direction, the relative centrifugal force set to 2400G, and the flow rate set to 30 ml / min, the cells remaining in the circulation channel are collected in the centrifugation chamber (Figure 10(c)). The pinch point 400H in front of the syringe for collecting the liquid culture medium and cells to be removed by elutriation is opened, the liquid circulation direction in the circulation channel is reversed, the relative centrifugal force is set to 2400G, and the flow rate is set to 30 ml / min, and the liquid containing the cell layer that has accumulated on the outer periphery of the centrifugation chamber before elutriation is collected by manually pulling syringe S4 to a target volume of 40 ml (i.e., the inflow is stopped when the inflow reaches 40 ml, as set in the control program). The circulation direction of the liquid in the circulation channel is reversed, and after flowing in the reverse direction with a relative centrifugal force of 10G and a flow rate of 100 ml / min, the pinch point 30E1 is opened, and the remaining suspension after eltriation is placed in the sealed container 24, and the suspension is sampled with syringe S2 for verification of the effect of eltriation. The direction of liquid circulation in the circulation channel is set to the positive direction, and the relative centrifugal force is gradually changed to 2000G and the flow rate to 30 ml / min over 60 seconds to form a cell layer in the centrifugation chamber. The pinch point 30E1 is opened to move the elutriated suspension from the sealed circulation container 24 into the centrifugation chamber and concentrate the suspension.

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

[0093] (9) Injection of initialization factors into the circulation channel (process (s1)) The pinch point 30F1 of the connecting conduit 30F to which syringe S3 (a syringe containing SeV-containing liquid) shown in Figure 1 is connected is opened. As an example of releasing the pressure increase, the pinch point 30C1 of the connecting conduit 30C leading to the sealed container 22 that contained the raw materials is opened. With a relative centrifugal force of 1500G and a flow rate of 5ml / min, the plunger of syringe S3 is manually pushed to inject small amounts of SeV-containing liquid into the circulation channel. The increase due to the injection of the SeV-containing liquid is released into the sealed container 22. It is preferable that the length of the conduit into which the SeV-containing liquid is injected and the length of the conduit for releasing the increase in SeV-containing liquid are approximately the same. In this example, a sealed container 22 is used, but in principle, a sealed container 26 for waste liquid or the like may be used.

[0094] (10) Contact between white blood cells (especially lymphocytes) and reprogramming factors (process (s1)) Counterflow centrifugation is performed for 2 hours at a relative centrifugal force of 1500G and a flow rate of 5 ml / min while circulating the liquid medium containing the SeV vector (Figure 11). This causes the liquid medium containing the SeV vector to flow as a counterflow between lymphocytes that are concentrated in the outer periphery of the centrifugation chamber, thus increasing the probability of contact between lymphocytes and the SeV vector.

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

[0096] (11) Concentration (recovery) of leukocytes (especially lymphocytes) that have come into contact with (become infected with) the SeV vector. After closing the circulation channel and increasing the rotation speed to circulate and concentrate the suspension (Figure 12(a)), the channel to the culture medium and 30H1 are opened, and the flow is reversed to collect the suspension (leukocytes (especially lymphocytes) that have come into contact with the SeV vector) into a sealed container connected to channel 30H (Figures 12(b) and (d)). This is repeated twice. Furthermore, when collecting leukocytes (especially lymphocytes) that have come into contact with the SeV vector into other sealed containers such as CELLPET disposable culture vessels, it is desirable to pass them through a filter (such as Re-Strainer 200μm pluriSelect) to trap and remove dead cells (Figure 12(c)).

[0097] (12) Establishment of iPS cells (Step (s2)) Cytokines are added to the collected cell suspension in a sealed container according to the amount of cell suspension collected, and cell culture is performed to establish iPS cells.

[0098] (13) Establishment of iPS cells Replace or add liquid culture medium in the sealed container.

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

[0100] (Steps for expanding the culture of iPS cells (s3)) In this manufacturing method, after the step of establishing iPS cells (s2) described above, a step of expanding the iPS cell culture (s3) may be further included, from the viewpoint of iPS cell yield. Step (s3) may be carried out in a Rotea centrifugation chamber, or in a sealed container for expansion culture that is aseptically connected to the closed system portion while maintaining a closed system. Step (s3) is carried out while maintaining the closed nature of the closed system portion, similar to steps (s0) to (s2).

[0101] Suitable sealed containers for expansion culture include sealed containers connected to connecting lines (30A~30H), sealed containers included in external expansion culture equipment (e.g., G-Rex (Wilson Wolf), etc.), and sealed containers equipped with vent pipes, culture medium exchange pipes, and CO2 gas pipes. Connecting line 30H shown in Figure 1 is a line suitable for output, and the sealed container connected to this connecting line 30H, like the sealed containers (21~26) connected to the other connecting lines (30A~30G), constitute a closed system. Whether all sealed containers and equipment connected aseptically to each connecting line to maintain a closed system belong to the closed system or to external equipment can be determined as appropriate for ease of management. Expansion culture of iPS cells can be performed by supplying a liquid culture medium suitable for expansion culture to elements for expansion culture (such as a centrifuge chamber or an expansion culture sealed container).

[0102] The culture medium used in step (s3) can be appropriately selected by those skilled in the art from the liquid culture media described above. In one embodiment, if the intention is to expand the culture of the established iPS cells, an undifferentiated maintenance factor may be added. In this specification, "undifferentiated maintenance factor" 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 undifferentiated maintenance factors commonly used by those skilled in the art include bFGF, FGF2, FGF4, FGF8, EGF, Nodal, ActivinA, ActivinB, TGFβ1, and TGFβ2. The undifferentiated maintenance factor used in the present invention is preferably isolated. "Isolation" means that an operation has been performed to remove the target component or factors other than cells, and that it has moved away from its naturally occurring state.

[0103] In step (s3), the number of expansion cultures performed is not particularly limited as long as the desired number of iPS cells can be obtained, but in normal processing operations, 1 to 5 times is preferred, and more preferably 2 to 5 times. The duration of step (s3) is not particularly limited as long as the desired number of iPS cells can be obtained, but typically it is, 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 continue expansion culture until the marker is removed.

[0104] (Quality assessment of iPS cells) The quality of the obtained iPS cells may be evaluated by a known method after step (s2) or after step (s3) of the manufacturing method of the present invention.

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

[0106] The process (s4) is carried out, for example, as in (I) or (II) below. (I) Materials necessary for differentiation induction are supplied into a centrifugation chamber, and iPS cells are differentiated within the centrifugation chamber. (II) The iPS cells present in the centrifugation chamber are transferred to a sealed container (not shown) for differentiation induction, which is aseptically connected to the closed system portion in a way that maintains the closed system. Materials necessary for differentiation induction are supplied to the sealed container for differentiation induction, and the iPS cells are differentiated within the sealed container. In Figures 1, 8, etc., the sealed containers for supplying materials necessary for differentiation induction and the sealed container for differentiation induction are not shown.

[0107] As sealed containers for differentiation induction, similar to the sealed containers for expansion culture, sealed containers connected to connecting conduits (30A~30H), sealed containers included in external differentiation induction equipment, G-Rex (Wilson Wolf), and sealed containers equipped with vent pipes, culture medium exchange pipes, and CO2 gas pipes can be used as appropriate.

[0108] The step (s4) of inducing differentiation of iPS cells may be performed after the establishment step (s2) in the iPS cell production method according to the present invention, without performing the step (s3) of expanding the culture of iPS cells, or it may be performed after the establishment step (s2) and after performing the step (s3) of expanding the culture of 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 the cell sorting step and the gene transfer step may be further performed. The cell sorting step can be carried out by connecting a known cell sorting device to the closed system portion described above. The gene transfer step may be performed, for example, with the intention of using iPS cells derived from patients with hereditary diseases as therapeutic agents for the disease, by repairing genes by genome editing or the like and then differentiating them into target cells, and electroporation, lipofection, or viral vectors can be used. If electroporation is used, 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 to be introduced.

[0110] (differentiated cells) In this specification, "differentiated cells" refers to cells or organoids obtained by differentiating induced pluripotent stem cells. The resulting cells may be undifferentiated cells such as stem cells or progenitor cells, or they may be terminally differentiated cells. In this specification, the term "differentiated cells" may be used to encompass both undifferentiated cells and terminally differentiated cells obtained by differentiating induced pluripotent stem cells. In this specification, "undifferentiated cells" means cells that have not reached terminal differentiation in the cell lineage, and examples of undifferentiated cells include stem cells other than pluripotent stem cells, progenitor cells, etc. Examples of stem cells 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, and mammary gland 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, and renal progenitor cells; and endodermal cells such as hepatic stem cells, hepatic progenitor cells, intestinal stem cells, and airway stem cells.

[0111] In this specification, "terminally differentiated cells" means cells that have reached terminal differentiation in a cell lineage. Terminally differentiated cells are not particularly limited, but examples include osteoblasts, chondrocytes, adipocytes, hepatocytes, hepatic mesothelial cells, cholangiocarpal epithelial cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, Kupffer cells, pit cells, vascular endothelial cells, hematopoietic cells, pancreatic ductal epithelial cells, pancreatic ductal cells, acinar central cells, acinar cells, islets of Langerhans, cardiomyocytes, 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, Krutschki cells, tubular epithelial cells, urothelial cells, columnar epithelial cells, glomerular epithelial cells, glomerular endothelial cells, podocytes, mesangial cells, nerve cells, and glial cells. Examples of white blood cells include lymphocytes, granulocytes, and monocytes.

[0112] In one embodiment, the cells or organoids (target cells or organoids) obtained by differentiating induced pluripotent stem cells are neural crest cells, neural progenitor cells, nerve cells, cerebral cortical organoids, hematopoietic progenitor cells, platelets, T cells, or cardiomyocytes.

[0113] (Substance that induces differentiation of iPS cells) In this specification, "differentiation-inducing substance" means a substance that can induce differentiation from induced pluripotent stem cells into the differentiated cells or organoids described above. The differentiation-inducing substance may be a substance that is already known, or it may be appropriately selected from those commonly used to induce differentiation of desired differentiated cells or organoids. Specifically, the substances described below are examples.

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

[0115] (differentiation induction method) In the manufacturing method of the present invention, known methods can be used for the differentiation induction method to obtain the target cells or organoids. For example, differentiation induction from pluripotent stem cells to neural crest cells can be carried out by methods such as those described in Fukuta M. et al., PLoS One, 2014, 9(12): e112291 and 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 cultured in adherent culture (suspension culture using scaffold material), and then differentiated into neural crest cells by culturing in adherent culture (suspension culture using 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, osteocytes, chondrocytes, corneal cells, and melanocytes. For example, differentiation induction into these cells can be carried out 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 can be seeded on a fibronectin-coated plate, replaced with DMEM / F12 supplemented with N-2 Supplement, BDNF, GDNF, NT-3, and NGF, and cultured at 37°C under 5% CO2 for approximately 14 days to obtain neural progenitor cells and neurons. Alternatively, neural crest cells can be seeded on a plate, cultured for 1 day in CDM medium containing SB431542 and CHIR99021, then replaced with Neurobasal medium supplemented with B-27 Supplement, N-2 Supplement, L-glutamine, Penicillin / Streptomycin, BDNF, GDNF, NT-3, and NGF, and cultured for approximately 35 days at 37°C under 5% CO2 to obtain neural progenitor cells and neurons.

[0117] Furthermore, differentiation induction into mesenchymal stromal cells can be performed using methods such as the following: Seed neural crest cells in a culture vessel and cultured for 1 day in CDM medium containing SB431542 and CHIR99021. After 1 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] Examples of methods for differentiating pluripotent stem cells into T cells include a method comprising (1) differentiating pluripotent stem cells into hematopoietic progenitor cells, and (2) differentiating the hematopoietic progenitor cells into T cells. Step (1) may be a step of culturing pluripotent stem cells in an induction medium for hematopoietic progenitor cells, as described in, for example, WO2013 / 075222, WO2016 / 076415, Liu S. et al., Cytotherapy, 17 (2015); 344-358. Step (2) may be a step of (2-1) inducing CD4 and CD8-positive T cells from hematopoietic progenitor cells, and (2-2) inducing CD8-positive T cells from CD4 and CD8-positive T cells, as described in WO2016 / 076415.

[0119] Examples of methods for inducing differentiation from pluripotent stem cells into cardiomyocytes include those described in WO2015 / 141827 and Laflamme MA and Murry CE, Nature. 473(7347):326-35 (2011). Other methods include, for example, producing cardiomyocytes by forming embryoid bodies from induced pluripotent stem cells in suspension culture, producing cardiomyocytes in the presence of a substance that suppresses BMP signaling (WO2005 / 033298), producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), and 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 (cardiac-specific transcription factor) and TNNT2 (troponin T), while marker proteins for cardiomyocyte progenitor cells include KDR (vascular endothelial growth factor (VEGF) receptor) and ISL1 (LIM homeodomain transcription factor).

[0120] Induction of monocyte differentiation can be performed using the method described in Di Cui., et al. Frontiers in Cell and Developmental Biology; vol 9, Article 656867 April 2021.

[0121] Furthermore, organoids can be produced using multiple types of cells. For example, in the case of liver organoids, as described in WO2013 / 047639, liver organoids can be produced by inducing liver progenitor cells (organ cells), mesenchymal stem cells, and vascular endothelial cells from pluripotent stem cells, and then culturing a mixture of these cells in suspension.

[0122] The manufacturing method of the present invention may involve culturing under feeder-free and / or xeno-free conditions for all or part of the period. From the viewpoint of clinical use, it is preferable that the differentiation induction method of the present invention be carried out 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. Alternatively, the cells recovered in a container may be counted using a cell counter, labeled with an antibody against a cell surface marker, and purified by flow cytometry, mass cytometry, magnetic cell separation, or the like.

[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 those produced by the cell production method of the present invention, and can be carried out by adding known undifferentiated cell removal agents to the culture medium (for example, 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, Yoshiki Nakashima, et. al., Molecular Therapy Vol. 26 No 7 July 2018, etc.).

[0125] Quality inspections may be performed as appropriate to determine whether the cells, organoids, etc., obtained by the manufacturing method of the present invention are as desired. The items for quality inspection are not particularly limited, but examples include basic tests such as the morphology of cells and organoids, the presence or absence of cell surface marker expression, sterility tests, endotoxin tests, and evaluation of cell viability, and testing equipment appropriate for each item can be used. [Examples]

[0126] In the following example, steps (s0) to (s4) of the manufacturing method according to the present invention described above were performed using the reagents, apparatus, and materials listed below, and the obtained iPS cells and differentiated cells were evaluated.

[0127] (reagent) Whole blood: Blood from healthy volunteer donors (unprocessed, unfrozen, collected 1-2 days prior) Hemolytic agent: ACK lysing buffer Gibco TM Product 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 (which contains growth factors). StemSpan is also acceptable.) SeV:SRV™ iPSC-4 (manufactured by 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), equipped with 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 equipment: Rotea Counterflow Centrifugation System (Thermo Fisher Scientific Inc.) Sealed container: Terumo isolation bag Sterile splicer: TERUMO TSCD-II CELLPET: Disposable culture vessel, JTEC Corp. CELLPET iPS / 3S / MA-2.1 (JTEC Corporation) Safety cabinet: phcbi ClassII type A2 (PHC Corporation) Cell counter: celldrop FL (manufactured by DeNovix) Incubator: Thermo Scientific FORMA STERI-CYCLE i160 (manufactured by 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 Corporation) Centrifuge: tomy lcx-100 (Tomy Seikou Co., Ltd.)

[0129] Example 1 1. Preparation (1) Mixture of whole blood and hemolytic agent Inside a safety cabinet, whole blood and hemolytic agent were manually mixed into a Terumo isolation bag, which was used as a sealed container 22. The bag was aseptically filled with DNase at a volume of 10 units / ml or more, so that the volume mixing ratio (whole blood:hemolytic agent) was (1:2) to (1:3), and the mixture was left to stand at room temperature for 15 minutes. The hemolytic agent was added to dissolve red blood cells and to reduce the viscosity of the viscous blood.

[0130] (2) Preparation of the closed system Inside the safety cabinet, liquid culture medium and hemolytic agent were manually filled into sealed containers 21 and 23, respectively. Inside the safety cabinet, SeV (2 vials, 0.2 ml) was mixed with 1 ml of liquid culture medium and filled into syringe (S3). Sealed container 24 for circulation, sampling syringes (S1, S2), sealed container 26 for waste liquid, harvest syringe (S5 in Figure 7), and syringe for collecting the liquid removed by eltriation (S5 in Figure 7) were connected to the flow path. Sealed container 25, filled with liquid culture medium for injecting the 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 collecting the liquid removed by eltriation (S4 in Figure 7) was connected to the sealed container 25 side of 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 joined to the connecting conduits (flexible tubing) of Rotea's sterile single-use kit using a sterile connector. The circulating sealed container 24 has two ports, with connecting conduits leading to the liquid inlet and outlet, which are connected to the flow path. The other sealed containers have only one port connected to the single-use kit via a connecting conduit. All sealed containers were suspended, and the tubes were checked for kinks or blockages. The QR code (registered trademark) on the single-use kit was scanned, and the Rotea mechanism automatically closed all pinch points.

[0131] 2. Separation of leukocytes from whole blood The Rotea control program was executed, the following protocol was set up, and a hemolytic agent was processed to wash away plasma, platelets, and red blood cells from whole blood, thereby obtaining white blood cells. The protocol for the CTS Rotea system was designed in the protocol builder software to perform all the processes described below.

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

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

[0134] (3) Wash with a hemolytic agent A small amount of hemolytic agent was introduced from the sealed container 22, and the process of switching to the circulation channel was repeated. This was because the cell layer would be disturbed due to the difference in liquid properties. The circulation channel is a circular path in the closed system portion of Figure 1, connecting the outlet of the centrifuge chamber 10, connecting conduit 35, branching point 36, connecting conduit 37, pinch point 30J1, pinch point 30K1, connecting conduit 38, confluence point 32, connecting conduit 33, connecting conduit 34, and the inlet of the centrifuge chamber 10 in that order. Rotation speed: 2000G Flow rate: 30 mL / min Replace in stages: (1) 3 mL, (2) 4 mL, (3) 5 mL Circulation time: 25-35sec Number of processing cycles: 3 times each

[0135] (4) Complete replacement with hemolytic agent While draining the liquid in the circulating channel into the sealed container 26, a hemolytic agent was introduced into the circulating channel from the sealed container 23, replacing the liquid in the circulating channel with the hemolytic agent, completely lysing the red blood cells in the whole blood, and removing the hemolytic components from the suspension. Rotation speed: 2000G Flow rate: 30 mL / min Volume of hemolytic agent delivered: 80 mL

[0136] (5) Replace the liquid (hemolytic agent) in the circulation channel with liquid culture medium. A small amount of liquid culture medium was introduced from the sealed container 21 into the centrifuge chamber, and then the process was repeatedly switched to the circulation channel. This was done to stabilize the cell layer, which had been disrupted by the difference in liquid properties. Rotation speed: 2000G Flow rate: 30 mL / min Replace in stages: (1) 3 mL and (2) 5 mL. Circulation time: 45-55sec Number of processing cycles: 3 times each

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

[0138] (7) Eltriation Except for the test group conducted without elutriation to verify the effect of elutriation, elutriation was performed as follows: The relative centrifugal force of the centrifuge chamber was increased to 2000G and a flow rate of 30 ml / min was maintained for 30 seconds to form a layer of cells in the chamber, after which the cells were returned to the chamber from the sealed circulation container 24. The relative centrifugal force was reduced to 800G and the cell layer was spread in the chamber at a flow rate of 30 ml / min for 50 seconds. At this time, lymphocytes are relatively lighter than larger cells such as granulocytes and monocytes, so they are thought to be located on the side of the center of rotation of the chamber. The cells were circulated in the forward direction, the valve between the sealed circulation container 24 and the flow path was opened, and the cell layer located on the side of the center of rotation of the chamber was collected into the sealed circulation container 24 at a relative centrifugal force of 800G and a flow rate of 50 ml / min for 60 seconds.

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

[0140] After flowing in the reverse direction with a relative centrifugal force of 10G and a flow rate of 100 ml / min, the pinch point 30A1 between the sealed circulation container 24 and the circulation path was opened, and the remaining post-eltriation suspension was placed into the sealed circulation container 24, and the suspension was sampled with syringe S4. The system was circulated in the forward direction at 2000G and 30 ml / min for 60 seconds to form a cell layer, and the pinch point 30E1 between the sealed circulation container 24 and the circulation path was opened, transferring the post-eltriation suspension from the sealed circulation container 24 into the centrifugation chamber. For spinoculation, the solution was concentrated at 1500G, 5 ml / min, for 60 seconds.

[0141] 3. SeV vector infection (contact between leukocytes (especially lymphocytes) and SeV vectors) SeV-containing liquid was manually added in small amounts from syringe S3 into the circulation channel at a relative centrifugal force of 1500G and a flow rate of 5 ml / min. Lymphocytes and SeV vectors were brought into contact by spinocuration at 1500G and 5 ml / min for 120 minutes. After contact, the SeV vectors were removed by washing with culture medium at a relative centrifugal force of 1500G and a flow rate of 5 ml / min.

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

[0143] 5.3D culture iPS cells were cultured up to the 8th passage in the CELLPET disposable culture vessel using an incubator. The following six types of cytokines were added according to the amount of cell suspension recovered 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 Up until day 14 after establishment, the culture medium was changed every 2-3 days, half the volume at a time, using StemFit AK03 medium. Furthermore, we confirmed that iPS cells can be established using a water bath or plate heater, without the need for an incubator.

[0144] Establishment results (1) Morphological observation of iPS cells The iPS cells established and cultured in 3D 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 day5, 23 days after SeV infection).

[0145] (2) Confirmation of GFP After harvesting, the cell saturation for the cultured cells was set aside, and the GFP-evaluated cells were seeded onto plates. FACS was performed on day 3 or 4 after seeding. The results are shown in Tables 1 and 2 below. From Tables 1 and 2, it can be seen that iPS cells were produced under various conditions using this manufacturing method, as evidenced by the detection of GFP fluorescence or the presence of undifferentiated markers.

[0146] (3) Number of spheroids after establishment The results for each test area were as shown in Tables 1 and 2 below.

[0147] [Table 1]

[0148] [Table 2]

[0149] (4) Confirmation of undifferentiated markers The expression of iPS cell markers TRA-1-60, OCT3 / 4, and SSEA4 was examined by flow cytometry in iPS cells 53 days after infection with Rotea0302 2D p6 vector (April 24) and 27 days after infection with Rotea0411 2D p2 vector (May 8). As shown in the graph in Figure 14, the reprogramming markers were expressed. 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. The fixed cells were permeabilized with 0.5% Triton X-100 (Roche) and blocked with Blocking One (Nacalai Tesque). The cells were incubated with primary antibody diluted in D-PBS containing 2% FBS at room temperature for 30-60 minutes. 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 in the dark at room temperature. 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).

[0150] (4) Immune cell staining Cell staining was performed to confirm that iPS cells produced by Rotea expressed the pluripotent cell markers NANOG and OCT3 / 4. Cells 67 days after (5 / 8) infection with the Rotea0302 2D p8 vector were used. The following reagents were used, the nuclei were stained with DAPI, and observations were made using a fluorescence microscope. The results were as shown in the photographic figure of Fig. 15 (scale bar: 100 μm). In the actual image, among the photographic figures of Fig. 15, NANOG is shown in green, OCT3 / 4 is shown in magenta, and DAPI is shown in blue.

[0151] BD Cytofix TM Fixation 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 white blood cell counts isolated by Rotea depending on the whole blood (donor) Using 20 ml (n = 11) or 40 ml (n = 5) of different whole blood (from different donors, etc.) as starting materials, lymphocytes were separated using Rotea under the above separation conditions. The separated white blood cells (living cells) were counted using a cell counter. The results were as shown in the graph of Fig. 16 (left graph: number of living cells, right graph: viability).

[0153] Example 3 Verification of the effects of Eltriation Using 40 ml of whole blood from donor 1 as the starting material, leukocytes were separated from the whole blood using Rotea under the separation conditions described above. One sample was washed (elutriation) after separation to increase the degree of multiple infection (MOI) of cells, and iPS cells were established from the other sample without elutriation. The number of recovered leukocytes was counted. The number of cells before and after isolation per 1 ml of whole blood was 230404 in Table 1 and the upper left graph in Figure 17 (dark bars represent pre-isolation, light bars represent post-isolation. + indicates ethylation, - indicates no ethylation). After harvesting, the cell saturation for the cultured cells was set aside, and the GFP-evaluated cells were seeded onto plates. FACS was performed on day 3 or 4 after seeding. The number of GFP(+) cells per 1 ml of whole blood was counted, and the difference in the number of GFP(+) cells with and without elutriation was compared. The results are shown in the upper right graph of Figure 17. Donor 2 underwent a similar experiment using 35 ml. The results are shown in Table 1, test group 230502, and in the lower left and lower right graphs in Figure 17 (+ indicates eltriation, - indicates no eltriation). 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 could have the effect of drastically reducing larger cells such as granulocytes and monocytes.

[0154] Example 4 Examination of SeV infection time The study examined SeV infection times of 30 minutes or 120 minutes. Specifically, 28 ml of whole blood from donor 1 was used as the starting material, and leukocytes were separated from the whole blood using the CTS Rotea System under the isolation conditions described above, including elutriation. The number of leukocytes separated and recovered was counted. The cell count before isolation and the leukocyte count after isolation are shown in the upper left graph of Figure 18. Furthermore, SeV infection was performed using the CTS Rotea System, in the same manner as the above SeV infection, except that the infection time was set to 30 minutes. The number of GFP(+) cells per 1 ml of whole blood was counted. The results were as shown in Table 1, for test group 230421, and in the upper right graph of Figure 18. Furthermore, a similar experiment was conducted using a SeV infection time of 120 minutes. In addition, a similar experiment was conducted using 35 ml of whole blood from donor 2 as the starting material. The results are shown in Table 1, for test group 230516, and in the lower left and lower right graphs in Figure 18.

[0155] Example 5 Investigation of the ratio f1 / Q of relative centrifugal force f1 to flow rate Q The study was conducted in the same manner as in Example 3, except that the SeV infection time remained 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 are shown in Table 1 for test plots 230421 and 230509, and in the graph in Figure 19.

[0156] Example 6 Induction of differentiation into the three germ layers We confirmed that iPS cells produced using Rotea can be induced to differentiate. (1) Three germ layer differentiation For iPS cells produced using 230307 3D p12 in Table 1, STEMdiff TMUsing the Trilineage Differentiation Kit (STEMCELL Technologies), differentiation into the three germ layers was performed according to the manufacturer's protocol. Differentiation between the ectoderm and mesoderm involved 3 × 10⁶ germ layers. 5 Using individual iPS cells, 4 × 10⁶ cells were used for endoderm differentiation. 5 One iPS cell was used. SB431542 (Nacalai Tesque) was added to the culture medium for ectoderm differentiation induction to a final concentration of 10 μM. The medium was changed daily, and induction culture was performed for 7 days for ectoderm differentiation and 5 days for mesoderm and endoderm differentiation.

[0157] (2) Confirmation of trigerm-differentiated cells by flow cytometry After dispersing the three germ layer differentiated cells into single cells, they were washed once with D-PBS and fixed with 4% paraformaldehyde (Nacalai Tesque) at room temperature for 15 minutes. The fixed cells were permeabilized with 0.5% Triton X-100 (Roche) and blocked with Blocking One (Nacalai tesque). The 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 in the dark at room temperature. The antibodies used are 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), anti-Mouse IgG Alexa Fluor TM Plus 555 (Thermo Fisher Scientific), anti-Rabbit IgG CF(registered trademark)640R (BIOTIUM). After a short wash with D-PBS containing 2% FBS, the stained cells were passed through a 45 μm cell strainer (BD Japan) and applied to a SA3800 cell analyzer (SONY). As a result, the three germ layer markers PAX6, Nestin (ectoderm), NCAM, Brachyury (mesoderm), FOXA2, and SOX17 (endoderm) were identified, as shown in the photographic and graphical diagrams of Figure 20.

[0158] Example 7 Differentiation and evaluation of cardiomyocytes (1) Differentiation into cardiomyocytes Rotea0307 3D 12th passage iPS cells were differentiated into cardiomyocytes. The differentiation induction method was performed 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 heart using optimized human iPSC-derived cardiomyocytes. Scientific reports 6. 10.1038 / srep19111, with only minor modifications. Specifically, it is as follows: Undifferentiated iPSCs are detached, and TrypLE TM The cells were incubated with Select Enzyme for 5 minutes and then dissociated into single cells. Single cells were treated with 10 μl / ml GlutaMax (Thermo Fisher Scientific) and 50 μg / ml ascorbic acid (SIGMA), in a 4x10 ratio. -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 germ layers were suspended in -34 SFM (Thermo Fisher Scientific), placed in a low-adhesion 6-well dish, and cultured for 24 hours to form germ layers. On day 1, culture medium containing human recombinant activin A (R&D Systems), BMP4, and bFGF (R&D Systems) was added to the wells. The final concentrations were activin A 6 ng / ml, BMP4 10 ng / ml, and bFGF 5 ng / ml. On day 3, the culture medium was 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) in StemPro TM -34 The culture medium was replaced. On day 7, 10 μl / ml GlutaMax, 50 μg / ml ascorbic acid, 4 × 10 -4 The medium was replaced with StemPro™-34 medium supplemented with M monothioglycerol, 150 μg / ml transferrin, and 5 ng / ml VEGF. To maintain the iPS cell-derived cardiomyocytes, the culture medium was changed every 2-3 days. On the day of cell injection, the germ cells were dissociated using collagenase II (Worthington Biochemical Company) over 3-6 hours, and TrypLE select over 30 minutes. As a result, cardiomyocytes shown in the upper photograph (40x magnification) of Figure 21 were observed 15 days after differentiation.

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

[0160] Example 8 iPS cell culture in a centrifuge chamber After infecting the cells with SeV using Rotea, the Rotea was stopped while the cells remained in the Rotea chamber (centrifugation chamber) without harvesting. The in and out tubes connected to the chamber were cut using a sterile conjugate (TERUMO TSCD-II), and the ends of the tubes were further clamped as a precaution. The chamber was fixed to a CO2 incubator with the front of the chamber facing downwards, and the cells were cultured at 37°C in 5% CO2 for 15 days. For medium changes, syringes were connected to both ends of the tubes, and the liquid was changed without aspirating cells from the front of the chamber. The 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 the incubator: 5% • Inverted microscope: ix83 p2zf (Evident Co., Ltd.) The timing of medium changes was the same as in Example 1. Until day 14 after establishment, the medium was changed every 2-3 days, half the volume of which was replaced with StemFit AK03 medium.

[0162] Photographs of spheroids (left in Figure 22) and fluorescence microscope images (right in Figure 22) of iPS cells cultured on the 15th day after SeV infection are shown. It was found that the reprogramming GFP marker emitted fluorescence, indicating that the iPS cells could be maintained or proliferated. Therefore, it was shown that iPS cells could also be cultured in a Rotea chamber (centrifugation chamber).

Industrial Applicability

[0163] By the production method of the present invention, the use of expensive reagents such as viral vectors for introducing reprogramming factors into somatic cells can be reduced, and thus the production cost of iPS cells can be reduced.

[0164] This application is based on Japanese Patent Application No. 2024-052336 (filing date: March 27, 2024), the content of which is entirely incorporated herein.

Explanation of Signs

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

Claims

[Claim 1] The invention described in the specification.

Images