Method for producing induced pluripotent stem cells

Abstract

The present invention provides a method for producing iPS cells by performing the following steps in the following order: a step for centrifugally separating blood cells from whole blood by using a cell processing device, wherein the cell processing device has a closed system part in which a sealed container for material supply and a closed chamber rotatable so as to perform centrifugation are coupled to each other via a coupling pipe and the centrifugal separation is performed in the rotatable closed chamber while maintaining the closed state of the closed system part; a step for bringing a reprogramming factor into contact with the blood cells; and a step for establishing iPS cells from the blood cells.

Description

Method for producing induced pluripotent stem cells

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

[0002] In recent years, research into regenerative medicine using differentiated cells derived from induced pluripotent stem cells (hereinafter also referred to as iPS cells) has been actively conducted. In particular, a therapy in which iPS cells are established from a patient's somatic cells (e.g., peripheral blood mononuclear cells, etc.) and then various differentiated cells or organoids induced to differentiate from the iPS cells are transplanted into the patient (autotransplantation) has attracted attention as a therapy that can reduce the risk of rejection (Non-Patent Documents 1 and 2). Patent Document 1 (JP 2017-195905 A) discloses a system capable of producing stem cells. Furthermore, CAR-T therapy and other therapies using the patient's own somatic cells have begun to be applied clinically, and Patent Document 2 (JP 2022-8735 A) discloses a cell production system and cell production method that can reduce costs by reducing the number of steps in a therapy using CAR-T cells.

[0003] Japanese Patent Application Laid-Open No. 2017-195905 Japanese Patent Application Laid-Open No. 2022-8735 International Publication No. 2020 / 040135 Japanese Patent Application Laid-Open No. 2020-182488 Japanese Patent Application Laid-Open No. 2014-018185 Special Publication No. 2011-505890

[0004] Shinsuke Yoshida., et al., CLINICAL AND TRANSLATIONAL RESOURCE AND TECHNOLOGY INSIGHTS VOLUME 4, ISSUE 1, P51-66.E10, JANUARY 13, 2023Madrid, M., et al., Current Protocols,1, e88. doi: 10.1002 / cpzl.88

[0005] One of the features of iPS cells is that they can be produced from a patient's own somatic cells, which could be an effective means of minimizing the risk of rejection. However, generating iPS cells from somatic cells requires a multi-stage, lengthy processing process, and the costs of numerous specialized devices and the labor costs for operating and managing them are high.

[0006] If the multi-step process of isolating somatic cells from peripheral blood collected from a patient, establishing iPS cells, and then expanding and culturing them to form differentiated cells could be performed automatically within a single closed device, it would be possible to significantly reduce the cost of producing autologous iPS cells compared to conventional methods. However, there has not been a cell processing device optimized to automatically and consistently perform all of the above-mentioned multi-step processes, and there have been no attempts to make an existing cell processing device perform the above-mentioned multi-step processes.

[0007] For example, Patent Document 3 (WO 2020 / 040135) describes a process for establishing iPS cells from somatic cells separated from whole blood, but the process for separating somatic cells from whole blood and the process for establishing iPS cells are carried out in different containers. Patent Document 4 (JP 2020-182488 A) describes transduction in a centrifugation chamber provided in a cell processing device, but does not describe performing the multi-step process of separating somatic cells from whole blood and establishing iPS cells from the somatic cells using a single cell processing device. Patent Document 4 also does not describe adhesion culture, which is useful for culturing iPS cells, which are adherent cells. Patent Document 5 (JP 2014-018185 A) describes adhesion culture of iPS cells in a closed system, but does not describe separating somatic cells from whole blood and establishing iPS cells from the somatic cells in the same container within the same closed system.

[0008] Meanwhile, cell processing devices designed to automatically perform sequential cell processing while maintaining a closed system have been proposed by device manufacturers (e.g., the CliniMACS Prodigy® manufactured by Miltenyi Biotec). Figure 12 is a photograph showing an example of such a cell processing device in use. The cell processing device shown in Figure 12 has a closed system section for processing cells. In this closed system section, a sealed container (material supply bag) X20 containing cell processing materials is connected to a rotatable sealed chamber X10 for centrifugation via a connecting pipeline (flexible tubing) X30. The connecting pipeline X30 passes through multiple pinch valves X40 that can be controlled to open and close and a peristaltic pump X50, enabling control of the pipeline connection and cutoff and material feed while maintaining a closed system. The sealed chamber X10 is also configured to be rotatable for centrifugation while the connecting pipeline remains connected.

[0009] However, conventional cell processing using cell processing devices such as those shown in Figure 12 (especially the CliniMACS Prodigy) has not yet demonstrated a consistent procedure for performing the multi-step process required for the production of iPS cells. Furthermore, the centrifugation function of the sealed chamber in conventional cell processing devices is intended for separation at the end of the processing process to harvest the processed cells. Furthermore, the material constituting the sealed chamber in the CliniMACS Prodigy is polycarbonate resin, which provides increased mechanical strength for the centrifugation function and makes the walls more transparent for internal observation. However, due to the properties of polycarbonate resin, the inner wall surface of the sealed chamber is non-adhesive to cells, making it unsuitable for the efficient establishment of iPS cells, which are adhesive cells, through adherent culture using the inner wall surface.

[0010] An object of the present invention is to provide a method for establishing iPS cells from whole blood using the conventional cell processing device described above, and in particular to provide a method for establishing iPS cells from whole blood in a single sealed container (sealed chamber for centrifugation) within a single closed system.

[0011] The main features of the present invention are as follows: [1] A method for producing induced pluripotent stem cells using a cell processing device, wherein the cell processing device has a closed system part in which a sealed container for supplying materials and a rotatable sealed chamber for performing centrifugation are connected via a connecting pipe, and the production method comprises, in the rotatable sealed chamber while maintaining the closed nature of the closed system part, performing the following steps in this order: (s1) centrifuging blood cells from whole blood; (s2) contacting the blood cells with reprogramming factors; and (s3) establishing induced pluripotent stem cells from the blood cells. [2] The sealed chamber has an inlet port and an outlet port, and is configured to allow materials to flow in and out through the inlet port and outlet port while rotating for centrifugation or while the rotation is stopped, and wherein in step (s1), a fraction containing the centrifuged blood cells is left in the sealed chamber, and other fractions are discharged to the outside of the sealed chamber. [3] The method for producing induced pluripotent stem cells according to [2], wherein the cell processing apparatus further comprises a magnetic separation column connected to the sealed chamber via a connecting pipeline as part of the closed system portion, and in the step (s1), after the centrifugation, a fraction containing blood cells is left in the sealed chamber, lymphoid cells among the blood cells are further labeled with magnetic beads and sent to the magnetic separation column to be magnetically separated from other blood cells, and the lymphoid cells labeled with magnetic beads are returned to the sealed chamber. [4] The method for producing induced pluripotent stem cells according to any one of [1] to [3], wherein the step (s2) is performed with the sealed chamber in one of a rotating state and a stationary state, or a combination of these states. [5] The shape of the internal space defined by the side wall surfaces surrounding the internal space of the sealed chamber is cylindrical, and the geometric center line of rotation of the cylinder coincides with the central axis of rotational motion of the sealed chamber during centrifugation, and the base area of ​​the cylinder is 90 to 200 cm ２The method for producing induced pluripotent stem cells according to any one of [1] to [4], wherein the relative centrifugal force acting on the contents of the sealed chamber during centrifugation is 100 to 500 G. [6] The method for producing induced pluripotent stem cells according to any one of [1] to [5], wherein the volume of the sealed chamber is 300 to 700 ml. [7] The method for producing induced pluripotent stem cells according to any one of [1] to [6], wherein the centrifugation in step (s1) is density gradient centrifugation performed by adding a centrifugation medium for density gradient to the sealed chamber. [8] The method for producing induced pluripotent stem cells according to any one of [1] to [7], wherein at least the inner wall surface of the sealed chamber has a polycarbonate resin portion. [9] The method for producing induced pluripotent stem cells according to any one of [1] to [8], wherein steps (s2) and (s3) are performed in the presence of a scaffold material.

[10] The method for producing induced pluripotent stem cells according to [9], wherein step (s1) is performed in the presence of a scaffold material.

[11] The method for producing induced pluripotent stem cells according to [9] or

[10] , wherein a scaffold material is adhered to the inner wall surface of the sealed chamber.

[12] The method for producing induced pluripotent hepatocytes according to any one of [9] to

[11] , wherein the scaffold material comprises a laminin fragment, a cell adhesive peptide and a polyvinyl acetal resin, or a cell adhesive peptide and a poly(meth)acrylate resin.

[13] The method for producing induced pluripotent hepatocytes according to

[12] , wherein the cell adhesive peptide comprises Arg-Gly-Asp.

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

[13] , further comprising, after step (s3), step (s4) of expanding the induced pluripotent stem cells in the sealed chamber while maintaining the closed system.

[15] A method for producing differentiated cells, the method comprising steps (s1) to (s3) of the method for producing induced pluripotent stem cells according to any one of [1] to

[13] , and further comprising, after step (s3), a step (s5) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closed system, wherein in step (s5), a material necessary for differentiation induction is supplied into the sealed chamber, and step (s5) is carried out in the sealed chamber.

[16] The method according to

[15] , further comprising, between steps (s3) and (s5), a step (s4) of expanding the induced pluripotent stem cells in the sealed chamber while maintaining the closed system.

[0012] The production method of the present invention uses a closed-system cell processing device with a sealed chamber equipped with a centrifuge function as a container for processing cells. In the production method of the present invention, this centrifuge function is used at the beginning of the entire processing process to centrifuge blood cells, the source of iPS cells, from whole blood. The blood cells are then initialized and established in the same sealed chamber. Furthermore, the iPS cells are expanded and differentiated in the same sealed chamber. This allows for a single closed-system cell processing device to consistently perform multiple steps, from separating blood cells from whole blood to establishing iPS cells (and even forming differentiated cells). This reduces cell contamination during production, and by automating the process, it is possible to produce iPS cells with fewer personnel than conventional CPCs. Furthermore, the use of a closed-system device eliminates the need for facilities requiring Grade A certification, thereby reducing the cost of iPS cell production.

[0013] FIG. 1 is a block diagram showing an example of the closed system portion of a cell processing apparatus used in the manufacturing method of the present invention. Each thick black line designated by the symbol A10 represents a connecting pipe such as a flexible tube (the same applies to FIG. 5). To clearly illustrate the movement and stoppage of materials within the closed system portion, a pinch valve that opens and closes the connecting pipe and a peristaltic pump that delivers materials are depicted on the connecting pipe. These pinch valves and peristaltic pumps are located outside the connecting pipes and do not belong to the closed system portion, but rather belong to the mechanical portion of the cell processing apparatus (the same applies to FIG. 5). FIG. 2 is a schematic diagram showing centrifugation in the sealed chamber of a cell processing apparatus used in the manufacturing method of the present invention, showing the interior of the sealed chamber. FIG. 3 is a photograph illustrating an example of the mechanical portion of a cell processing apparatus used in the manufacturing method of the present invention. FIG. 4 is a photograph illustrating the state in which a closed system portion is attached to the mechanical portion of a cell processing apparatus according to the present invention. FIG. 5 is a block diagram showing another example of the closed system portion of a cell processing apparatus used in the manufacturing method of the present invention. FIG. 6 shows the expression results of CD14- and CD45-positive cells, monocyte differentiation markers, in Example 2. Figure 7 shows microscopic images taken on days 24 and 28 after establishment of iPS cells in Example 9, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 harbored in the vector one week after each passage at passages 1 and 3. Figure 8 shows microscopic images taken on days 12 and 16 after establishment of iPS cells in Example 10, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 harbored in the vector one week after each passage at passages 1 and 3. Figure 9 shows microscopic images taken on days 21 and 28 after establishment of iPS cells in Example 13, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 harbored in the vector one week after each passage at passages 1 and 3. Figure 10 shows microscopic images of differentiation-induced monocytes in Example 14 (days 17 and 20 after differentiation induction) and the results of the expression of cells positive for CD14 and CD45, monocyte differentiation markers. Figure 11 shows microscopic images taken on days 14 and 20 after the establishment of iPS cells in Example 16, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 carried in the vector at passage 1, one week after passage.Figure 12 is a photograph showing an example of a conventional cell processing device. Figure 13 shows the results of an investigation into the coating method of scaffold materials in Examples 23 and 24. Figure 14 shows the results of an investigation into the amount of Sendai virus vector (SeV) in Examples 25 to 27. Figure 15 shows an overview of medium exchange in the monocyte differentiation process investigated in Examples 28 to 30. Figure 16 shows the results of an investigation into the monocyte differentiation process in Example 28. Figure 17 shows the results of an investigation into the monocyte differentiation process in Example 29. Figure 18 shows the results of an investigation into the monocyte differentiation process in Example 30.

[0014] A method for producing iPS cells according to the present invention is described in detail below. This method produces iPS cells using a cell processing apparatus. The cell processing apparatus has a closed system portion as shown in FIG. 1. This closed system portion is configured such that a sealed container for supplying materials (in the example shown in the figure, the portions designated by the reference numerals 210 to 250) and a rotatable sealed chamber 100 for centrifugal separation are connected via a connecting pipeline A10. In this production method, the following steps (s1) to (s3) are carried out in this order within the same sealed chamber 100 while maintaining the closed nature of the closed system portion: Step (s1): Centrifuging blood cells from whole blood; Step (s2): Contacting the blood cells with reprogramming factors; and Step (s3): Establishing iPS cells from the blood cells. Other processing steps may be added between steps (s1) to (s3). Furthermore, between steps (s1) to (s3), blood cells may be transferred from the sealed chamber to another element within the closed system and then returned to the sealed chamber. Furthermore, depending on additional processing, such as quality control measurements, between steps (s1) to (s3), the blood cells being processed may be aseptically transferred to an external sealed container, where additional filtering or measurement may be performed, and then the cells may be returned to the closed system for the next step. These additional processes may be performed manually, automatically, or semi-automatically. In either case, the main steps (s1) to (s3) of the present invention are automatically performed within the closed system according to instructions from a control unit of the cell processing device (e.g., Prodigy). The instructions from the control unit are preset for the present invention so that steps (s1) to (s3) are automatically performed sequentially. Through the steps (s1) to (s3), whole blood is processed in a single sealed container (sealed chamber for centrifugation) in a single closed system to obtain raw blood cells, from which iPS cells are established.

[0015] (Cell Processing Apparatus) First, the configuration of each part of a cell processing apparatus that can be used in the manufacturing method will be described. The cell processing apparatus itself may be a conventionally known apparatus as shown in FIG. 12. The cell processing apparatus has a closed system part as shown in FIG. 1 and a mechanical part as shown in FIG. 3. The closed system part is attached to and detached from the mechanical part in a disposable manner. An example of the state in which the closed system part is attached to the mechanical part is shown in FIG. 4. The mechanical part automatically operates according to a predetermined control program (computer program), acts on the closed system part, and automatically performs the above steps (s1) to (s3) in order.

[0016] A preferred cell processing device is the CliniMACS Prodigy (registered trademark) manufactured by Miltenyi Biotec, which is also shown in Figure 12. The CliniMACS Prodigy is described in detail, for example, in Patent Document 3. Hereinafter, the CliniMACS Prodigy will also be simply referred to as Prodigy. Hereinafter, the production method will be described while referring to Prodigy as a cell processing device.

[0017] (Mechanical parts of the cell processing device) The mechanical parts include parts other than the closed system part, such as various actuators, a magnetic separation device, a computer (including a computer program) that serves as a control unit, a power source, a gas supply source, a heat source, a display device, a support part, a housing part, etc. Important parts of the actuators include the pinch valve V10 and the peristaltic pump F10 shown in Figures 1 and 3, and a motor (not shown) that rotates the sealed chamber 100.

[0018] The pinch valve V10 is a type of electromagnetic valve that applies pressure to the connecting pipe (soft tubing) of the closed system from the outside to block the connecting pipe, and releases the pressure to open the connecting pipe. The peristaltic pump F10, also known as a tube pump or peristaltic pump, is a pump that moves a fluid in the connecting pipe (soft tubing) by moving the external pressure on the connecting pipe. The pinch valve V10 and the peristaltic pump F10 make it possible to move materials inside the closed system from the outside as intended while maintaining the closed nature of the closed system.

[0019] The control unit (including a computer and computer program) of the mechanical part selectively operates the necessary pinch valves V10 to open or close specific connecting lines A10, operates the peristaltic pump F10 to move the necessary materials and blood cells being processed within the closed system, and rotates the sealed chamber 100 to perform centrifugation, thereby sequentially carrying out each step of the manufacturing method. The computer program executed by the control computer can be pre-programmed to suit the present invention by inputting or modifying parameters, such as the timing of opening and closing any pinch valves, the operation period and feed rate of the peristaltic pump, the operation period and relative centrifugal force of the sealed chamber, etc. Thus, by setting the sealed containers for supplying materials required for each step and empty sealed containers as the closed system parts and executing the pre-programmed computer, the steps of the novel manufacturing method of the present invention are automatically carried out sequentially. Furthermore, the execution of the computer program (i.e., the operation of Prodigy) can be temporarily stopped by pre-programming or by inputting a stop command using a stop switch, and can be restarted at will. This allows for additional manual processing (such as sampling for quality control or filtering) during or between each step.

[0020] (Closed system portion: sealed container for supplying materials) Sealed containers for supplying materials may be prepared in the same number as the number of types of materials used in processing the cells to be produced. By pre-mixing the materials, the number of types of materials used may differ from the number of sealed containers. Furthermore, for materials required in large quantities, multiple sealed containers containing the same material may be prepared. The capacity of the sealed container is not particularly limited and varies depending on the material, but is generally around 20 to 3000 ml.

[0021] In FIG. 1 , as an illustrative example, a sealed container 210 containing whole blood diluted with PBS (e.g., a mixture of whole blood and PBS in a 1:1 volume ratio), a sealed container 220 containing a liquid culture medium, a sealed container 230 containing a centrifugation medium, a sealed container 240 containing a fluid containing a reprogramming factor, and an empty sealed container 250 used as a temporary transfer location for materials are each connected to the sealed chamber 100 via connecting pipelines. However, without being limited to the example of FIG. 1 , sealed containers containing materials appropriate for the process may be connected as part of a closed system for supply. Each material will be described later.

[0022] The structure of the sealed container for supplying materials is not particularly limited, and reference can be made to sealed containers used in conventional cell processing devices such as Prodigy. A preferred example is a flexible bag made of a flexible film or flexible sheet. The flexible film is flexible enough to be deformed according to the amount of material contained in the bag. The flexible film is flexible enough to contain the O necessary for cell culture. ２ YaCO ２ In the Prodigy, a required number of bags, which are airtight containers for supplying materials, are hung from the support B10 shown in Fig. 3, and the bag is in the state of use shown in Fig. 4.

[0023] (Closed System Portion: Sealed Chamber with Centrifugal Function) The sealed chamber 100 is configured so that it can rotate for centrifugation while maintaining a closed system, i.e., while the connecting pipeline A10 remains connected and even while material is flowing in or out. The centrifugation may be based on density gradient centrifugation. To perform density gradient centrifugation, the necessary centrifugation medium (described below) may be supplied to the sealed chamber from a sealed container for supplying material.

[0024] The material used to prepare the sealed chamber is not particularly limited, but examples of materials that can be used include commonly used glass, polystyrene, polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polyethylene, polypropylene, polyethylene methacrylate, and other polymeric compounds, ceramics, and metals.

[0025] (Centrifugal separation mechanism) Figure 2 is a schematic diagram for explaining the centrifugal separation process within the sealed chamber of the Prodigy. The shape defined by the side wall surfaces (inner wall surfaces) surrounding the internal space of the sealed chamber is generally cylindrical. A support pillar is located in the center of the internal space, and a flow path is provided inside. As shown in Figure 1, the geometric rotation center line y1 of the cylinder coincides with the central axis of rotational motion during centrifugation of the sealed chamber. The base area of ​​the cylinder is 90 to 200 cm ２ The actual measured inner diameter of the cylindrical shape (inner diameter of the sealed chamber) is, for example, about 12 cm or 14 cm, but is not limited to these. In centrifugation, the relative centrifugal force acting on the contents in the sealed chamber is 100 to 500 G, and the relative centrifugal force used in this example is 400 G.

[0026] 2(a) and 2(b), an opening 100a for the inflow and outflow of material is provided at the bottom of the sealed chamber 100, at a position a predetermined distance from the side wall surface toward the center. The opening 100a is connected to the external connecting pipe shown in FIG. 1 through a flow path 110 that passes through the bottom and the central support 120. Meanwhile, an opening 100b for the outflow of material is provided at the upper side of the sealed chamber 100, at a position close to the side wall surface. The opening 100b communicates with the external connecting pipe shown in FIG. 1 through a flow path 130 on the upper side.

[0027] In centrifugation, as shown in FIG. 2( a), first, the object to be centrifuged (e.g., whole blood) K10, and optionally a centrifugation medium, are injected into the sealed chamber 100 through the flow channel 110. Next, as shown in FIG. 2( b), the entire sealed chamber rotates at the rotation speed required for centrifugation. In density gradient centrifugation, this rotation separates the object in the sealed chamber into multiple fractions with different densities. In the example shown in FIG. 2( b), from the inside, the object is separated into a fraction K11 containing primarily the centrifugation medium, a fraction K12 containing primarily peripheral blood mononuclear cells (PBMCs), and a fraction K13 containing primarily red blood cells. While maintaining this separation state (i.e., while the sealed chamber is rotating), fraction K11 is discharged from the bottom opening 100 a, fraction K13 is discharged from the top opening 100 b, and fraction K12 containing primarily PBMCs is left in the sealed chamber, completing the desired centrifugation (isolation). In this way, the centrifuged target fraction can be left in the sealed chamber, while the other fractions can be discharged outside the sealed chamber. If necessary, it is also possible to discharge only the centrifuged target fraction from the sealed chamber. This series of processes is performed automatically by operating the pinch valve, peristaltic pump, and sealed chamber drive motor in response to commands from a preset control unit.

[0028] The capacity of the sealed chamber is not particularly limited, but is preferably about 300 to 700 ml, for example. Alternatively, sealed chambers of various capacities may be prepared depending on the scale of processing, such as a regular-sized sealed chamber with a capacity of 400 ml and a large-sized sealed chamber with a capacity of 600 ml.

[0029] The amount of fluid centrifuged at one time in the closed chamber is not particularly limited, but is about 10 to 250 ml, preferably about 30 to 130 ml.

[0030] (Closed System Portion: Connecting Pipeline) The pipe members constituting the connecting pipeline A10 are preferably tubes (soft tubes) made of soft materials (such as silicone or vinyl chloride) because the Prodigy uses the pinch valve and peristaltic pump described above. The connecting pipeline may also include pipe members made of hard materials at joints and other portions.

[0031] (Closed or airtight) The closed or airtight interior of a closed system part, airtight container, or airtight chamber means not only a state in which the interior is kept airtight or liquidtight, but also a state in which the interior is isolated from the outside world to the extent that microorganisms or viruses cannot enter from the outside, i.e., to the extent that sterility of the interior is maintained. For example, a sealed chamber provided with a porous filter (e.g., pore size of about 0.2 μm or less, particularly about 0.1 to 0.2 μm) that does not allow bacteria or viruses to pass through but allows fluids (particularly gases) to pass through, allows outside air to pass through the porous filter and into the sealed container, but does not allow bacteria or viruses to enter, and therefore maintains sterility within the closed system part, and is therefore airtight. Also, if the wall of the container is made of a gas-permeable membrane, bacteria, viruses, etc. cannot pass through, and O ２ Gas molecules and CO ２ A container that is permeable to gas molecules is also considered airtight. Therefore, a "closed system" is not only an airtight or liquid-tight system, but also a system whose interior is isolated from the outside world to the extent that microorganisms and viruses cannot enter from the outside.

[0032] Next, we will explain the iPS cells to be produced, as well as the raw materials and ingredients required for production.

[0033] (Induced pluripotent stem cells) As used herein, "induced pluripotent stem cells (iPS cells)" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells through the introduction of reprogramming factors. Induced pluripotent stem cells can differentiate into tissues and cells with a variety of different morphologies and functions in the body, and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm).

[0034] In the present specification, iPS cells may be cells derived from a patient. Producing iPS cells from somatic cells derived from a patient and using them for clinical treatment can be an effective means of minimizing the risk of rejection.

[0035] This production method can be used to produce any currently available iPS cells.

[0036] In this specification, induced pluripotent stem cells may be cells derived from a patient with a genetic disease. Cells induced to differentiate from pluripotent stem cells derived from a patient with a genetic disease can serve as disease models that reflect the pathology of the disease, and are therefore suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from a patient with a genetic disease can be genetically repaired by genome editing using a CRISPR-Cas system or the like, and then differentiated into the desired cells, making it possible to use the cells as a therapeutic drug for the disease.

[0037] (Whole Blood) As used herein, "whole blood" refers to blood collected from a subject such as a human, from which blood cells and the like have not been separated. Furthermore, as used herein, "whole blood" may include blood diluted by mixing with an appropriate buffer or the like, or blood to which additives such as blood coagulation inhibitors (e.g., heparin, EDTA, citric acid, etc.) or protease inhibitors have been added. The buffer used to dilute whole blood is not particularly limited as long as it does not cause hemolysis or other effects on blood cell components in the whole blood, and examples thereof include phosphate buffer solution (PBS), physiological saline, etc.

[0038] (Amount of Whole Blood Supplied) In the present invention, the amount of whole blood supplied to the sealed chamber is not particularly limited, but may be about 5 to 100 ml, preferably 10 to 50 ml, more preferably 10 to 40 ml, and even more preferably 10 to 20 ml. The whole blood may be stored in a sealed container for supplying material in the state in which it is collected and then sent to the sealed chamber, or may be diluted with a buffer solution or the like as described above, stored in a sealed container for supplying material, and then sent to the sealed chamber.

[0039] (Blood cells) As used herein, the term "blood cells" refers to all cells at various stages, from hematopoietic stem cells, through hematopoietic progenitor cells (including pluripotent hematopoietic progenitor cells and unipotent hematopoietic progenitor cells), to finally functional blood cells. Examples of blood cells include peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMNCs).

[0040] (Lymphoid Cells) As used herein, the term "lymphoid cells" refers to cells including CD34-positive hematopoietic progenitor cells (lymphoid progenitor cells) and their differentiated cells such as B cells, NK cells, and T cells.

[0041] In the present specification, the species from which blood cells originate is not particularly limited, and the preferred species from which blood cells originate is human.

[0042] In this specification, unless otherwise specified, the term "cell" includes a "cell population." A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

[0043] As used herein, "processing" cells means subjecting cells to treatments such as culturing the cells, diluting a solution containing the cells, washing the cells, separating the target cells from a solution containing the cells, etc. It also means subjecting cells to chemical treatment, modification of biological properties, combination with non-cellular components, genetic engineering modification, etc. for the purpose of artificial proliferation / differentiation of the cells, establishment of a cell line, or activation of the cells.

[0044] (Liquid medium) The liquid medium delivered from the sealed container can be used not only for cell cultivation but also for various purposes in the production process, such as washing cells, diluting chemical solutions, etc. Examples of liquid media that can be used in the present invention include the following:

[0045] The liquid medium is not particularly limited, but may be, for example, Essential 8 medium (CTS TM Essential 8 TM Medium, Essential 8 TM Medium, Essential 8 TM Flex Medium, Essential 6 TMExamples of suitable media include StemFit® AK02 Medium (Thermo Fisher Scientific), StemFit® AK03 Medium (Ajinomoto Co., Inc.), StemFit® Basic03 Medium, CTS® KnockOut SR XenoFree Medium (Gibco), mTeSR1 Medium, TeSR1 Medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), and Improved MEM (Thermo Fisher Scientific). These media can also be used for culture under feeder-free and xeno-free conditions. Other examples include, but are not limited to, MSCBM-CD, MSCGM-CD (both manufactured by Lonza) and mixtures thereof.

[0046] If necessary, physiologically active substances and nutritional factors necessary for cell survival or proliferation can be added to the medium.

[0047] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the medium as needed.

[0048] In this specification, known serum can be used as the medium.

[0049] As used herein, the medium may or may not contain serum substitutes, as well as serum.

[0050] (Scaffold Material) In this specification, the term "scaffold material" refers to a material or substrate that functions as a scaffold for cells in cell culture. The scaffold material is not particularly limited as long as it can be used for adhesion culture or suspension culture using the scaffold material (in other words, it may be free in the medium), and examples include those containing or made of synthetic resin, and those made of flexible materials such as collagen. Microcarriers may also be used as scaffold materials. As an example, the scaffold material may contain atelocollagen. The scaffold material may be manufactured by a known method, or a commercially available product may be used. Examples of commercially available products include Cytodex-1 (manufactured by GE Healthcare) and Corning® Low Concentration Synthemax® II Microcarrier (manufactured by Corning).

[0051] The scaffold material may also contain or consist of an extracellular matrix, such as a basement membrane preparation (e.g., Matrigel (manufactured by Corning), Geltrex matrix (manufactured by Thermo Fisher Scientific), etc.), fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, vitronectin, etc., or a combination thereof. The exemplified extracellular matrices may be natural products, artificially synthesized by genetic engineering techniques, etc., fragments obtained by cleavage with restriction enzymes, etc., or synthetic proteins or synthetic peptides based on these biological substances.

[0052] Laminin is a heterotrimeric molecule consisting of α, β, and γ chains, and is an extracellular matrix protein that exists in isoforms with different subunit chain compositions. Specifically, laminin has approximately 15 isoforms, consisting of heterotrimeric combinations of five α chains, four β chains, and three γ chains. The names of laminins are determined by combining the numbers of the α chains (α1 to α5), β chains (β1 to β4), and γ chains (γ1 to γ3). For example, laminin composed of a combination of α5, β1, and γ1 chains is called laminin-511. Laminins may be natural products, artificially synthesized using recombinant DNA technology, or synthetic proteins or peptides based on the laminins.

[0053] The laminin or fragments thereof used in the present invention include laminin-111 and a fragment containing its E8 region (e.g., iMatrix-111), laminin-211 and a fragment containing its E8 region (e.g., iMatrix-211), laminin-121 or a fragment containing its E8 region, laminin-221 or a fragment containing its E8 region (e.g., iMatrix-221), laminin-332 or a fragment containing its E8 region (e.g., iMatrix-332), laminin-3A11 or a fragment containing its E8 region, laminin-411 or a fragment containing its E8 region (e.g., iMatrix-411), laminin-421 or a fragment containing its E8 region, and laminin-511 or a fragment containing its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment comprising its E8 region, laminin-213 or a fragment comprising its E8 region, laminin-423 or a fragment comprising its E8 region, laminin-523 or a fragment comprising its E8 region, laminin-212 / 222 or a fragment comprising its E8 region, and laminin-522 or a fragment comprising its E8 region.

[0054] Vitronectin may be a natural product, artificially synthesized by genetic recombination technology, or a synthetic protein or peptide based on vitronectin. Commercially available products that are readily available include human plasma-derived vitronectin (Sigma-Aldrich), human vitronectin (20-398 aa), recombinant human solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Gibco).

[0055] Fibronectin may be a natural product, artificially synthesized by genetic recombination technology, or a synthetic protein or peptide based on the fibronectin. Commercially available products include, for example, fibronectin solution, derived from human plasma (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and Retronectin (registered trademark) (manufactured by Takara Bio Inc.).

[0056] The type of collagen is not particularly limited, and for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, may be artificially synthesized using genetic engineering techniques, or may be a synthetic peptide based on the collagen. Commercially available collagens include, for example, collagen I, human (manufactured by Corning) and collagen IV, human (manufactured by Corning) due to their ease of availability.

[0057] The scaffold material may contain or consist of the above-mentioned synthetic resin and extracellular matrix. Examples of polymers (resins) used in the scaffold material include polystyrene, polyolefin, polyethylene terephthalate, polyether, polyvinyl alcohol, polyvinyl acetal (e.g., polyvinyl butyral), polyester, poly(meth)acrylic acid ester (poly(meth)acrylate), epoxy resin, polyamide, polyimide, polyurethane, polycarbonate, cellulose, dextran, and polypeptides (e.g., gelatin).

[0058] Specific examples of scaffold materials containing a synthetic resin and an extracellular matrix or consisting of a synthetic resin and an extracellular matrix include those described in, for example, WO 2023 / 127777, JP 2022-077840, JP 2021-023287, JP 2021-003103, JP 2021-003102, JP 2021-003099, JP 2021-003062, JP 2021-003061, JP 2020-174682, JP 2019-118345, JP 2019-115323, JP 2019-115322, WO 2019 / 131982, WO No. 2019 / 131981, International Publication No. 2019 / 131978, JP 2016-106545, JP 2015-221851, International Publication No. 2015 / 098919, International Publication No. 2009 / 099555, International Publication No. 2010 / 138486, International Publication No. 2010 / 138687, International Publication No. 2010 / 138702, US2011152455A1, International Publication No. 2011 / 014594, International Publication No. 2011 / 014605, International Publication No. 2012 / 150475, International Publication No. 2013 / 116432, and the like.

[0059] In one aspect, the scaffold material comprising a synthetic resin and a peptide comprises a polyvinyl acetal resin, preferably a polyvinyl butyral resin, and a cell adhesive peptide, or a poly(meth)acrylate resin (poly(meth)acrylic acid ester resin) and a cell adhesive peptide, as described in JP 2021-23287 A, JP 2022-077840 A, WO 2023 / 127777 A, WO 2011 / 014594 A, etc. As used herein, the term "cell adhesive peptide" refers to a peptide whose cell adhesive activity has been confirmed by phage display, Sepharose bead, or plate coating. The cell adhesive peptide is preferably a peptide consisting of at least three amino acids, more preferably 3 to 20 amino acids (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids), even more preferably 3 to 15 amino acids, and particularly preferably 3 to 10 amino acids. The cell adhesive peptide preferably contains an RGD sequence (Arg-Gly-Asp). An example of a commercially available scaffold material is Corning® Synthemax® Vitronectin Substrate (manufactured by Corning).

[0060] The method of supplying the scaffold material to a sealed chamber, etc. is not particularly limited as long as it can appropriately process the desired cells, and the supply conditions (e.g., supply timing, volume, etc.) and type of scaffold material can be appropriately set by a person skilled in the art.

[0061] In one embodiment, the scaffold material as described above may be coated in the sealed chamber used in the production method of the present invention before performing at least one of steps (s1), (s2), (s3), (s4), and (s5). In another embodiment, when iMatrix is ​​used as the scaffold material, the scaffold material as described above may be added to the culture medium used in at least one of steps (s1), (s2), (s3), (s4), and (s5) to coat the sealed chamber. In another embodiment, the scaffold material may be added to the culture medium used in at least one of steps (s1), (s2), (s3), (s4), and (s5) and then supplied into the sealed chamber.

[0062] (Centrifugation Medium) In step (s1), density gradient centrifugation is useful for separating blood cells from whole blood. In this case, a centrifugation medium for the density gradient centrifugation is supplied from a sealed container for supplying materials to the sealed chamber. As the centrifugation medium, a hydrophilic polysaccharide (Ficoll ＴＭ etc.) are examples.

[0063] In the density gradient centrifugation in step (s1), whole blood (which may be diluted) and a centrifugation medium are supplied to a sealed chamber, and density gradient centrifugation is performed, leaving a fraction containing blood cells in the sealed chamber.

[0064] (Reprogramming Factors) In step (s2), the pinch valve and peristaltic pump are activated to send a fluid containing reprogramming factors from a sealed container for supplying materials into the sealed chamber. Separated blood cells are present in the sealed chamber through step (s1), and the reprogramming factors come into contact with the blood cells. When the reprogramming factors are brought into contact with the blood cells, the sealed chamber may be stationary, may be rotating, or may be a combination of these.

[0065] As used herein, examples of "reprogramming factors" include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, ESrrb, Nr5a2, Tbx3, and Glis1, and these reprogramming factors may be used alone or in combination. Any known combination of reprogramming factors may be used.

[0066] The reprogramming factor introduced into the somatic cell may be in the form of a protein, a nucleic acid (RNA or DNA) encoding the protein, or an expression vector containing the nucleic acid. When the reprogramming factor is introduced in the form of RNA, immunogenic RNA introduced into the cell may activate the cellular defense mechanism, so RNA for circumventing the defense mechanism may be introduced into the somatic cell.

[0067] Examples of expression vectors include viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, as well as plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors (piggyBac, piggyBat, TolII).

[0068] Nucleic acids, expression vectors containing the nucleic acids, or proteins (e.g., reprogramming factors) can be introduced into cells by various known methods, including calcium phosphate-mediated transfection, electroporation, liposome transfection, lipofection, gene guns, microinjection, viral vector methods, virus-like particle methods, Agrobacterium methods, agroinfiltration methods, PEG-calcium methods, sonoporation methods, and lipid nanoparticle methods.

[0069] (Materials Constituting the Sealed Chamber and Cell Culture) The material of the sealed chamber of Prodigy includes a portion made of polycarbonate resin. The majority of the internal space of the actual sealed chamber (the internal sidewall surface and the internal bottom surface) is made of polycarbonate resin. Therefore, these surfaces are non-cell-adhesive and unsuitable for culturing iPS cells, which are adherent cells (adherent culture using the inner wall surface). Therefore, in a preferred embodiment of the manufacturing method, an appropriate scaffold material is supplied to the sealed chamber, and steps (s2) and (s3) are performed in the presence of the scaffold material. Furthermore, the scaffold material may be supplied to the sealed chamber at step (s2) to simplify coating.

[0070] In a preferred embodiment of this production method, a scaffold material is attached to the inner wall surface of the sealed chamber. This is preferable because it enables adherent culture even when the inner wall surface of the sealed chamber is made of polycarbonate resin. Examples of such scaffold materials include those containing laminin fragments (e.g., laminin-511, laminin-332, etc.), cell adhesive peptides and polyvinyl acetal resins (e.g., polyvinyl butyral resins), and those containing cell adhesive peptides and poly(meth)acrylate resins. In a preferred embodiment, the cell adhesive peptide contains the RGD sequence (Arg-Gly-Asp).

[0071] The scaffold material can be supplied by dissolving powdered Synthemax in 1 vial (10 ml) of sterile water in a safety cabinet, sealing the solution in an airtight container, removing the container from the safety cabinet, connecting it to Prodigy in an aseptic manner, and pumping it into a sealed chamber using a peristaltic pump. The solution can then be kept overnight at 22°C for pre-coating. When only a small amount of scaffold material is used, it can be mixed with PBS or sterile water to facilitate supply. Liquid iMatrix can be supplied as is.

[0072] (Other Substances to be Supplied) In addition to the above-mentioned materials, substances necessary for cell processing may be added as appropriate. The added materials can be stored in a sealed container or the like and connected to the sealed chamber, similar to the above-mentioned materials. Examples of other materials to be added include liquids, powders, additives, release agents, cryoprotectants, CO ２ Examples include:

[0073] (Regarding the step (s3) of establishing iPS cells) Step (s3) in the production method of the present invention is a step of culturing blood cells in a liquid medium to establish iPS cells, and the establishment of induced pluripotent stem cells can be appropriately confirmed by the expression of reprogramming factors introduced by a method known per se (e.g., Oct3 / 4, SOX2, Nanog, TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase, etc.). The period for which step (s3) is performed is not particularly limited as long as iPS cells are established, but is typically, for example, 10 days or more, and preferably 14 days or more. There is also no particular upper limit, but it is typically 30 days or less, and preferably 40 days or less.

[0074] A preferred example of the operation of Prodigy for carrying out steps (s1) to (s3) is as follows: (e1) In FIG. 1 , diluted whole blood (e.g., 20 ml of whole blood diluted with 30 ml of phosphate-buffered saline (PBS)) contained in the sealed container 210 for supplying material is sent to the sealed chamber 100. (e2) Ficoll, a density gradient centrifugation medium contained in the sealed container 230 for supplying material, is sent to the sealed chamber 100. (e3) The sealed chamber 100 is rotated, density gradient centrifugation is performed, and a fraction containing mainly PBMCs is left in the sealed chamber. (e4) The fraction containing mainly PBMCs is sent to an empty sealed container 250. The connecting pipes connected to the sealed container 250 are sealed by heat sealing, and the sealed container 250 is aseptically disconnected from the Prodigy. (e5) The sealed container 250 and another sealed container containing a filter are aseptically joined, and the PBMCs contained in the sealed container 250 are placed into the other sealed container containing a filter through the top opening. Debris (like clumps of unwanted cells) is removed through the filter inside the sealed container. (e6) A portion of the fraction containing mainly PBMCs is manually sampled, stained with trypan blue, and the cell count is counted using a cell counter. Steps (e4) to (e6) are not essential steps for the present invention, but are preferably performed. (e7) Optionally, at this point, a scaffold material (e.g., Synthemax®) is delivered from a material supply sealed container into the sealed chamber 100, and the inner wall surface of the sealed chamber is coated with the scaffold material. (e8) The sealed container with the filter built in is manually and aseptically connected to the closed system portion, and the fraction mainly containing PBMCs is returned to the sealed chamber 100 by a peristaltic pump, and a fluid containing a Sendai virus vector as a reprogramming factor is sent to the sealed chamber 100 from the sealed container 240 for supplying material. (e9) The Sendai virus vector is brought into contact with the PBMCs in the sealed chamber (2 hours). (e10) Liquid medium is added to the sealed chamber from the sealed container 220 for supplying material. (e11) A portion of the contents in the sealed chamber is manually sampled and the cell count is counted.This step (e11) is not essential to the present invention, but is a preferred step. (e12) The liquid medium in the sealed chamber is reduced, cytokines are added, and cell culture is initiated (establishment of iPS cells is initiated). (e13) Optionally, at this point, a scaffold material is supplied to the sealed chamber 100 from a sealed container for supplying materials and mixed by rotating the sealed chamber (if the inner wall surface of the sealed chamber is to be coated, the mixing conditions are 37°C and approximately 30 to 60 minutes). (e14) The liquid medium in the sealed chamber is replaced or added. (e15) 14 to 21 days after the start of the cell culture (start of establishment of iPS cells), colonies (in the case of adherent culture) or spheroids (in the case of suspension culture) are observed.

[0075] (Expansion Culturing) From the viewpoint of iPS cell yield, the production method may further include a step (s4) of expanding the iPS cells in the sealed chamber after the step (s3) of establishing iPS cells. Similar to steps (s1) to (s3), step (s4) is performed while maintaining the closed system. The expansion of the iPS cells in the sealed chamber can be achieved by supplying a medium suitable for expansion to the sealed chamber. The medium used in step (s4) can be appropriately selected by those skilled in the art from the liquid media described above. In one embodiment, if expansion of the established induced pluripotent stem cells is intended, a factor for maintaining undifferentiated states can be added. As used herein, the term "factor for maintaining undifferentiated states" refers to a substance that has the effect of suppressing differentiation of induced pluripotent stem cells, and is not particularly limited as long as it is such a substance. Factors for maintaining undifferentiation commonly used by those skilled in the art include bFGF, FGF2, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, and TGFβ2. The factors for maintaining undifferentiation used in the present invention are preferably isolated. "Isolated" means that they have been subjected to a procedure to remove factors other than the target component or cells, and are no longer in a naturally occurring state.

[0076] In step (s4), the number of times expansion culture is performed is not particularly limited as long as the desired number of iPS cells is obtained, but in a typical processing procedure, it is preferably about 1 to 5 times, more preferably about 2 to 5 times. The period for performing step (s4) is not particularly limited as long as the desired number of iPS cells is obtained, but is typically, for example, 1 to 40 days, 3 to 20 days, or 5 to 10 days. If the Sendai virus vector used for reprogramming contains a GFP marker, it is desirable to perform expansion culture until the marker is lost.

[0077] (Magnetic Separation Step) In the production method of the present invention, when the blood cells to be separated in step (s1) are the lymphoid cells described above (e.g., CD34-positive cells (more specifically, hematopoietic progenitor cells)), the required lymphoid cells can be isolated from PBMCs by centrifuging the PBMCs from whole blood and then adding a magnetic separation step. In this case, as shown in FIG. 5 , a magnetic separation column 300 connected to the sealed chamber 100 via a connecting pipeline A10 is used as part of the closed system configuration. The magnetic separation column 300 is attached to the magnetic separation unit M10 in the mechanism portion shown in FIG. 3 . In FIG. 5 , the lymphoid cells to be separated are labeled with magnetic beads in the sealed chamber 100. In the example of FIG. 5 , the magnetic beads are sent to the sealed chamber from a material supply sealed container 260. When the suspension containing the lymphoid cells (with magnetic beads) is sent from the sealed container 100 into the magnetic separation column 300, only the lymphoid cells (with magnetic beads) are trapped (i.e., magnetically separated) in the magnetic separation column 300 by the magnetic field applied by the magnetic separation unit M10. Other blood cells and foreign matter pass through the magnetic separation column 300 and are sent to a bag for negative selection (not shown). Thereafter, the application of the magnetic field by the magnetic separation unit M10 is stopped, the pinch valve is switched, and the lymphoid cells (with magnetic beads) trapped in the magnetic separation column 300 exit the magnetic separation column 300 and are sent to the bag for positive selection 310. Next, the pinch valve is switched, and the lymphoid cells (with magnetic beads) in the bag for positive selection 310 are returned to the sealed chamber 100. The magnetic beads may be actively removed by a detachment process and then sent to the next step, but if the magnetic beads are extremely fine, for example, about 50 nm, they will naturally detach from the lymphatic cells by continuing processing in the next step (s2).

[0078] (Magnetic beads, detachment of magnetic beads) The process of magnetic separation itself and the necessary materials can be appropriately set or selected with reference to known techniques. Magnetic beads include CliniMACS CD1c (BDCA-1) GMP Biotin, CliniMACS CD3 GMP MicroBeads, CliniMACS CD4 GMP MicroBeads, CliniMACS CD8 GMP MicroBeads, CliniMACS CD14 GMP MicroBeads, CliniMACS CD19 GMP MicroBeads, CliniMACS CD25 GMP MicroBeads, CliniMACS CD34 GMP MicroBeads, CliniMACS45RA GMP MicroBeads, CliniMACS Prodigy CD45RA CD304(BDCA-4) GMP MicroBeads, CliniMACS Examples include Anti-Biotin GMP MicroBeads, CliniMACS TCRα / β GMP Biotin, and CliniMACS TCRα / β GMP Kit. One method for detaching magnetic beads is to allow the magnetic beads to naturally detach during cell culture. Materials for modification with magnetic beads and their detachment can be appropriately delivered to the sealed chamber from sealed containers for supplying materials, which are added as needed.

[0079] A preferred example of the operation of Prodigy when steps (s1) to (s2) include magnetic separation of lymphoid cells is as follows, and is performed according to the magnetic separation program "T cell transduction process" of Prodigy. (f1) In FIG. 1, 50 ml of diluted whole blood (20 ml of whole blood diluted with 30 ml of phosphate buffered saline (PBS)) contained in the sealed container 210 for supplying material is sent to the sealed chamber 100. (f2) Ficoll, a density gradient centrifugation medium contained in the sealed container 230 for supplying material, is sent to the sealed chamber 100. ＴＭ 150 ml is sent to the sealed chamber 100. (f3) The sealed chamber 100 is rotated, and density gradient centrifugation is performed, leaving a fraction containing mainly PBMCs in the sealed chamber. (f4) The fraction containing mainly PBMCs is sent to an empty sealed container 250. (f5) The fraction is sent to a labeling column (not shown) in the Prodigy, and lymphoid cells in the PBMCs are labeled with magnetic beads. (f6) The lymphoid cells (with magnetic beads) are returned to the sealed chamber and incubated. (f7) Washing is performed in the sealed chamber to remove excess magnetic beads. (f8) The lymphoid cells (with magnetic beads) are sent to a magnetic separation column and magnetically trapped within the magnetic separation column. (f9) They are washed within the magnetic separation column. (f10) The magnetic application to the magnetic separation unit is stopped. (f11) The lymphoid cells (with magnetic beads) are returned to the sealed chamber 100. Because the magnetic beads are extremely small, they are directly subjected to the next initialization step. (f12) A portion is manually sampled and stained with trypan blue (filtered Gibco Trypan Blue Strain (0.4%), 15250-061), and the cell count is measured using a cell counter. This step (f12) is not essential to the present invention, but is a preferred step. (f13) Optionally, at this point, a scaffold material (Synthemax, etc.) is delivered from a sealed material supply container to the sealed chamber 100, and the inner wall surface of the sealed chamber is coated with the scaffold material. (f14) A fluid containing a Sendai virus vector as a reprogramming factor is delivered from a sealed material supply container to the sealed chamber 100. (f15) Within the sealed chamber, the Sendai virus vector is brought into contact with lymphoid cells (with magnetic beads) (for 2 hours). (f16) A liquid medium is added to the sealed chamber from a sealed material supply container. (f17) A portion of the contents in the sealed chamber is sampled and the cell count is counted. (f18) The liquid medium in the sealed chamber is reduced, cytokines are added, and cell culture is initiated (establishment of iPS cells is initiated).(f19) Optionally, at this point, a scaffold material is supplied from a sealed container for supplying materials into the sealed chamber and mixed (if the inner wall surface of the sealed chamber is to be coated, the mixing conditions are, for example, 37°C and approximately 30 to 60 minutes). (f20) The liquid medium in the sealed chamber is replaced or added. (f21) Colonies (in the case of adherent culture) or spheroids (in the case of suspension culture) are observed 14 to 21 days after the start of the cell culture (start of iPS cell establishment).

[0080] (Quality Evaluation of iPS Cells) After step (s3) or step (s4) of the production method of the present invention, the quality of the obtained iPS cells may be evaluated by a known method.

[0081] (Method for Producing Differentiated Cells) Next, a method for producing differentiated cells according to the present invention will be described. This method for producing differentiated cells comprises steps (s1) to (s3) of the method for producing iPS cells according to the present invention described above, and further comprises a step (s5) of inducing differentiation of iPS cells after these steps. Like steps (s1) to (s3) and step (s4), step (s5) is carried out while maintaining the closed nature of the closed system. In step (s5), materials necessary for differentiation induction are supplied into the sealed chamber from a sealed container (not shown) for supplying materials, and step (s5) is carried out within the sealed chamber. The sealed container for supplying the materials necessary for differentiation induction is not shown in Figures 1 and 5.

[0082] The step (s5) may be performed after the step (s3) in the method for producing iPS cells according to the present invention described above, without performing the step (s4) of expanding the iPS cells, or may be performed after the step (s4) of expanding the iPS cells after the step (s3).

[0083] (Differentiated cells) As used herein, "differentiated cells" refers to cells or organoids obtained by inducing differentiation of induced pluripotent stem cells. The cells obtained may be undifferentiated cells such as stem cells or progenitor cells, or may be terminally differentiated cells. As used herein, the term "differentiated cells" may be used to encompass both undifferentiated cells and terminally differentiated cells obtained by inducing differentiation of induced pluripotent stem cells. As used herein, "undifferentiated cells" refers to cells that have not reached terminal differentiation in the cell lineage, and examples of undifferentiated cells include stem cells excluding pluripotent stem cells, progenitor cells, and the like. Examples of stem or progenitor cells include ectodermal cells such as neural crest cells, neural stem cells, neural progenitor cells, glial progenitor cells, retinal stem cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary stem cells, mesodermal cells such as hematopoietic progenitor cells, myeloid stem cells, lymphoid stem cells, B progenitor cells, T progenitor cells, mesenchymal stem cells, cardiac stem cells, cardiac progenitor cells, vascular endothelial progenitor cells, vascular pericytes, platelet progenitor cells, skeletal muscle stem cells, adipose stem cells, kidney progenitor cells, and endodermal cells such as hepatic stem cells, liver progenitor cells, intestinal stem cells, and airway stem cells.

[0084] As used herein, the term "terminally differentiated cells" refers to cells that have reached terminal differentiation in a cell lineage. Examples of terminally differentiated cells include, but are not limited to, osteoblasts, chondrocytes, adipocytes, hepatic mesothelial cells, bile duct epithelial cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, Kupffer cells, pit cells, vascular endothelial cells, blood cells, pancreatic duct epithelial cells, pancreatic duct cells, acinar centro-cells, acinar cells, islets of Langerhans, cardiac myocytes, fibroblasts, smooth muscle cells, type I alveolar epithelial cells, type II alveolar epithelial cells, Clara cells, ciliated epithelial cells, basal cells, goblet cells, neuroendocrine cells, Kruczykki cells, renal tubular epithelial cells, urothelial cells, columnar epithelial cells, glomerular epithelial cells, glomerular endothelial cells, octopus podocytes, mesangial cells, neurons, and glial cells. Examples of leukocytes include lymphocytes, granulocytes, and monocytes.

[0085] In one aspect, the cells or organoids (target cells or organoids) obtained by inducing differentiation of induced pluripotent stem cells are neural crest cells, neural progenitor cells, neurons, cerebral cortical organoids, hematopoietic progenitor cells, platelets, T cells, or cardiomyocytes.

[0086] (iPS cell differentiation inducer) In this specification, the term "differentiation inducer" refers to a substance that can induce differentiation from induced pluripotent stem cells into the differentiated cells or organoids described above. The differentiation inducer may be a known substance, or may be selected from those that are commonly used to induce differentiation of the desired differentiated cells or organoids. Specific examples include the substances described below.

[0087] The fluid used as a medium for containing the reprogramming factors, undifferentiated maintenance factors, and differentiation inducers is not particularly limited, but preferred examples include buffer solutions, culture media, and cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerin.

[0088] (Differentiation Induction Method) In the production method of the present invention, known methods can be used as the differentiation induction method for obtaining the target cells or organoids. For example, differentiation induction from pluripotent stem cells to neural crest cells can be performed by the methods described in Fukuta M. et al., PLoS One, 2014, 9(12): e112291 or Kamiya D, et al., NPJ Regen Med., 2022 Sep 15;7(1):47. Specifically, pluripotent stem cells can be seeded in a culture vessel and subjected to adhesion culture (suspension culture using a scaffold material), and then differentiated into neural crest cells by adhesion culture (suspension culture using a scaffold material) in a medium containing a TGFβ inhibitor and a GSK3β inhibitor.

[0089] Neural crest cells can also be used to produce cells such as mesenchymal stem cells, neural progenitor cells, neurons, glial cells, bone cells, chondrocytes, corneal cells, and melanocytes. For example, differentiation into these cells can be performed based on the methods described in Fukuta M. et al., PLoS One, 2014, 9(12): e112291, Horikiri T. et al., PLoS One, 2017, 12(1): e0170342, and Kamiya D, et al., NPJ Regen Med., 2022 Sep 15;7(1):47. Specifically, for example, neural crest cells are seeded onto a fibronectin-coated plate, and the medium is replaced with DMEM / F12 supplemented with N-2 Supplement, BDNF, GDNF, NT-3, and NGF, and then incubated at 37°C and 5% CO. ２ Alternatively, neural crest cells can be plated and cultured in CDM medium containing SB431542 and CHIR99021 for 1 day, after which the medium is replaced with neurobasal medium supplemented with B-27 supplement, N-2 supplement, L-glutamine, penicillin / streptomycin, BDNF, GDNF, NT-3, and NGF, and the medium is incubated at 37°C, 5% CO ２ By culturing the cells under the conditions described above for about 35 days, neural progenitor cells and neural cells can be obtained.

[0090] Differentiation into mesenchymal stromal cells can be induced, for example, by the following method: Neural crest cells are seeded in a culture vessel and cultured for one day in CDM medium containing SB431542 and CHIR99021. After one day, the medium is replaced with αMEM containing FBS. Mesenchymal stromal cells can be obtained approximately 14 days after the start of differentiation induction.

[0091] Methods for differentiating pluripotent stem cells into T cells include, for example, methods comprising: (1) differentiating pluripotent stem cells into hematopoietic progenitor cells; and (2) differentiating the hematopoietic progenitor cells into T cells. Step (1) can be, for example, culturing pluripotent stem cells in a medium for inducing hematopoietic progenitor cells, as described in WO 2013 / 075222, WO 2016 / 076415, Liu S. et al., Cytotherapy, 17 (2015); 344-358, etc. Step (2) can be, for example, (2-1) inducing CD4 / CD8 bipositive T cells from hematopoietic progenitor cells, or (2-2) inducing CD8 / positive T cells from CD4 / CD8 bipositive T cells, as described in WO 2016 / 076415, etc.

[0092] Examples of methods for inducing differentiation of pluripotent stem cells into cardiomyocytes include those described in WO2015 / 141827, Laflamme MA and Murry CE, Nature. 473(7347):326-35 (2011), etc. Other methods include, for example, a method for producing cardiomyocytes by forming embryoid bodies through suspension culture of induced pluripotent stem cells, a method for producing cardiomyocytes in the presence of a substance that suppresses BMP signaling (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), and a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical (classical) Wnt signaling pathway (WO2007 / 126077). Typically, for example, marker proteins for cardiomyocytes include NKX2.5 (a cardiac muscle-specific transcription factor) and TNNT2 (troponin T), and marker proteins for cardiac progenitor cells include KDR (a receptor for vascular endothelial growth factor (VEGF)) and ISL1 (a LIM homeodomain transcription factor).

[0093] Differentiation into monocytes can be induced by the method described in Di Cui., et al. Frontiers in Cell and Developmental Biology; vol 9, Article 656867 April 2021.

[0094] In addition, organoid can be produced by using multiple kinds of cells.For example, in the case of hepatic organoid, as described in WO2013 / 047639 etc., hepatic progenitor cells (organ cells), mesenchymal stem cells and vascular endothelial cells are induced from pluripotent stem cells, and these mixtures are subjected to suspension culture, thereby hepatic organoid can be produced.

[0095] In the production method of the present invention, the culture may be performed under feeder-free conditions and / or xeno-free conditions for all or part of the period. From the viewpoint of clinical use, the differentiation induction method of the present invention is preferably performed under feeder-free and xeno-free conditions for the entire period.

[0096] The manufacturing method of the present invention may include a step of recovering the obtained target cells or organoids. The recovered cells may be cryopreserved using a cell cryopreservation solution. The recovered cells may also be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and purified by flow cytometry, mass cytometry, magnetic cell separation, etc.

[0097] In the cell production method of the present invention, undifferentiated cells may be removed as appropriate. The method for removing undifferentiated cells is not particularly limited as long as it can remove cells other than cells produced by the cell production method of the present invention, and can be performed by adding a known agent for removing undifferentiated cells to the medium (e.g., Di Mao., et al., AngewandteChemie International Edition; 9 January 2017; Ben-David, U., et al., Cell Stem Cell, 12, 167 (2013); WO2019 / 187918; JP 2016-93178 A; Yoshiki Nakashima, et al., Molecular Therapy Vol. 26 No. 7 July 2018, etc.).

[0098] Quality testing may also be performed as appropriate to determine whether the cells, organoids, etc. obtained by the production method of the present invention are desirable. The test items for quality testing are not particularly limited, but include basic tests such as the morphology of the cells or organoids, the presence or absence of expression of cell surface markers, sterility tests, endotoxin tests, and evaluation of cell viability, and testing devices suitable for each test item can be used.

[0099] In the examples shown below, steps (s1) to (s5) of the production method according to the present invention were carried out using the following reagents, equipment, and materials, and the resulting iPS cells and differentiated cells (monocytes) were evaluated. The closed system portion of the Prodigy used had a configuration similar to that shown in FIG. 1, with the main portion of the sealed chamber being made of polycarbonate resin. This closed system portion was attached to the mechanism shown in FIG. 3 and placed in an operable state as shown in FIG. 4. The operation of the Prodigy to carry out steps (s1) to (s3) is similar to steps (e1) to (e15) or steps (f1) to (f25) shown above as preferred examples of the operation of the Prodigy. Although the conditions, such as the materials and amounts supplied, differ in each example, the basic operation steps of the Prodigy are similar to the above steps (e1) to (e15) or (f1) to (f21). Furthermore, although the expansion culture step of the established iPS cells in step (s4) and the differentiation induction step in step (s5) differ in the conditions such as the materials and amounts supplied into the sealed chamber, the basic operation method of Prodigy is the same as (s1) to (s3).

[0100] Reagents Whole blood: Peripheral blood from healthy volunteer donors: Whole blood, received the day before the experiment and stored horizontally at room temperature, protected from light. Leukocyte reduction filter: Sepacell, RZ-2000N, Asahi Kasei Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) 2% HSA: CSL Behring, Albuminar 25% Intravenous Injection 12.5 g / 50 mL HSC Brew GMP Medium: MiltenyiBiotec, 170-076-310 Six cytokines used for reprogramming SCF: Recombinant human SCF (Fujifilm Wako Pure Chemical Industries, Ltd., 197-15511) TPO: Recombinant human TPO (Fujifilm Wako Pure Chemical Industries, Ltd., 207-17581) Flt-3L: Recombinant human Flt3L (Fujifilm Wako Pure Chemical Industries, Ltd., 061-05391) IL-6: Recombinant human IL-6 (Fujifilm Wako Pure Chemical Industries, Ltd., 098-06041) IL-3: Recombinant Human IL-3 (Fujifilm Wako Pure Chemical Industries, 090-05761) G-CSF: Recombinant human G-CSF (Fujifilm Wako Pure Chemical Industries, 072-06101) iMatrix-511: MATRIXOME iMatrix-322: MATRIXOME Peptide-containing acrylic resin with RGD sequence Synthemax II-SC Corning, 3535, 10mg Laminin 521: BLA-LN521-05 Human recombinant laminin 521 500μg SeV: SRVTM iPSC-4, Tokiwa Bio Co., Ltd. Equipped with GFP.StemFit (registered trademark) AK03N: Ajinomoto Healthy Supply Co., Ltd. TrypLE: TrypLE™ Select (1X) Gibco, 12563-029 0.5 mmol / L-EDTA / PBS solution: Nacalai Tesque, Inc. ROCK inhibitor: CultureSure 10 mmol / L Y-27632 solution, animal-derived-free, Wako Pure Chemical Industries, Ltd., 035-24593 StemSpan-AOF: Stemcell Technologies, British Columbia, Canada BMP4: 314-BP-010 VEGF: R&D Systems, Minneapolis, MN, 293-VE-050 CHIR: Miltenyi Biotec, 130-103-926 Essential 8 medium: Gibco, A1517001 Materials Blood bag (FLEXBOY (registered trademark) BAG 50 ml (LUER LOCKS, sartorius stedim Biotech) Reagent bag: MiltenyiBiotec, 20mL Regent Bag, 170-076-631 Terumo separation bag Microbeads: CliniMACS CD34 GMP MicroBeads, MiltenyiBiotec Equipment Cell processing equipment: CliniMACS Prodigy, MiltenyiBiotec Sterile splicer: TERUMO TSCD-II 5% CO. ２ Incubator: Panasonic Healthcare, MCO-170AICUVH-PJ Microscope: Olympus Corporation, inverted routine microscope Trinocular tube phase contrast set (precenter) CKX41 Automated cell counter: CountessII, Thermo Fisher Scientific Flow cytometry: SA3800 Spectral Cell Analyzer, Sony

[0101] Example 1 (1) Installation of the tubing set The day before SeV infection, the barcode of the Prodigy tubing set (type: TS730) was scanned, the ACC program was selected, and the operation was performed according to the operation screen. In a safety cabinet, the above tubing set, including the Prodigy sealed chamber, was manually checked for looseness or damage during transportation, and after retightening, it was assembled into the Prodigy.

[0102] (2) Preparation of Starting Material (PBMC Separation from Whole Blood) 70 ml of whole blood was manually aseptically filled into a blood bag in a safety cabinet. 150 ml of Ficoll-Paque was aseptically filled into a bag, and HSC Brew was aseptically filled into a bag filled with 2% HSA. These bags were manually aseptically connected to the Prodigy via tubing using a sterile connector and suspended from the Prodigy. The Adherent Cell Culture Density Gradient program (LP-34) was selected on the Prodigy screen, conditions were set and entered, and the program was started. Using this program, whole blood was transferred from the blood bag to the chamber, and then Ficoll was introduced from the suspended Ficoll bag into the chamber and centrifuged to obtain PBMCs. Using this program on the Prodigy, PBMCs were transferred from the chamber to the bag. The program was paused, and the PBMCs in the bag were manually transferred from the top opening of another bag equipped with a filter using a sterile transfer device (TERUMO TSCD-II), and debris was removed from the PBMCs through the filter in the bag. (3) During the PBMC incubation program, the chamber was washed with 500 ml of HSC Brew. The bag containing PBMCs suspended in 2% HSA HSC Brew was detached and transferred to a cell expansion bag containing 108 μl (20 mL) of six cytokines (SFC (final concentration 50 ng / mL), TPO (final concentration 10 ng / mL), Fit3L (final concentration 20 ng / mL), IL-6 (final concentration 50 ng / mL), IL-3 (final concentration 20 ng / mL), and G-CSF (final concentration 10 ng / mL)) in a safety cabinet. PBMCs were cultured in a CO2 incubator. The Prodigy chamber was washed using a program. The bag was manually filled with 0.35 mL x 3 tubes (3.5-52.5 volumes) of iMatrix-511 (MATRIXOME) and PBS / MgCl2 (25 mL) in a safety cabinet, and then aseptically connected to the Prodigy.iMatrix-511 and PBS / MgCl2 were injected into the chamber, and the chamber was precoated overnight at 22°C.

[0103] (4) SeV infection The next day, a reagent bag was filled with 8 ml of 2% HSA HSC brew, 0.3 ml of SeV, and 18 ml of six cytokines in a safety cabinet, and then aseptically connected to Prodigy. The bag containing PBMCs was connected to Prodigy. The coating solution was removed from the chamber. PBMCs (2 ml: 5 × 10 ) were added to the chamber by setting the conditions using the Activate Shaker type 1 and Deactive Shaker modules of the Prodigy's Activity Matrix program. 6 cells), and the culture medium containing SeV (8 ml) was added, and the cells were shaken (rotated) for 1 minute every 30 minutes and left to stand (37°C) for 2 hours for infection.

[0104] (5) Culture After SeV infection, 10 mL of 2% HSA HSC Brew was transferred using the Prodigy program (Culture module of the Adherent Cell Culture (ACC) System) to initiate culture (day 0 after establishment). On day 5 after establishment, 200 mL of StemFit® AK03N (Ajinomoto Healthy Supply Co., Inc.) and 200 μL of iMatrix-511 were filled into a Terumo separation bag in a safety cabinet. The HSC Brew bag was aseptically disconnected from the Prodigy, and the StemFit-containing bag was manually connected. 100 mL of StemFit was added to the chamber using the Prodigy program as a feed. The Prodigy program automatically discharged 50 mL of medium and added 20 mL of StemFit to the chamber for medium replacement. Culture was then continued. On day 7 after establishment, a Terumo isolation bag was manually filled with 200 mL of StemFit® AK03N and 200 μL of iMatrix-511 in a safety cabinet. The StemFit-containing bag previously connected to the Prodigy was manually detached using a heat sealer and replaced with a new Terumo isolation bag using a sterile connector. The medium was automatically changed by adding 100 mL of StemFit using the Feed command in the Prodigy program, discharging 129 mL of medium, and then adding 5 mL of StemFit. Culture continued. From day 7 after establishment, medium changes were automatically performed using the Prodigy program by removing a portion of the old medium from the chamber by centrifugation and adding new medium. Medium changes were performed every two days until day 16 after establishment.

[0105] (6) Harvesting. On day 19 after establishment, 25 ml of TrypLE™ Select (1X) mixed 1:1 with 0.5 mmol / L EDTA / PBS solution was added to the chamber and incubated at 39°C for 10 minutes to detach the cells from the chamber. The cells were seeded into a 48-well plate, and the detached and recovered cells were added to an appropriate amount of AK03N, a final concentration of 10 μM ROCK inhibitor, and 1 μl of iMatrix-511. The cells were then cultured in the 48-well plate. The next day, the medium was replaced with AK03N containing no ROCK inhibitor, and the ROCK inhibitor was removed. On day 25 after establishment, iPS cell colonies were observed under a microscope.

[0106] (7) Results: iPS cell colonies were observed under a microscope on the 23rd day after establishment, and 6.6 x 10 4 iPS cells were observed. Therefore, it was confirmed that the process of PBMC isolation from whole blood, SeV infection, and culture followed by iPS cell establishment could be performed consistently in a closed system within a single device. On the 24th day after establishment, 500 μL / well of AK03N medium was replaced. On the 26th day after establishment, 1.3 × 10 iPS cells were transferred from a 48-well plate to a 6-well plate. 4 The cells / well were divided into 1 well, and all remaining cells in each well were subcultured into 1 well.

[0107] Example 2 (1) Preparation A tubing set was attached in the same manner as in Example 1. A leukocyte removal filter was prepared and placed horizontally at room temperature, sealed and protected from light. In a safety cabinet, a 60 mL HSA bag was filled with 3 L of PBS / EDTA buffer, and a 20 mL HSA bag was filled with saline. Saline was run through the Prodigy using the Prodigy program.

[0108] (2) Isolation of CD34-positive cells from PBMCs. After whole blood was filtered through a leukocyte removal filter, the blood remaining on the filter was manually eluted with 50 mL of PBS (-) in a safety cabinet and separated with Ficoll to prepare PBMCs. The resulting PBMCs were 2 x 10 8The cells were manually mixed with 200 ml of 0.5% HSA / PBS(-) in a safety cabinet and used as the starting material. PBMC debris was manually removed as in Example 1. Using the Prodigy program "T cell transduction process," CD34-positive cells were magnetically separated using microbeads that had been left at room temperature beforehand. 40 ml of the resulting liquid containing CD34-positive cells was eluted using the program, and 4 ml of the liquid containing CD34-positive cells was aseptically extracted from the bag by manually squeezing a sampling bag that was part of the tubing set to reduce pressure. The extracted CD34-positive cells were counted using an automatic cell counter, yielding 1.52 x 10 5 The total volume was approximately 1.52 × 10 cells / 36 mL. 5 The cells were then subjected to infection with 1.5 ml of Sendai virus vector. The medium was replaced with StemSpan-AOF by programming.

[0109] After fixing the cells with DAPI, the nuclei were stained, and the percentages of CD45-positive cells and CD34-positive cells were examined by FACS. The results are shown in Table 1.

[0110]

[0111] Of these, 1% are CD34-positive cells, and the theoretical cell number is 1.52 × 10 3 cells were considered.

[0112] (3) SeV infection The chamber was aseptically removed and placed in a safety cabinet. The tubing set was reconnected from the TS730 to the T520, and the removed chamber was reconnected to the Prodigy in an aseptic manner. In the safety cabinet, 1 L of StemSpan was filled into a bag and aseptically connected to the Prodigy. Approximately 1.52 × 10 CD34-positive cells were obtained using the program. 3 The liquid containing cells was concentrated in the chamber until the internal volume reached 10 ml. The program then concentrated 5.1 x 10 SeV into the chamber. 71.5 mL of 1 / mL of PBS and 20 mL of StemSpan were added, bringing the total volume in the chamber to 20 mL. Using the spin module in the Prodigy's Activity Matrix program, the infection was carried out with centrifugation at 32°C for 2 hours. The program washed the culture twice using 600 mL of StemSpan, and then reduced the volume to 10 mL.

[0113] (4) Culture: StemSpan, six cytokines, and iMatrix-511 were simultaneously added to the chamber at a concentration of 1 μg / cm 2 The final volume was adjusted to 30 mL. 200 μL of iMatrix and 180 μL of the six cytokines were added to a final volume of 30 mL. 200 mL StemSpan + 200 μL iMatrix + 180 μL of the six cytokines were added as a feed, resulting in a final volume of 30 mL. - Day 1 after establishment: 20 mL of StemFit + 200 μL of iMatrix - Day 3 after establishment: 30 mL of StemFit was added as a feed to the chamber - Day 5 after establishment: 50 mL of StemFit was added as a feed to the chamber - Days 7, 9, 12, 14, 16, 19, 21, and 23 after establishment: 50 mL of StemFit medium was removed and then added, bringing the culture medium in the chamber to 130 mL.

[0114] (5) Harvesting On the 26th day after establishment, the tubing connected to the chamber was cut with a tube sealer and heat-sealed to aseptically separate the chamber from the Prodigy. A lid and a sterile filter were attached to the part of the chamber that came into contact with the outside, and the cells were observed under a microscope in a sterile environment with the chamber still in place.

[0115] (6) Results Aseptic observation of the cells under a microscope revealed the formation of numerous spheroids within the chamber. Counting with a tally counter or counter revealed approximately 308 spheroids 28 days after establishment. One-eighth of the resulting cell culture medium was taken and treated with TrypLE for 30 minutes to break down into single cells, and the number of cells was counted. The number of single cells in 1 / 8 of the culture medium was 1.26 x 106 cells, the obtained iPS cells were 1.0 × 10 7 cells, the number of viable cells in a single cell in 1 / 8 of the culture medium was 1.18 × 10 6 cells, the number of viable iPS cells obtained was 9.44 × 10 6 The viability of the cells was 94%.

[0116] (7) Differentiation Induction Using the protocol described in Noriko Shimasaki, et al., Cytotherapy 25 (2023) 1338-1348, the obtained iPS cells were induced to differentiate into monocytes using the Prodigy program (TCT, Empty Matrix) while maintaining a closed system in a Prodigy chamber and leaving the scaffold intact. Specifically, on the first day of differentiation, the AK03N medium was removed and Essential 8 medium supplemented with 160 ng / mL BMP4, 160 ng / mL VEGF, and 8 μM CHIR was added to the chamber. After 2 days, the medium was replaced with Essential 6 medium (Gibco) supplemented with 160 ng / mL VEGF, 100 ng / mL SCF, 100 ng / mL FGF-2 (MiltenyiBiotec, 130-093-841), and 4 μM SB431542 (1614 / 10, TOCRIS). Four days after initiation, the medium was replaced with StemPro-34 medium (Gibco, 10640-019) containing 80 ng / mL VEGF, 100 ng / mL SCF (Miltenyi Biotec), 20 ng / mL Tpo (288-TPN-025, R&D Systems), 100 ng / mL IL-3 (203IL-050, R&D Systems), and 100 ng / mL FLT-3 (308-FK-025 / CF, R&D Systems). Three days later, the medium was replaced with the same medium containing 100 ng / mL M-CSF (316-MC-100, R&D Systems) instead of VEGF. Ten days after the start of differentiation, the medium was replaced with StemPro-34 medium supplemented with 100 ng / mL FL, 100 ng / mL M-CSF, and 50 ng / mL GM-CSF (215-GM-050, R&D). An equal volume of fresh medium was added every two days. Monocytes were observed 10 days after the start of differentiation induction. Therefore, we confirmed that monocytes were obtained by performing a series of steps in the Prodigy system, including PBMC isolation from whole blood, SeV infection (spin infection), iPSC establishment, and monocyte differentiation, all within the same closed system.

[0117] (8) Evaluation of Monocyte Differentiation by Flow Cytometry On the final day of differentiation, cells were detached from the chamber using TrypLE Select and counted by trypan blue staining. The percentage of CD14-positive cells was analyzed by flow cytometry. The results show the expression of CD14 and CD45-positive cells, which are markers of monocytic differentiation (Figure 6). The percentage of monocytic differentiated cells was 48.2% of all live cells.

[0118] Example 3 (1) Preparation The procedure was the same as in Example 2, except that the tubing set was T520.

[0119] (2) Pre-coating of the scaffold material into the chamber The chamber was manually pre-coated with an acrylic resin containing a peptide having an RGD sequence in a safety cabinet. 2 PBS and iMatrix-511 (0.5 μg / μL) were mixed to prepare a 100 μL solution of iMatrix 511. The iMatrix-511 was allowed to warm to room temperature. A 40 mL solution was prepared containing 200 μL of iMatrix-511 and 200 μL of iMatrix-332. The Prodigy program was programmed to coat the chamber automatically with a coating volume of 25 mL, a coating temperature of 22°C, and a coating time of 24 hours. 45 mL of coating solution was then removed.

[0120] (3) Preparation of starting material: A leukocyte separation filter was manually washed with PBS in a safety cabinet, and PBMCs were separated from the resulting blood-containing liquid using Ficoll. The cells were counted to 1 × 10 8 The cells were to be infected with SeV, and the obtained PBMCs were manually aseptically loaded into a bag together with 100 mL of StemSpan in a safety cabinet. The bag was then aseptically connected to the Prodigy. The Prodigy program replaced the StemSpan. The PBMCs were concentrated using the program, and the volume of the chamber was adjusted to 10 mL.

[0121] (4) SeV infection 1×10 8 The procedure was the same as in Example 1, except that 1.5 mL of SeV was used and the total volume in the chamber was 20 mL.

[0122] (5) Cultivation Volume reduction was performed to reduce the culture medium to 10 mL. Preparations up to the start of culturing were the same as in Example 1. The following culturing steps were entered on the Prodigy screen, and culturing was performed automatically using the Prodigy program. Day 0 after establishment: Stemspan 20 mL + 6 cytokines 180 μL + iMatrix-511 200 μL + iMatrix-332 200 μL (iMatrix 1 μg / cm²), final volume 30 mL. Day 1 after establishment: StemFit AK03N 20 mL + iMatrix-511 200 μL + iMatrix-332 200 μL. Day 3 after establishment: 30 mL of StemFit AK03N was added to the chamber as a feed. Day 6 after establishment: 50 mL of StemFit AK03N was added to the chamber as a feed. From day 7 onwards, medium changes were performed by centrifuging the chamber to remove a portion of the old medium and adding new medium. Medium changes were performed every 2 or 3 days (chamber volume 30–130 mL). The cells were cultured in Prodigy from establishment until 13 days after establishment, and on day 14 after establishment, the chamber was aseptically removed from Prodigy, and the cells were continued to be cultured in a 5% CO 2 incubator.

[0123] (6) Harvesting The cells were aseptically removed from the Prodigy chamber without detaching them from the chamber, and the cells were observed under a microscope.

[0124] (7) Results: Microscopic observation 14 days after establishment revealed that spheroids were not observed in the chamber, but that many adherent colonies had formed. Counting with a tally counter or counter revealed approximately 284 colonies and 4.88 x 10 live cells 28 days after establishment. 7 It was cells.

[0125] Example 5: The same procedure as in Example 3 was repeated, except that twice the amount of scaffold material used in Example 3 was used for pre-coating, and no additional coating was applied. As a result, when observed under a microscope on day 18 after establishment, adherent colonies were observed to have formed in the chamber. Counting with a tally counter or counter revealed approximately 482 colonies on day 21 after establishment, and 6.4 x 10 live cells on day 28 after establishment. 7 cells were counted.

[0126] Example 7 (1) Preparation Preparation was carried out in the same manner as in Example 1, except that the TS730 and TS520 tubing sets were checked and tightened in a safety cabinet the day before, and the following buffers were prepared: 150 mL of Ficoll, 0.5% HSA PBS / EDTA: 60 mL of Albuminer / 3 L of PBS / EDTA, and 20 mL of 0.5% HSA / physiological saline: 1 L of physiological saline.

[0127] (2) Preparation of Starting Material Whole blood from a volunteer donor was filtered through a leukocyte removal filter, and the blood remaining on the filter (filtered blood) was eluted with 50 mL of PBS (-) to prepare a starting material. Separation was carried out in the same manner as in Example 1, except that 500 mL of 0.5% HSA / physiological saline was used.

[0128] (3) SeV infection The same chamber as that used in the separation process from whole blood to PBMC was used, and the tubing set was changed from TS730 to TS520. The chamber was washed with 0.5% HSA PBS / EDTA. 1 × 10 8 The cells were infected with SeV (MOI 5.1 × 10 7 1.5 mL of 1000mg / mL of SeV was used, and the SeV was diluted with 10 mL of StemSpan to a total volume of 20 mL. The "T cell culture 3" program was started. SeV infection was carried out in the same manner as in Example 1, except for the procedures described above. 20 mL of StemSpan was added as a feed. The mixture was concentrated, and the final chamber volume was adjusted to 10 mL.

[0129] (4) Culture The same procedure as in Example 2 was used except that when cells were seeded on day 0 after establishment, 200 μL of iMatrix-511 was added instead of 1 mL, and no scaffold material was added on day 1 after establishment. The medium was automatically changed using the Prodigy program on days 7, 9, and 11 after establishment. From day 15 onwards, the medium was changed manually.

[0130] (5) Harvesting The cells were not detached from the chamber, but were aseptically removed from the Prodigy along with the chamber on day 15 after establishment, and the cells were observed under a microscope.

[0131] (6) Results: Microscopic observation 29 days after establishment revealed the formation of spheroids in the chamber. Counting with a tally counter or counter revealed approximately 86 spheroids and 6.98 x 10 live cells. 6 cells were counted.

[0132] Example 8: Using filtered blood from the same donor as in Example 7, PBMCs were isolated from the filtered blood, infected with SeV, and cultured in the same manner as in Example 7, except that 200 μL of iMatrix was added on day 1 after establishment. As a result, microscopic observation on day 29 after establishment revealed the formation of spheroids in the chamber. Counting with a tally counter or counter revealed approximately 28 spheroids and 1.09 × 10 live cells on day 29 after establishment. 6 cells were counted.

[0133] Example 9 (1) Isolation of PBMCs from Whole Blood, SeV Infection, and Culturing Preparations were the same as in Example 7. PBMCs were isolated from whole blood in the same manner as in Example 1, except that 40 mL of peripheral blood (whole blood) from a volunteer donor and 40 mL of PBS were used as starting materials. SeV infection was carried out in the same manner as in Example 7. Culture was carried out in the same manner as in Example 1, except that 2 mL of iMatrix-511 was added instead of 1 mL when seeding the cells on day 0 after establishment, and that no scaffold material was added on day 1 after establishment. The cells were harvested without detaching them from the chamber.

[0134] (2) Results Microscopic observation 28 days after establishment revealed the formation of spheroids in the chamber. Photographs of spheroids on days 24 (Fig. 7) and 28 (Fig. 7) after establishment are shown. Counting with a tally counter revealed approximately 424 spheroids and 4.21 x 10 live cells on day 28 after establishment. 6 On day 28 after establishment, 2 mL of iMatrix332 was added, resulting in some adhesion. On day 31 after establishment, 3 mL of iMatrix332 was added, but no further adhesion occurred, and the adhesion rate was visually estimated at about 30%.

[0135] (3) Passage The cells were passaged 28 days after establishment.

[0136] (4) Flow cytometry The expression of GFP harbored in the vector and representative iPS cell markers SSEA4 and TRA-1-60 was confirmed at passages 1 and 3 (Figure 7). SSEA4 and TRA-1-60 were confirmed by immunostaining using antibodies. These results confirmed that iPS cells were established.

[0137] Example 10: Preparation was the same as in Example 7. Pre-coating of the scaffold material into the chamber was performed as in Example 5, preparation of the starting material (PBMC isolation from whole blood) was performed as in Example 9, SeV infection was performed as in Example 7, culture was performed as in Example 5, and harvesting was performed as in Example 7. As a result, colonies were observed in the chamber when observed under a microscope 21 days after establishment. Counting with a tally counter or counter revealed approximately 402 colonies and 5.48 x 10 live cells 21 days after establishment. 7 Photographs of the cells on days 12 and 16 after establishment are shown in Figure 8.

[0138] (2) Passage The cells were passaged 21 days after establishment.

[0139] (3) Flow cytometry The expression of GFP, SSEA4, and TRA-1-60 carried in the vector was confirmed one week after each passage at passages 1 and 3 (Figure 8). These results confirmed that iPS cells were established.

[0140] Example 12 (1) Isolation of PBMCs from Whole Blood, SeV Infection, and Culture PBMCs were isolated from whole blood, infected with SeV, and cultured in the same manner as in Example 9, except that 1 ml of iMatrix-332 was used as a scaffold material during culture. As a result, when observed under a microscope 28 days after establishment, spheroids were observed to have formed in the chamber. Counting with a tally counter or counter revealed approximately 555 spheroids (some colonies included) and 2.59 × 10 live cells 28 days after establishment. 6 cells were counted.

[0141] Example 13 (1) Isolation of PBMCs from Whole Blood, SeV Infection, and Culturing PBMCs were isolated from whole blood, infected with SeV, cultured, and harvested in the same manner as in Example 12, except that the starting materials were 20 mL of whole blood and 20 mL of PBS, and 2 mL of the scaffold material iMatrix-322 was added instead of 1 mL.

[0142] (2) Results: Microscopic observation 28 days after establishment revealed the formation of spheroids in the chamber. Counting with a tally counter or counter revealed approximately 518 spheroids (including some colonies) and 7.46 x 10 live cells. 6 Photographs of the cells on days 12 and 16 after establishment are shown in Figure 9.

[0143] (3) Passaging: Passaging was performed on day 21 after establishment. (4) Flow cytometry: The expression of GFP, SSEA4, and TRA-1-60 harbored in the vector was confirmed one week after each passage at passages 1 and 3 (Figure 9). These results confirmed that iPS cells had been established.

[0144] Example 14 (1) Isolation of PBMCs from whole blood, SeV infection, and culture The preparation was performed as in Example 9, pre-coating of the scaffold material inside the chamber was performed as in Example 5, preparation of the starting material (PBMC isolation from whole blood) and SeV infection were performed as in Example 13, and culture was performed as in Example 10. The cells were not detached from the chamber, and the harvested material was directly subjected to differentiation induction.

[0145] (2) Differentiation induction Differentiation induction was performed from day 21 after establishment using the same method as in Example 2. iPS cells were differentiated until day 17 after establishment. The cells were placed in the chamber and observed under a microscope under sterile conditions. As a result, monocytes were observed on day 17 and day 20 after the start of differentiation induction, as shown in Figure 10 and Figure 10, respectively. Therefore, it was confirmed that monocytes were obtained by performing a series of steps using Prodigy, including PBMC isolation from whole blood, SeV infection, iPSC establishment, and differentiation into monocytes, all while maintaining a closed system in the same chamber.

[0146] (3) Evaluation of Monocyte Differentiation by Flow Cytometry Differentiation was evaluated by flow cytometry as in Example 2. The expression of CD14 and CD45 positive cells, which are monocyte differentiation markers, is shown in Figure 10. The percentage of monocytes differentiated into 1.21% of all live cells.

[0147] (4) Differentiation of monocytes into macrophages. On day 20 after the start of differentiation induction, cells were seeded onto 6-well plates and cultured for 5-7 days in TexMACS medium (MiltenyBiotec) containing 50 ng / mM L-CSF to induce differentiation from monocytes into macrophages. FACS analysis revealed that the ratio of CD14-positive cells, a marker for M-CSF, to viable cells was 1.21% on day 5 and 15.4% on day 7 after M-CSF stimulation.

[0148] (5) Confirmation of macrophage phagocytic activity. Cells were seeded in a 96-well plate. Red fluorescent latex beads (L3030; Sigma-Aldrich) with an average diameter of 2 μm were diluted in 50 μL of TexMACS medium and added to the cells at a final concentration of 5 μL / mL. The cells were incubated at 37°C for 4 hours before harvesting. The harvested cells were washed with Cell Staining Buffer and labeled with an APC-conjugated anti-CD14 antibody (BD Pharmingen, San Diego, CA, USA). The cells were analyzed by flow cytometry to determine the percentage of CD14-positive cells that had taken up L3030, which reflects nonspecific phagocytic activity. Phagocytic activity was confirmed.

[0149] Example 15 (1) Isolation of PBMCs from whole blood, infection with SeV, and culture The number of cells used for SeV infection was 5 × 10 7 PBMCs were isolated from whole blood, infected with SeV, cultured, and harvested in the same manner as in Example 13, except that cells were used, spin infection was performed as in Example 2, and 5 mL of iMatrix-332 was used as the scaffold material for seeding the cells. As a result, when observed under a microscope 28 days after establishment, colonies and spheroids were observed to have formed in the chamber.

[0150] Example 16: As a preliminary step, a scaffold material was pre-coated inside the chamber. Specifically, 5 vials of Synthemax were aseptically filled into a bag and sealed in a safety cabinet. The bag was then aseptically connected to the Prodigy via tubing. The chamber was injected using the Prodigy program (the Coating module of the Adherent Cell Culture (ACC) System) and pre-coated overnight. PBMC isolation from whole blood, SeV infection, culture, and harvesting were performed in the same manner as in Example 15, except that the scaffold material iMatrix-332 was not added when the cells were seeded. As a result, colonies were observed to have formed in the chamber when observed under a microscope 21 days after establishment. Counting using a tally counter or counter revealed approximately 202 colonies and 1.74 × 10 live cells 21 days after establishment. 7 The cells were counted as 100 cells. Photographs of the cells on days 14 and 20 after establishment are shown in Figure 11. They were passaged on day 21 after establishment. The expression of GFP, SSEA4, and TRA-1-60 carried in the vector at passage 1 was confirmed one week after passage (Figure 11). These results confirmed that iPS cells had been established.

[0151] Example 17: After separating PBMCs from whole blood, the Tubing Set was transferred to the TS520, including the chamber, and Synthemax 3 vials were injected into the chamber. The chamber was then pre-coated at room temperature for 2 hours. The procedure was the same as in Example 16. Microscopic observation 21 days after establishment revealed colony formation within the chamber. Counting with a tally counter or counter revealed approximately 297 colonies and 1.18 x 10 live cells 21 days after establishment. 7 cells were counted.

[0152] Example 18: The same procedure as in Example 17 was repeated, except that the coating time was 30 minutes. 21 days after establishment, colonies were observed under a microscope. Counting with a tally counter or counter revealed that approximately 189 colonies and 1.37 x 10 live cells were found 21 days after establishment. 7 cells were counted.

[0153] Example 19: After Ficoll separation, 3 vials of Synthemax were coated on a TS520 chamber overnight at 22°C without pre-coating, and then transferred to the chamber after SeV infection. 7 The experiment was carried out in the same manner as in Example 17, except that the cells were infected with iPS cells. As a result, colonies of iPS cells were observed. The experiment was carried out twice, and the same results were obtained both times.

[0154] Example 22: The same procedure as in Example 17 was performed, except that 6 vials were coated with 500 μg of Laminin 521 overnight at 6°C. As a result, iPS cell colonies were observed. The results of the examples are summarized below (Tables 2 to 4).

[0155]

[0156]

[0157]

[0158] Examples 23 and 24 A method for coating a scaffold material was investigated by establishing iPS cells in the same manner as in Example 13, except for the conditions shown in Table 5 below.

[0159]

[0160] As a result of our investigation, we found that when the scaffold material was mixed with the cells and applied at the time of cell seeding, the iPS cells that were established had a 3D (spheroid) shape rather than a 2D shape. When the scaffold material was applied before cell seeding, the iPS cells that were established had a 2D (colony) shape. Furthermore, from the viewpoint of the amount of scaffold material used, it was suggested that applying the scaffold material before cell seeding was preferable (Figure 13).

[0161] Examples 25 to 27 The amount of Sev was examined by establishing iPS cells in the same manner as in Example 13, except for the conditions shown in Table 6 below.

[0162]

[0163] As a result of the investigation, it was found that even with the Sev amounts shown in Table 6, iPS cells can be sufficiently established using the production method of the present invention (FIG. 14).

[0164] Examples 28-30 (Summary) The process of monocyte differentiation using the cell processing device (Prodigy) was investigated. (1) The medium and cytokines are as shown in Tables 7-10. While it is possible to manually remove all of the medium and replace it with new differentiation medium, the specifications of the cell processing device (Prodigy) do not allow for complete removal of the medium. Therefore, the old medium was diluted with a large amount of base medium, the volume was reduced by centrifugation, and an equal volume of differentiation medium containing twice the amount of cytokines was added. This adjustment was made so that the medium in the chamber containing the cells would ultimately be 1x the volume of differentiation medium, completing the exchange from the establishment medium to the differentiation medium (Figure 15).

[0165]

[0166]

[0167]

[0168]

[0169] (2) The process is as follows: <Day 0> (i) 30ml AK03N <Feed> (ii) 200mL of Essential 8 was added (total 130mL) (iii) AK03N was diluted (iv) The volume was reduced to 10mL (AK03N was present at 3 / 23 of the 10mL) (v) This was repeated twice to dilute the AK03N as much as possible (vi) The volume was reduced to 10mL (vii) The amount of Essential 8 was reduced to 10mL <Feed> (viii) 10mL of Essential 8 containing 2x BMP4, 2x VEGF, and 2x CHIR was added to this (ix) This was mixed with 10mL of Essential 8 in the chamber, and the cells were immersed in Essential 8 medium containing 1x BMP4, 1x VEGF, and 1x CHIR (x) Differentiation into monocytes (iMonocytes) using a cell processing device (Prodigy) (similar to the differentiation induction protocol in Example 2)

[0170] Example 28: An RGD sequence-coated scaffold material was applied to the chamber (Prodigy chamber) of a cell processing device in a volume twice the usual amount. After infection with a Sendai virus vector, on day 21, without removing the chamber from the cell processing device (Prodigy), differentiation induction into monocytes was initiated within the chamber using the mechanism of the cell processing device (Prodigy). On day 20 after the start of differentiation induction into monocytes, cells (cell number 1.12 x 10 7 The FACS results were GFP: 88.3%, SSEA4: 29.2%, and TRA-1-60: 3.81% (Figure 16). Differentiation into macrophages was induced using 8x10 cells. 6 The cells were harvested on day 6 after the start of the experiment, and the cell count was 1.85 x 10 6The viability of the cells was 64%. The FACS results were GFP: 77.7%, SSEA4: 26.5%, and TRA-1-60: 8.64% (Figure 16). The percentage of live cells positive for CD14, a marker for M0 macrophages, was 15.4% on day 6 of culture in a 6-well plate after M-CSF stimulation.

[0171] Example 29: Synthemax was applied as a scaffold material to the chamber (Prodigy chamber) of a cell processing device. After infection with the Sendai virus vector, on day 28, differentiation induction into monocytes was initiated within the chamber using the mechanism of the cell processing device (Prodigy) without removing the chamber from the cell processing device (Prodigy). On day 20 after the start of differentiation induction into monocytes, cells (cell number 5.43 x 10 7 Viability: 86% (cells). Expression of CD14 and CD45-positive cells, markers of monocytic differentiation, indicated that 6.85% of all live cells were monocytic (Figure 17). FACS analysis revealed that the GFP+ (62.5%) cell group, in which reprogramming factors remained, had very low CD14+ (0.077%) and were difficult to differentiate into monocytes (Figure 17). On the other hand, the GFP- (34.8%) cell group, in which no reprogramming factors remained, differentiated into monocytes (CD14+ (18.4%)), indicating that the cells differentiated into monocytes. This result is considered reasonable (Figure 17). These results suggest that the timing of induction initiation is important.

[0172] Example 30: The cells of Example 29 were seeded again in the chamber used in Example 29, and macrophage differentiation was carried out. Based on the expression of CD14 and CD45 positive cells, which are markers of monocyte differentiation, the percentage of macrophage differentiated cells was 71.9% of all live cells ( FIG. 18 ). Differentiation into macrophages was induced using a cell count of 5.32×10 7 The cells were harvested on day 6, and the cell count was 9.8 x 10 6The viability was 62%. When GFP was examined by FACS, the GFP+ cell group, which had remaining reprogramming factors, was very low at 1.17%, while the GFP- cell group, which had no remaining reprogramming factors, was 95.1%. The CD14+ cell group was 66.4%, indicating that the cells had differentiated into macrophages (Figure 18).

[0173] The manufacturing method of the present invention makes it possible to perform the entire process from centrifuging whole blood to establishing iPS cells in a single closed chamber using an automated cell processing device, thereby reducing the cost of producing iPS cells.

[0174] This application is based on patent application No. 2023-204958 filed in Japan (filing date: December 4, 2023), the contents of which are incorporated in their entirety herein.

[0175] 100 Sealed chamber 210-250 Sealed container for supplying material A10 Connecting pipe V10 Pinch valve F10 Peristaltic pump

Claims

1. A method for producing induced pluripotent stem cells using a cell processing apparatus, the cell processing apparatus having a closed system portion in which a sealed container for supplying materials and a rotatable sealed chamber for performing centrifugation are connected via a connecting pipe, the method for producing induced pluripotent stem cells comprising the steps of: (s1) centrifuging blood cells from whole blood within the rotatable sealed chamber while maintaining the closed nature of the closed system portion; (s2) contacting the blood cells with reprogramming factors; and (s3) establishing induced pluripotent stem cells from the blood cells, in that order.

2. The method for producing artificial pluripotent stem cells described in claim 1, wherein the sealed chamber has an inlet port and an outlet port, and is configured to allow materials to flow in and out through the inlet port and outlet port while the chamber is rotating for centrifugation or while the rotation is stopped, and in step (s1), a fraction containing the centrifuged blood cells is left in the sealed chamber, and other fractions are discharged outside the sealed chamber.

3. The method for producing artificial pluripotent stem cells described in claim 2, wherein the cell processing apparatus further has, as part of the configuration of the closed system portion, a magnetic separation column connected to the sealed chamber via a connecting pipeline, and in the step (s1), after the centrifugation, a fraction containing blood cells is left in the sealed chamber, lymphatic cells among the blood cells are further labeled with magnetic beads and sent to the magnetic separation column where they are magnetically separated from other blood cells, and the lymphatic cells labeled with the magnetic beads are returned to the sealed chamber.

4. A method for producing artificial pluripotent stem cells described in any one of claims 1 to 3, wherein step (s2) is carried out with the sealed chamber rotating or stationary, or in a combination of these states.

5. The shape of the internal space defined by the side wall surface surrounding the internal space of the sealed chamber is cylindrical, the geometric center line of rotation of the cylinder coincides with the central axis of the rotational motion of the sealed chamber during centrifugation, and the base area of ​​the cylinder is 90 to 200 cm ２ The method for producing artificial pluripotent stem cells according to any one of claims 1 to 4, wherein a relative centrifugal force acting on the contents in the sealed chamber during centrifugation is 100 to 500 G.

6. A method for producing artificial pluripotent stem cells described in any one of claims 1 to 5, wherein the volume of the sealed chamber is 300 to 700 ml.

7. A method for producing artificial pluripotent stem cells described in any one of claims 1 to 6, wherein the centrifugation in step (s1) is density gradient centrifugation performed by adding a centrifugation medium for density gradient to the sealed chamber.

8. A method for producing artificial pluripotent stem cells described in any one of claims 1 to 7, wherein at least the inner wall surface of the sealed chamber has a polycarbonate resin portion.

9. A method for producing artificial pluripotent stem cells described in any one of claims 1 to 8, wherein steps (s2) and (s3) are carried out in the presence of a scaffold material.

10. The method for producing artificial pluripotent stem cells described in claim 9, wherein step (s1) is carried out in the presence of a scaffold material.

11. A method for producing artificial pluripotent stem cells as described in claim 9 or 10, wherein a scaffold material is adhered to the inner wall surface of the sealed chamber.

12. A method for producing artificial pluripotent hepatocytes described in any one of claims 9 to 11, wherein the scaffold material comprises laminin fragments, a cell adhesive peptide and a polyvinyl acetal resin, or a cell adhesive peptide and a poly(meth)acrylate resin.

13. The method for producing artificial pluripotent hepatocytes according to claim 12, wherein the cell adhesive peptide comprises Arg-Gly-Asp.

14. A method for producing artificial pluripotent stem cells described in any one of claims 1 to 13, further comprising, after step (s3), a step (s4) of expanding and culturing the artificial pluripotent stem cells in the sealed chamber while maintaining the closed nature of the closed system portion.

15. A method for producing differentiated cells, comprising steps (s1) to (s3) of the method for producing induced pluripotent stem cells described in any one of claims 1 to 13, and further comprising, after step (s3), a step (s5) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closed nature of the closed system portion, wherein in step (s5), materials necessary for differentiation induction are supplied into the sealed chamber, and step (s5) is carried out in the sealed chamber.

16. The method according to claim 15, further comprising a step (s4) between steps (s3) and (s5) of expanding the induced pluripotent stem cells in the sealed chamber while maintaining the closed nature of the closed system portion.

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