Method for producing induced pluripotent stem cells

Abstract

The present invention provides a method for producing iPS cells using a cell processing device having a closed system in which a sealed container for supplying materials and a rotatable sealed chamber for performing centrifugation are connected via a connecting pipeline, and the method involves carrying out the following steps in that order within the rotatable sealed chamber while maintaining the closed nature of the closed system: centrifuging blood cells from whole blood; contacting the blood cells with reprogramming factors; and establishing iPS cells from the blood cells.

Description

[Technical Field]

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

[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 that establishes iPS cells from a patient's somatic cells (e.g., peripheral blood mononuclear cells) and then transplants various differentiated cells or organoids differentiated from the iPS cells 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. Patent Document 2 (JP 2022-8735 A) discloses a cell production system and method that can reduce costs by reducing the number of steps in a treatment method using CAR-T cells. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2017-195905 [Patent Document 2] Japanese Patent Publication No. 2022-8735 [Patent Document 3] International Publication No. 2020 / 040135 [Patent Document 4] Japanese Patent Publication No. 2020-182488 [Patent Document 5] Japanese Patent Application Laid-Open No. 2014-018185 [Patent Document 6] Special Publication No. 2011-505890 [Non-patent literature]

[0004] [Non-Patent Document 1] Shinsuke Yoshida., et al., CLINICAL AND TRANSLATIONAL RESOURCE AND TECHNOLOGY INSIGHTS VOLUME 4, ISSUE 1, P51-66.E10, JANUARY 13, 2023 [Non-patent document 2] Madrid, M., et al., Current Protocols,1, e88. doi: 10.1002 / cpzl.88 Summary of the Invention [Problem to be solved by the invention]

[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-step, lengthy processing process, and the cost 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, expanding them, and forming 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 been no cell processing device optimized to automatically and consistently perform all of the above multi-step processes, and there have been no attempts to make existing cell processing devices perform these multi-step processes.

[0007] For example, Patent Document 3 (International Publication No. 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 centrifuge chamber equipped 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. Furthermore, Patent Document 4 does not describe adherent culture, which is useful for culturing iPS cells, which are adherent cells. Patent Document 5 (JP 2014-018185 A) describes the adherent culture of iPS cells in a closed system, but does not describe the isolation of somatic cells from whole blood in the same container within the same closed system and the establishment of iPS cells from the somatic cells.

[0008] Meanwhile, some cell processing manufacturers have proposed cell processing devices that can automatically perform sequential cell processing while maintaining a closed system (e.g., the CliniMACS Prodigy (registered trademark) manufactured by Miltenyi Biotec). Figure 12 is a photograph showing an example of such a cell processing device in use. The cell processing device illustrated in Figure 12 has a closed system portion for processing cells. In this closed system portion, 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, thereby enabling control of pipeline connection and cutoff and material feed while maintaining a closed system. Furthermore, the sealed chamber X10 is configured to be rotatable for centrifugation while the connecting pipeline remains connected.

[0009] However, conventional cell processing using a cell processing device such as that shown in Figure 12 (especially the CliniMACS Prodigy) has not yet demonstrated a procedure for consistently performing the multi-step process required for the production of iPS cells as described above. Furthermore, the centrifugal separation function of the sealed chamber in conventional cell processing devices is intended for use at the end of the processing process to separate the processed cells and harvest them. Furthermore, the material constituting the sealed chamber in the CliniMACS Prodigy is polycarbonate resin, which provides increased mechanical strength for the centrifugal separation function and makes the walls more transparent for observing the interior. 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. [Means for solving the problem]

[0011] The main configuration of the present invention is as follows. [1] A method for producing induced pluripotent stem cells using a cell processing device, the cell processing device has a closed system portion in which a sealed container for supplying material and a rotatable sealed chamber for performing centrifugation are connected via a connecting pipe line; The manufacturing method includes, while maintaining the closed system portion closed, A step (s1) of centrifuging blood cells from whole blood; a step (s2) of contacting the blood cells with a reprogramming factor; and (s3) a step of establishing induced pluripotent stem cells from the blood cells, The method for producing induced pluripotent stem cells is carried out in this order. [2] The sealed chamber has an inlet port and an outlet port, and is configured to allow a material to flow in and out through the inlet port and the outlet port while the chamber is rotating for centrifugation or while the rotation is stopped; In the step (s1), a fraction containing centrifuged blood cells is left in the sealed chamber, and other fractions are discharged to the outside of the sealed chamber. [1] A method for producing induced pluripotent stem cells. [3] The cell processing apparatus further includes a magnetic separation column connected to the sealed chamber via a connecting pipe as part of the closed system portion; 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, sent to the magnetic separation column, and magnetically separated from other blood cells; The magnetic bead-labeled lymphoid cells are returned to the sealed chamber. [2] A method for producing induced pluripotent stem cells. [4] A method for producing artificial pluripotent stem cells according to any one of [1] to [3], wherein step (s2) is carried out with the sealed chamber rotating or stationary, or 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, and the geometric rotation center line of the cylinder coincides with the central axis of rotational motion of the sealed chamber during centrifugation; The cylindrical base area is 90 to 200 cm 2 and During centrifugation, the relative centrifugal force acting on the contents in the sealed chamber is 100 to 500 G. A method for producing induced pluripotent stem cells according to any one of [1] to [4]. [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] A method for producing artificial pluripotent stem cells described in 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 carried out in the presence of a scaffold material.

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

[11] The method for producing induced pluripotent stem cells described in [9] or

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

[12] The artificial pluripotent stem cell 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. stem A method for producing cells.

[13] The artificial pluripotency method according to

[12] , wherein the cell adhesive peptide comprises Arg-Gly-Asp. stem A method for producing cells.

[14] A method for producing artificial pluripotent stem cells described in any one of [1] to

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

[15] A method for producing differentiated cells, comprising: The manufacturing method includes: [1] to

[13] , the method for producing induced pluripotent stem cells comprising steps (s1) to (s3), and After the step (s3), the method further comprises a step (s5) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closed system portion; In the step (s5), a material necessary for differentiation induction is supplied into the sealed chamber, and the step (s5) is carried out in the sealed chamber. The manufacturing method.

[16] The method described in

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

[0012] In the production method of the present invention, a closed-system cell processing device with a sealed chamber equipped with a centrifugal separator is used as a container for processing cells. In the production method of the present invention, this centrifugal separator is used at the beginning of the entire processing process to separate blood cells, the source of iPS cells, from whole blood by centrifugation. 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 to upgrade facilities to Grade A, thereby reducing the cost of iPS cell production. [Brief explanation of the drawings]

[0013] [Figure 1]Figure 1 is a block diagram showing an example of the configuration of the closed system portion of the cell processing apparatus used in the manufacturing method of the present invention. Each thick black line designated by the symbol A10 represents a connecting pipeline such as a flexible tube (the same applies to Figure 5). In addition, to clearly show the movement and stoppage of materials within the closed system portion, pinch valves that open and close the connecting pipelines and a peristaltic pump that sends materials are also drawn on the connecting pipelines. These pinch valves and peristaltic pumps are located outside the connecting pipelines and do not belong to the closed system portion, but belong to the mechanical portion of the cell processing apparatus (the same applies to Figure 5). [Figure 2] FIG. 2 is a schematic diagram showing the state of centrifugation in the closed chamber of the cell processing device used in the production method of the present invention, showing the inside of the closed chamber. [Figure 3] FIG. 3 is a photograph illustrating the mechanical parts of the cell processing device used in the production method of the present invention. [Figure 4] FIG. 4 is a photograph illustrating a state in which a closed system part is attached to a mechanism part of a cell processing device in the present invention. [Figure 5] FIG. 5 is a block diagram showing another example of the configuration of the closed system part of the cell processing device used in the production method of the present invention. [Figure 6] FIG. 6 shows the results of expression of CD14 and CD45 positive cells, which are markers of monocyte differentiation, in Example 2. [Figure 7] Figure 7 shows microscopic images taken 24 and 28 days after establishment of iPS cells in Example 9, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 carried in the vector at passages 1 and 3, one week after each passage. [Figure 8] Figure 8 shows microscopic images taken on days 12 and 16 after the establishment of iPS cells in Example 10, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 carried in the vector at passages 1 and 3, one week after each passage. [Figure 9]Figure 9 shows microscopic images taken on days 21 and 28 after the establishment of iPS cells in Example 13, and the results of confirming the expression of GFP, SSEA4, and TRA-1-60 carried in the vector at passages 1 and 3, one week after each passage. [Figure 10] FIG. 10 shows microscopic images of monocytes induced to differentiate in Example 14 (17th and 20th days after differentiation induction), and the results of expression of CD14- and CD45-positive cells, which are monocyte differentiation markers. [Figure 11] Figure 11 shows microscopic images taken 14 and 20 days after 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] FIG. 12 is a photograph showing an example of a conventional cell processing device. [Figure 13] FIG. 13 shows the results of investigation into the coating methods for the scaffold materials in Examples 23 and 24. [Figure 14] FIG. 14 shows the results of examining the amount of Sendai virus vector (SeV) in Examples 25 to 27. [Figure 15] FIG. 15 shows an overview of medium exchange in the process of monocyte differentiation examined in Examples 28-30. [Figure 16] FIG. 16 shows the results of investigation into the process of monocyte differentiation in Example 28. [Figure 17] FIG. 17 shows the results of investigation into the process of monocyte differentiation in Example 29. [Figure 18] FIG. 18 shows the results of investigation into the process of monocyte differentiation in Example 30. DETAILED DESCRIPTION OF THE INVENTION

[0014] The method for producing iPS cells according to the present invention will be described in detail below. This production method is a method for producing iPS cells using a cell processing apparatus. The cell processing apparatus has a closed system portion as shown in Figure 1. The closed system portion is configured such that a sealed container for supplying materials (parts designated by reference numerals 210 to 250 in the example shown in the figure) 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): A step of centrifuging blood cells from whole blood. Step (s2): A step of contacting the blood cells with a reprogramming factor. Step (s3): A step of 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 in the closed system portion 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 blood cells may be returned to the closed system portion for the next step. These additional processing steps 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 portion according to instructions from a control unit of a 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 equipment) First, the configuration of each part of a cell processing device 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 Figure 12. The cell processing apparatus has a closed system part as exemplified in Figure 1 and a mechanical part as exemplified in Figure 3. The closed system part is attached to and detached from the mechanical part in a disposable manner. Figure 4 shows an example of the state in which the closed system part is attached to the mechanical part. The mechanical part operates automatically 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 CliniMACS Prodigy (registered trademark) manufactured by Miltenyi Biotec, which is also shown in Figure 12. 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 manufacturing method will be described with reference 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, 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 shuts off the connecting pipe (soft tube) of the closed system portion by pressing the connecting pipe from the outside, and opens the connecting pipe by releasing the pressure. The peristaltic pump F10 is also called a tube pump or a peristaltic pump, and is a pump that moves a fluid in a connecting pipe line (soft tube) by moving an outer pressing part against the pipe line. The pinch valve V10 and the peristaltic pump F10 enable the internal material to be moved from the outside as intended while maintaining the closed nature of the closed system portion.

[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 program, 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 part: sealed container for supplying materials) The number of sealed containers for supplying materials may be equal to 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 about 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 the material 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 below.

[0022] The configuration of the sealed container for material supply 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 may be gas-permeable, allowing the permeation of O2 and CO2 necessary for cell culture. In Prodigy, the required number of bags, which are sealed containers for material supply, are hung from the support B10 shown in Figure 3, and are in the usage state shown in Figure 4.

[0023] (Closed system part: sealed chamber with centrifugal separator function) The sealed chamber 100 is configured so that rotation for centrifugation is possible while maintaining a closed system, i.e., while the connecting pipe A10 remains connected and even while material is being introduced or extracted. 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 materials.

[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) FIG. 2 is a schematic diagram illustrating 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 FIG. 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 this. 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 materials 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 materials 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 fraction K11, which mainly contains the centrifugation medium; fraction K12, which mainly contains peripheral blood mononuclear cells (PBMCs), which are blood cells; and fraction K13, which mainly contains red blood cells. While maintaining this separation state (i.e., while the sealed chamber continues to rotate), fraction K11 is discharged from the bottom opening 100a, fraction K13 is discharged from the top opening 100b, and fraction K12, which mainly contains 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. Also, sealed chambers of various capacities may be prepared depending on the scale of processing, such as preparing 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 part: connecting pipe) The tubing that constitutes the connecting pipeline A10 is preferably made of a soft material (such as silicone or vinyl chloride) because the Prodigy uses the pinch valve and peristaltic pump described above. The connecting pipeline may also include tubing made of a hard material at joints and other portions.

[0031] (occlusive or hermetic) The closed or hermetic nature of the interior of a closed system, sealed container, or sealed chamber refers not only to an airtight or liquidtight state of the interior, but also to a state in which the interior is isolated from the outside world to the extent that microorganisms and viruses cannot enter from the outside, i.e., to the extent that sterility of the interior is maintained. For example, a sealed chamber equipped with a porous filter (e.g., pore size of approximately 0.2 μm or less, particularly approximately 0.1 to 0.2 μm) that is impermeable to bacteria and viruses but allows fluids (e.g., gases) to pass through is considered hermetic. Outside air can pass through the porous filter and enter the sealed container, but bacteria and viruses cannot, thus maintaining the sterility of the closed system. A container whose wall is made of a gas-permeable membrane that does not allow bacteria or viruses to pass through but allows O2 gas molecules and CO2 gas molecules to pass through is also considered hermetic. Thus, a "closed system" is not limited to an airtight or liquidtight system, but refers to a system in which the interior is isolated from the outside world to the extent that microorganisms and viruses cannot enter from the outside.

[0032] Next, we will explain the iPS cells to be produced and 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 have the ability to differentiate into various tissues and cells with different morphologies and functions in the body, and into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm).

[0034] As used herein, iPS cells may be 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 other components have not been separated. Furthermore, as used herein, "whole blood" may include whole blood diluted by mixing with an appropriate buffer or the like, or blood containing additives such as blood coagulation inhibitors (e.g., heparin, EDTA, citric acid, etc.) or protease inhibitors. 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 include phosphate buffer solution (PBS), physiological saline, etc.

[0038] (whole blood supply) In the present invention, the amount of whole blood to be 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 the above-mentioned buffer solution or the like and then 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] As used herein, 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 cell-containing solution, washing the cells, and isolating the target cells from a cell-containing solution. It also means subjecting cells to chemical treatments, altering their biological properties, combining them with non-cellular components, or genetic engineering for the purpose of artificially increasing or differentiating the cells, establishing a cell line, or activating the cells.

[0044] (liquid medium) The liquid medium delivered from the sealed container can be used not only for culturing cells 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 8TM Medium, Essential 8 TM Medium, Essential 8 TM Flex Medium, Essential 6 TM Examples of suitable media include StemFit® AK02 Medium (Thermo Fisher Scientific), StemFit® 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 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] (Scaffolding materials) As used herein, 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 adherent culture or suspension culture using the scaffold material (i.e., it may be free in the medium), but examples include those containing or made of synthetic resin, and those made of flexible materials such as collagen. Microcarriers may also be used as scaffold materials. As an example, the scaffold material may contain atelocollagen. The scaffold material may be produced by known methods, or commercially available products may be used. Examples of commercially available products include Cytodex-1 (manufactured by GE Healthcare) and Corning® Low Concentration Synthemax® II Microcarriers (manufactured by Corning).

[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) or Geltrex matrix (manufactured by Thermo Fisher Scientific)), fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, vitronectin, or a combination thereof. The exemplified extracellular matrices may be natural products, artificially synthesized using genetic engineering techniques, fragments obtained by cleavage with restriction enzymes, or synthetic proteins or 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 with isoforms that differ in the composition of the subunit chains. 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] Examples of laminin or fragments thereof used in the present invention include laminin-111 and fragments containing its E8 region (e.g., iMatrix-111), laminin-211 and fragments containing its E8 region (e.g., iMatrix-211), laminin-121 or fragments containing its E8 region, laminin-221 or fragments containing its E8 region (e.g., iMatrix-221), laminin-332 or fragments containing its E8 region (e.g., iMatrix-332), laminin-3A11 or fragments containing its E8 region, laminin-411 or fragments containing its E8 region (e.g., iMatrix-411), laminin-421 or fragments containing its E8 region, and laminin-511 or fragments 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 using genetic recombination technology, or a synthetic protein or peptide based on vitronectin. Commercially available products include human plasma-derived vitronectin (Sigma-Aldrich), human recombinant vitronectin (20-398aa), 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. 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 products 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, International Publication No. 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, International Publication No. 2019 / 131982, and International Publication No. 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 embodiment, 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 analysis, or plate coating. The cell adhesive peptide is preferably a peptide consisting of at least three amino acids, more preferably 3 to 20 (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, and particularly preferably 3 to 10. 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 a 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 coated in the sealed chamber by being added to a culture medium used in at least one of steps (s1), (s2), (s3), (s4), and (s5). In another embodiment, the scaffold material may be added to a culture medium used in at least one of steps (s1), (s2), (s3), (s4), and (s5) and then supplied into the sealed chamber.

[0062] (Centrifugation media) 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 to the sealed chamber from a sealed container for supplying materials. The centrifugation medium is typically a hydrophilic polysaccharide (Ficoll TM etc.) are examples.

[0063] During 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] (initialization factor) In step (s2), the pinch valve and peristaltic pump are activated to send a fluid containing the 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, rotating, or 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 somatic cells may be in the form of a protein, a nucleic acid (RNA or DNA) encoding the protein, or an expression vector containing the nucleic acid. When the reprogramming factor is introduced in the form of RNA, immunogenic RNA introduced into cells may activate the cellular defense mechanism, so RNA for circumventing the defense mechanism may be introduced into somatic cells.

[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 that make up the sealed chamber and cell culture) The material of the sealed chamber of Prodigy includes a portion made of polycarbonate resin. The main portion of the actual internal space of the sealed chamber (the internal sidewall surface and the internal bottom surface) is made of polycarbonate resin, which makes these surfaces non-cell-adhesive and unsuitable for culturing iPS cells, which are adherent cells (adherent culture using the internal 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 carried out in the presence of the scaffold material. Furthermore, the scaffold material may be supplied to the sealed chamber at the stage of step (s2) for the purpose of simplifying 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 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 10 ml of powdered Synthemax in 1 vial (10 ml) of sterile water in a safety cabinet, sealing the container in an airtight container, removing the container from the safety cabinet, connecting it to the Prodigy aseptically, and pumping it into a sealed chamber using a peristaltic pump. The container can then be pre-coated overnight at 22°C. When only a small amount of scaffold material is used, it can be mixed with PBS or sterile water to facilitate delivery. Liquid iMatrix can be supplied as is.

[0072] (Other materials to be supplied) In addition to the materials described above, substances necessary for cell processing may be added as appropriate. The added materials, like the materials described above, can be housed in a sealed container or the like and connected to the sealed chamber. Examples of other materials that may be added include liquids, powders, additives, release agents, cryoprotectants, CO2, etc.

[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. The establishment of induced pluripotent stem cells can be appropriately confirmed by the expression of reprogramming factors introduced by methods known per se (e.g., Oct3 / 4, SOX2, Nanog, TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase, etc.). The period for performing step (s3) 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) to (e15). (e1) In FIG. 1, diluted whole blood (for example, 20 ml of whole blood diluted with 30 ml of phosphate buffered saline (PBS)) contained in a sealed container 210 for supplying material is sent to the sealed chamber 100. (e2) Ficoll, a density gradient centrifugation medium contained in a 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 transferred 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 Prodigy. (e5) The sealed container 250 and another sealed container with a built-in filter are aseptically joined, and the PBMCs contained in the sealed container 250 are placed into the other sealed container with a built-in filter from the top opening, and 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 number of cells 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 scaffolding material (such as Synthemax) is pumped from a sealed material supply container into the sealed chamber 100, and the inner wall surface of the sealed chamber is coated with the scaffolding material. (e8) The sealed container incorporating the filter is manually and aseptically connected to the closed system portion, and the fraction mainly containing the 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) PBMCs are exposed to Sendai virus vectors in a sealed chamber (2 hours). (e10) A liquid medium is added to the sealed chamber from the sealed container 220 for supplying materials. (e11) A portion of the contents in the sealed chamber is manually sampled and the number of cells is counted. This step (e11) is not essential to the present invention, but is a step that is preferably carried out. (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) In some cases, at this point, scaffold material is supplied from a sealed container for supplying materials to the sealed chamber 100 and mixed by rotating the sealed chamber (if coating is to be performed on the inner wall surface of the sealed chamber, the mixing conditions are 37°C and approximately 30 to 60 minutes). (e14) Replace or add liquid medium in the sealed chamber. (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 culture) 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 among the liquid media described above. In one embodiment, when expanding the established induced pluripotent stem cells, a factor for maintaining undifferentiated states can be added. As used herein, the term "factor for maintaining undifferentiated states" refers to a substance that inhibits differentiation of induced pluripotent stem cells, and is not particularly limited as long as it is such a substance. Examples of factors for maintaining undifferentiated states commonly used by those skilled in the art include bFGF, FGF2, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, and TGFβ2. The undifferentiated state maintenance factor used in the present invention is preferably isolated. "Isolated" means that it has been subjected to a procedure to remove the target component and factors other than the cells, and has been released from the 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 typical processing procedures, it is preferably performed 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 process) 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 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 section 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 sealed container 260 for supplying materials. 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, magnetic bead peeling) 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. The method for detaching the magnetic beads may be such that the magnetic beads are naturally released during cell culture. Materials for modification with magnetic beads and their removal may be appropriately fed into the sealed chamber from sealed containers for supplying materials that 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 Prodigy magnetic separation program "T cell transduction process." (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 a sealed container 210 for supplying material is sent to the sealed chamber 100. (f2) Ficoll, a density gradient centrifugation medium contained in a sealed container 230 for supplying materials TM 150 ml is delivered to the sealed chamber 100 . (f3) The sealed chamber 100 is rotated, density gradient centrifugation is performed, and a fraction containing mainly PBMCs is left in the sealed chamber. (f4) The fraction containing mainly PBMCs is transferred to an empty sealed container 250. (f5) The fraction is sent to a labeling column (not shown) in 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) In the sealed chamber, washing is carried out to remove excess magnetic beads. (f8) Lymphoid cells (with magnetic beads) are sent to a magnetic separation column and magnetically trapped within the magnetic separation column. (f9) Washing in the magnetic separation column. (f10) The magnetic application of 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, stained with trypan blue (filtered Gibco Trypan Blue Strain (0.4%), 15250-061), and the number of cells is counted 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 scaffolding material (such as Synthemax) is pumped from a sealed material supply container into the sealed chamber 100, and the inner wall surface of the sealed chamber is coated with the scaffolding material. (f14) A fluid containing a Sendai virus vector as a reprogramming factor is sent to the sealed chamber 100 from a sealed container for supplying materials. (f15) In a sealed chamber, lymphoid cells (with magnetic beads) are contacted with Sendai virus vectors (for 2 hours). (f16) A liquid medium is added to the sealed chamber from a sealed container for supplying materials. (f17) A portion of the contents in the sealed chamber is sampled and the number of cells is measured. (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) In some cases, 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) Replace or add liquid medium in the sealed chamber. (f21) 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.

[0080] (Quality assessment 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, the 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, with the addition of a step (s5) of inducing differentiation of iPS cells after these steps. Like steps (s1) to (s3) and step (s4), step (s5) is performed while maintaining 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 performed within the sealed chamber. The sealed container for supplying 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" is sometimes 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 yet reached terminal differentiation in a cell lineage, and examples of undifferentiated cells include stem cells excluding pluripotent stem cells, progenitor cells, and the like. Examples of stem or progenitor cells include 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, hepatocytes, 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, "differentiation inducer" refers to a substance that can induce the differentiation of induced pluripotent stem cells into the above-mentioned differentiated cells or organoids. Differentiation inducer may be a known substance, or may be selected from those that are commonly used to induce the differentiation of desired differentiated cells or organoids. Specifically, the following substances may be mentioned.

[0087] The fluid used as a medium for containing the reprogramming factors, undifferentiated maintenance factors, and differentiation-inducing substances is not particularly limited, but preferred examples include buffer solutions, culture media, and 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 to induce differentiation to obtain the desired cells or organoids. For example, differentiation of pluripotent stem cells into neural crest cells can be performed by the methods described in Fukuta M. et al., PLoS One, 2014, 9(12): e112291 or Kamiya D, et al., NPJ Regen Med., 2022 Sep 15;7(1):47. Specifically, pluripotent stem cells can be seeded in a culture vessel and subjected to adhesion culture (suspension culture using a scaffold material), 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 according to 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, neural crest cells can be seeded onto a fibronectin-coated plate, replaced with DMEM / F12 supplemented with N-2 Supplement, BDNF, GDNF, NT-3, and NGF, and cultured at 37°C under 5% CO for approximately 14 days to obtain neural progenitor cells and neurons. 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 containing B-27 Supplement, N-2 Supplement, L-glutamine, Penicillin / Streptomycin, BDNF, GDNF, NT-3, and NGF. Neural progenitor cells and neurons can be obtained by culturing the cells at 37°C under 5% CO2 for approximately 35 days.

[0090] Differentiation into mesenchymal stromal cells can be induced, for example, by the following method: Neural crest cells are seeded into 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 hematopoietic progenitor cell induction medium, as described in WO2013 / 075222, WO2016 / 076415, Liu S. et al., Cytotherapy, 17 (2015); 344-358, etc. Step (2) can be (2-1) inducing CD4 / CD8 bipositive T cells from hematopoietic progenitor cells, or (2-2) inducing CD8 bipositive T cells from CD4 / CD8 bipositive T cells, as described in WO2016 / 076415, etc.

[0092] Examples of methods for inducing differentiation of pluripotent stem cells into cardiomyocytes include those described in WO2015 / 141827 and Laflamme MA and Murry CE, Nature. 473(7347):326-35 (2011). Other methods include, for example, 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), while 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 cultured in suspension, thereby hepatic organoid can be produced.

[0095] In the production method of the present invention, the culture may be performed under feeder-free and / or xeno-free conditions for all or part of the period. From the viewpoint of clinical use, it is preferable that the differentiation induction method of the present invention is 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 undifferentiated cell removing agent to the culture 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. [Example]

[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, devices and materials, and the obtained iPS cells and differentiated cells (monocytes) were evaluated. The closed system part of the Prodigy used had the same configuration as in Figure 1, and the main part of the sealed chamber was made of polycarbonate resin. The closed system part was attached to the mechanism part shown in Figure 3 and was made operable as shown in Figure 4. The operation of 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 Prodigy. In each example, the conditions such as the materials and amounts supplied are different, but the basic operation steps of Prodigy are the same as the above steps (e1) to (e15) or (f1) to (f21). In addition, although the expansion culture process of the established iPS cells in step (s4) and the differentiation induction process in step (s5) differ in the conditions such as the materials and amounts supplied to the sealed chamber, the basic operating method of Prodigy is the same as (s1) to (s3).

[0100] reagent Whole blood: Peripheral blood from healthy volunteer donors: Whole blood, received the day before the experiment, stored horizontally at room temperature, protected from light. Leukocyte removal 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, 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, Ltd., 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 Inc., loaded with GFP. StemFit (registered trademark) AK03N: Ajinomoto Healthy Supply Co., Ltd. TrypLE: TrypLETM 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: MiltenyiBiotec, 130-103-926 Essential 8 Medium: Gibco, A1517001 Materials Blood bag (FLEXBOY (registered trademark) BAG 50ml (LUER LOCKS, sartorius stedim biotech)) Reagent bag: MiltenyiBiotec, 20mL Regent Bag, 170-076-631 Terumo Separation Bag Microbeads: CliniMACS CD34 GMP MicroBeads, MiltenyiBiotec Device Cell processing equipment: CliniMACS Prodigy, MiltenyiBiotec Sterile splicer: TERUMO TSCD-II 5% CO2 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) Installing 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, retightened, and then assembled into the Prodigy.

[0102] (2) Preparation of starting material (PBMC isolation from whole blood) In a safety cabinet, 70 ml of whole blood was manually aseptically filled into a blood bag. 150 ml of Ficoll-Paque was 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. The program transferred whole blood 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. The Prodigy program transferred PBMCs from the chamber to the bag. The program was temporarily stopped, and the PBMCs in the bag were manually aseptically connected from the top opening of another bag containing a built-in filter using a sterile connector (TERUMO TSCD-II). Debris was removed from the PBMCs through the filter in the bag. (3) PBMC incubation The chamber was washed with 500 ml of HSC Brew using a program. The bag containing PBMCs suspended in 2% HSA HSC Brew was separated, and in a safety cabinet, the PBMCs were transferred to a cell expansion bag containing 108 μl (20 ml equivalent) 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), G-CSF (final concentration 10 ng / mL)). The PBMCs were cultured in a CO2 incubator. The Prodigy chamber was washed using a program. In a safety cabinet, iMatrix-511 (MATRIXOME) 0.35 ml x 3 tubes (3.5 to 52.5 volumes) and PBS / MgCl2 (25 After filling the bag with iMatrix-511 (mL), the bag was aseptically connected to the Prodigy. The chamber was pre-coated overnight at 22°C with iMatrix-511 and PBS / MgCl.

[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 the Prodigy. The bag containing PBMCs was then connected to the 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 Deactivate 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, 2% HSA HSC Brew (10 mL) was transferred using the Prodigy program (Culture module of Adherent Cell Culture (ACC) System) to initiate culture (day 0 after establishment). On day 5 after establishment, a Terumo separation bag was filled with 200 mL of StemFit® AK03N (Ajinomoto Healthy Supply Co., Inc.) and 200 μL of iMatrix-511 in a safety cabinet. The HSC brew bag was aseptically disconnected from the Prodigy, and the StemFit-containing bag was manually connected. Using the Prodigy program, 100 mL of StemFit was added to the chamber via the Feed function. 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) Harvest 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 left 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 without 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 23 days 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 The 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 then run through the Prodigy using the Prodigy program.

[0108] (2) Isolation of CD34-positive cells from PBMCs After filtering whole blood through a leukocyte removal filter, the blood remaining on the filter was manually eluted with 50 mL of PBS (-) in a safety cabinet and subjected to Ficoll separation to prepare PBMCs. The resulting PBMCs were 2 × 10 8 The 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] [Table 1]

[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 aseptically. In the safety cabinet, 1 L of StemSpan was filled into the bag and aseptically connected to the Prodigy. Approximately 1.52 x 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. 7 1.5 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 1 μg / cm 2 The final volume was adjusted to 30 mL. 200 μL of iMatrix and 6 cytokines were added to a final volume of 180 μL. 200 mL of StemSpan, 200 μL of iMatrix, and 180 μL of 6 cytokines were added as a feed to bring the final volume to 30 mL. - 1st day after establishment: StemFit 20 mL + iMatrix 200μL - Day 3 after establishment: Add 30 mL of StemFit to the feed chamber. - 5 days after establishment: Add 50 mL of StemFit to the chamber. - Days 7, 9, 12, 14, 16, 19, 21, and 23 after establishment: StemFit medium was replaced by removing 50 mL and adding 50 mL, bringing the culture medium in the chamber to 130 mL.

[0114] (5) Harvest On day 26 after establishment, the tubing connected to the chamber was cut with a tube sealer and heat-sealed to aseptically separate the chamber from 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 left in the chamber and observed under a microscope in a sterile environment.

[0115] (6) Results Microscopic observation of the cells under sterile conditions revealed the formation of numerous spheroids within the chamber. Counting with a tally counter revealed approximately 308 spheroids 28 days after establishment. One-eighth of the obtained cell culture medium was taken, treated with TrypLE for 30 minutes to dissolve single cells, and the number of cells was counted. The number of single cells in 1 / 8 of the culture medium was 1.26 × 10 6 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 resulting iPS cells were differentiated 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, 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. Two days later, the medium was replaced with Essential 6 medium (Gibco) supplemented with 160 ng / mL VEGF, 100 ng / mL SCF, 100 ng / mL FGF-2 (Miltenyi Biotec, 130-093-841), and 4 μM SB431542 (1614 / 10, TOCRIS). The medium was replaced with fresh medium by centrifugation. Four days after the start of the experiment, 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, but with 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) Flow cytometric evaluation of monocyte differentiation On the final day of differentiation, cells were detached from the chamber using TrypLE Select and then counted by trypan blue staining. The percentage of CD14-positive cells was analyzed by flow cytometry. The expression of CD14 and CD45-positive cells, markers of monocytic differentiation, is shown in Figure 6. The percentage of monocytic differentiated cells was 48.2% of all live cells.

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

[0119] (2) Pre-coating of the scaffold material into the chamber The chambers were manually pre-coated in a safety cabinet with acrylic resin containing a peptide with an RGD sequence. 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 materials The leukocyte separation filter was washed manually with PBS in a safety cabinet, and PBMCs were isolated from the resulting blood-containing fluid 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)Culture Volume reduction was performed to reduce the culture medium to 10 mL. Preparations up to the start of culture were the same as in Example 1. The following culture steps were entered on the Prodigy screen, and culture was performed automatically using the Prodigy program. Day 0 after establishment: Stemspan 20 mL + 6 cytokines 180μL + iMatrix-511 200μL + iMatrix-332 200μL)400μL (iMatrix 1μg / cm2), final volume 30 ml 1 day after establishment: StemFit AK03N 20 mL + iMatrix-511 200μL + iMatrix-332 200μL 3 days after establishment: Add 30 mL of StemFit AK03N to the chamber. 6 days after establishment: Add 50 mL of StemFit AK03N to the chamber. After 7 days, medium changes were performed by centrifuging the chamber to remove the old medium and adding new medium. The medium was changed 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) Harvest The cells were not detached from the chamber, but the chamber was aseptically removed from Prodigy, 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 × 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, it was observed that adherent colonies had formed in the chamber. Counting with a tally counter or counter revealed approximately 482 colonies 21 days after establishment, and 6.4 x 10 live cells on day 28 after establishment. 7 cells were counted.

[0126] Example 7 (1) Preparation Preparations were carried out in the same manner as in Example 1, except that the TS730 and TS520 tubing sets were checked and retightened 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 0.5% HSA / physiological saline: 20 mL of Albuminer / 1 L of physiological saline.

[0127] (2) Preparation of starting materials 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 chamber used was the same as that used in the separation process from whole blood to PBMCs, but the tubing set was changed from TS730 to TS520. The chamber was washed with 0.5% HSA PBS / EDTA. 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 performed in the same manner as in Example 1, except for the above procedures. 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 procedure was the same as in Example 2, 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) Harvest The cells were not detached from the chamber, but on day 15 after establishment, the chamber was aseptically removed from Prodigy, 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 × 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) PBMC isolation from whole blood, SeV infection, and culture Preparation was 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 the cells were seeded 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 (Figure 7) and 28 (Figure 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 Subculture was carried out 28 days after establishment.

[0136] (4) Flow cytometry The expression of GFP carried by 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 had been established.

[0137] Example 10 The 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 Subculture was carried out 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 had been established.

[0140] Example 12 (1) PBMC isolation 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. 6cells were counted.

[0141] Example 13 (1) PBMC isolation from whole blood, SeV infection, and culture 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 (some colonies included) 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) Passage Subculture was carried out 21 days after establishment. (4) 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 9). These results confirmed that iPS cells had been established.

[0144] Example 14 (1) PBMC isolation from whole blood, SeV infection, and culture The preparation was performed as in Example 9, pre-coating of the scaffold material inside the chamber as in Example 5, preparation of the starting material (PBMC isolation from whole blood) and SeV infection as in Example 13, and culture 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 From day 21 after establishment, differentiation induction was performed using the same method as in Example 2. iPS cells were induced to differentiate until day 17 after establishment. The cells were left 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 consistently 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 within the same chamber.

[0146] (3) Flow cytometric evaluation of monocyte differentiation Differentiation was evaluated by flow cytometry in the same manner as in Example 2. The results of expression of CD14 and CD45 positive cells, which are markers of monocytic differentiation, are shown in Figure 10. The percentage of monocytic differentiated cells was 1.21% of all live cells.

[0147] (4) Differentiation of monocytes into macrophages On day 20 after the start of differentiation induction, the 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 to macrophages. FACS analysis revealed that the ratio of CD14-positive cells, a marker for M0 macrophages, to live 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). 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) PBMC isolation from whole blood, SeV infection, and culture The number of cells subjected to 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, the chamber was pre-coated with the scaffold material. Specifically, in a safety cabinet, 5 vials of Synthemax were aseptically filled into a bag and sealed, and the bag was aseptically connected to the Prodigy via tubing. The chamber was injected using the Prodigy program (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 with a tally counter or counter revealed approximately 202 colonies and 1.74 × 10 live cells 21 days after establishment. 7 cells were counted. Photographs of the cells on days 14 and 20 after establishment are shown in Figure 11. The cells 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 connected to the TS520, including the chamber, and Synthemax 3 vials were injected into the chamber, followed by pre-coating at RT for 2 hours, in the same manner as in Example 16. As a result, when observed under a microscope 21 days after establishment, colonies were observed to have formed in 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 in the chamber. 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 onto a TS520 chamber overnight at 22°C without pre-coating. After SeV infection, 5 × 10 cells were transferred to the chamber. 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 in the Examples are summarized below (Tables 2 to 4).

[0155] [Table 2]

[0156] [Table 3]

[0157] [Table 4]

[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] [Table 5]

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

[0161] Examples 25-27 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] [Table 6]

[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) We investigated the process of monocyte differentiation using the cell processing device (Prodigy). (1) The medium and cytokines are listed 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, we diluted the old medium with a large amount of base medium, reduced the volume by centrifugation, and added an equal volume of differentiation medium containing twice the amount of cytokines. We then adjusted the volume so that the medium in the chamber containing the cells was finally 1x the volume of differentiation medium, thereby completing the exchange from the establishment medium to the differentiation medium (Figure 15).

[0165] [Table 7]

[0166] [Table 8]

[0167] [Table 9]

[0168] [Table 10]

[0169] (2) The process is as follows: <day0> (i) AK03N 30ml <feed> (ii) 200 mL of Essential 8 was added (total 130 mL) (iii) Dilution of AK03N (iv) Volume reduction to 10 mL (AK03N was present at 3 / 23 of the 10 mL volume). (v) Repeat this process twice to dilute the AK03N as much as possible. (vi) Volume reduction to 10 mL (vii) Suppose Essential 8 becomes 10 mL. <feed> (viii) 10 mL of Essential 8 containing 2x BMP4, 2x VEGF, and 2x CHIR was added. (ix) The mixture was mixed with 10 mL 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 The chamber of the cell processing device (Prodigy chamber) was coated with twice the usual amount of RGD sequence-coated scaffold material. After infection with the 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 cell processing device (Prodigy) mechanism. 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). 8x10 differentiation induction into macrophages 6 The cells were harvested on day 6 after the start of the experiment, and the cell count was 1.85 x 10 6 The 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 ratio of CD14-positive cells, a marker of M0 macrophages, to live cells 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 of the cell processing device (Prodigy chamber). 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% of cells were recovered. Expression of CD14 and CD45-positive cells, markers of monocytic differentiation, indicated that 6.85% of all live cells were monocytic (Figure 17). When GFP was examined by FACS, the GFP+: 62.5% cell group, in which reprogramming factors remained, showed very low CD14+ (0.077%) and low differentiation into monocytes (Figure 17). On the other hand, the GFP-: 34.8% cell group, in which no reprogramming factors remained, showed CD14+: 18.4%, indicating differentiation into monocytes, a result considered reasonable (Figure 17). These results suggest that the timing of induction initiation is important.

[0172] Example 30 The cells of Example 29 were reseeded in the chamber used in Example 29 and subjected to macrophage differentiation. 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 5.32 x 10 cells. 7 The cells were harvested on day 6, and the cell count was 9.8 x 10 6 Viability: 62%. When GFP was examined by FACS, the GFP+ cell group, in which the reprogramming factors remained, was very low at 1.17%, while the GFP- cell group, in which no reprogramming factors remained, was 95.1%. CD14+: 66.4%, indicating that the cells had differentiated into macrophages (Figure 18). [Industrial Applicability]

[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 iPS cell production.

[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. [Explanation of symbols]

[0175] 100 sealed chamber 210~250 Sealed container for supplying materials A10 Connecting pipe V10 Pinch Valve F10 Peristaltic Pump< / feed> < / feed>

Claims

1. A method for producing induced pluripotent stem cells using a cell processing device, comprising: the cell processing device has a closed system portion in which a sealed container for supplying material and a rotatable sealed chamber for performing centrifugation are connected via a connecting pipe line; The manufacturing method includes, while maintaining the closed system portion closed, A step (s1) of centrifuging blood cells from whole blood; a step (s2) of contacting the blood cells with a reprogramming factor; and (s3) a step of establishing induced pluripotent stem cells from the blood cells. The method for producing induced pluripotent stem cells is carried out in this order.

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

3. The cell processing apparatus further includes, as a part of the closed system portion, a magnetic separation column connected to the sealed chamber via a connecting pipe line; 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, sent to the magnetic separation column, and magnetically separated from other blood cells; The magnetic bead-labeled lymphoid cells are returned to the sealed chamber. The method for producing induced pluripotent stem cells according to claim 2.

4. The method for producing induced pluripotent stem cells according to claim 1, wherein step (s2) is carried out with the sealed chamber rotating or stationary, or 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, and the geometric rotation center line of the cylinder coincides with the central axis of rotational motion of the sealed chamber during centrifugation; The cylindrical base area is 90 to 200 cm ２ and During centrifugation, the relative centrifugal force acting on the contents in the sealed chamber is 100 to 500 G. The method for producing induced pluripotent stem cells according to claim 1.

6. The method for producing induced pluripotent stem cells according to claim 1, wherein the volume of the sealed chamber is 300 to 700 ml.

7. The method for producing induced pluripotent stem cells according to claim 1, wherein the centrifugation in step (s1) is density gradient centrifugation performed by adding a centrifugation medium for density gradient into the sealed chamber.

8. The method for producing induced pluripotent stem cells according to claim 1 , 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 claim 1, wherein steps (s2) and (s3) are carried out in the presence of a scaffold material.

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

11. The method for producing induced pluripotent stem cells according to claim 9 , wherein a scaffold material is adhered to the inner wall surface of the sealed chamber.

12. The method for producing induced pluripotent stem cells according to claim 9, 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 induced pluripotent stem cells according to claim 12, wherein the amino acid sequence of the cell adhesive peptide comprises Arg-Gly-Asp.

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

15. A method for producing differentiated cells using a cell processing device, comprising: the cell processing device has a closed system portion in which a sealed container for supplying material and a rotatable sealed chamber for performing centrifugation are connected via a connecting pipe line; The manufacturing method includes, while maintaining the closed system portion closed, A step (s1) of centrifuging blood cells from whole blood; a step (s2) of contacting the blood cells with a reprogramming factor; A step (s3) of establishing induced pluripotent stem cells from the blood cells; A step (s5) of inducing differentiation of the induced pluripotent stem cells present in the sealed chamber while maintaining the closedness of the closed system portion, Do this in this order: In the step (s5), a material necessary for differentiation induction is supplied into the sealed chamber. The manufacturing method.

16. The method of 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 system portion.

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