Method for producing induced pluripotent stem cells

Abstract

To provide a novel method for producing induced pluripotent stem cells (iPS cells) employing a novel scaffold material that can reduce problems associated with conventional microcarriers, and a method for producing differentiated cells using the iPS cells obtained by the production method.SOLUTION: Somatic cells are brought into contact with reprogramming factors, and induced pluripotent stem cells are established in the presence of a sheet-like piece having a surface containing a polymer material as at least one principal surface.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing induced pluripotent stem cells and a method for producing differentiated cells using said 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 treatment in which iPS cells are established from a patient's somatic cells (e.g., peripheral blood mononuclear cells, etc.) and then various differentiated cells or organoids induced to differentiate from the iPS cells are transplanted into the patient (autotransplantation) has attracted attention as a treatment that can reduce the risk of rejection (Non-Patent Documents 1 and 2). In this context, several scaffolds have been developed for culturing iPS cells, taking into account factors such as their adhesive properties (Patent Documents 1 to 6). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2023 / 037986 [Patent Document 2] International Publication No. 2009 / 116951 [Patent Document 3] International Publication No. 2011 / 077035 [Patent Document 4] International Publication No. 2020 / 080561 [Patent Document 5] International Publication No. 2021 / 108243 [Patent Document 6] International Publication No. 2021 / 180781 [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] As mentioned above, iPS cells are cells that require adhesion to a scaffold (so-called adhesive cells or anchorage-dependent cells), and a scaffold is required when establishing iPS cells, except when the cells are established in a suspended state.

[0006] Therefore, the following methods (a) and / or (b) are mainly considered as methods for introducing reprogramming factors into somatic cells to establish iPS cells: (a) A method of using the inner surface (bottom surface, side wall surface, etc.) of a culture vessel as a scaffold; (b) A method in which tiny scaffolding materials (called microcarriers, or larger ones called macrocarriers) are dispersed in a liquid medium.

[0007] In the method (a) using the inner surface of a culture vessel as a scaffold, the establishment of large quantities of iPS cells requires the use of a large number of culture vessels or the use of culture vessels with a complex internal structure (such as multi-tiered shelves) to increase the total internal surface area of ​​the vessel. However, methods using a large number of culture vessels have problems such as poor space efficiency. Furthermore, methods using culture vessels with complex internal structures have problems such as difficulty in handling and poor workability.

[0008] On the other hand, in the method of establishing iPS cells using a minute scaffold as described in (b) above, available scaffolds include microbeads, pieces of nonwoven fabric (such as BioNOC II manufactured by ESCO), and atelocollagen (Patent Document 1). However, to the inventors' knowledge, atelocollagen is the only scaffold that has been reported to actually establish iPS cells.

[0009] According to the research of the present inventors, scaffold materials such as microbeads and nonwoven fabric pieces have the problem of obstructing microscopic observation of iPS cells, hindering detailed observation. Furthermore, microbeads have the problem of being broken down by agitation, depending on the material. Furthermore, nonwoven fabric pieces have the problem of fibers loosening and collapsing upon agitation, releasing many of the constituent fibers into the liquid medium. Furthermore, when nonwoven fabric pieces are used for the expansion and culture of iPS cells, some of the proliferated iPS cells penetrate between the constituent fibers, making it difficult to detach the iPS cells from the nonwoven fabric piece.

[0010] Furthermore, Patent Documents 2 to 6 describe scaffolds for use in culturing iPS cells, but do not describe the use of such scaffolds for establishing iPS cells. The reason for this is that, for example, when establishing iPS cells from peripheral blood mononuclear cells, the peripheral blood mononuclear cells are nonadhesive, and the iPS cells adhere to the scaffold after establishment (at least several days after introduction of reprogramming factors). Therefore, those skilled in the art would not have thought of adding a scaffold to a cell suspension before establishing iPS cells (using a scaffold during establishment). Furthermore, each of the scaffolds described in Patent Documents 2 to 6 has at least one of the problems described above.

[0011] The object of the present invention is to provide a new method for producing iPS cells using a new scaffold material that can alleviate the above-mentioned problems, and a method for producing differentiated cells using iPS cells obtained by said method. [Means for solving the problem]

[0012] The main configuration of the present invention is as follows. [1] A method for producing induced pluripotent stem cells, comprising: A step (s1) of contacting a somatic cell with a reprogramming factor; a step (s2) of establishing induced pluripotent stem cells from the somatic cells in the presence of a sheet-like piece having a surface containing a polymer material as at least one main surface; The method comprising: [2] The area of ​​the main surface of the sheet-like piece is 0.007 to 80 mm 2 The method according to [1], wherein the thickness of the sheet-like piece is 0.005 to 3 mm. [3] The method according to [1] or [2], wherein the overall shape of the sheet-like piece is a disk, one of the main surfaces of the disk is entirely curved, or one of the main surfaces of the disk is bent in two at the diameter portion. [4] The method according to any one of [1] to [3] above, wherein the sheet-like piece has a single-layer structure made of the polymer material. [5] The method according to any one of [1] to [4] above, wherein the polymer material is polyethylene terephthalate. [6] The method according to any one of [1] to [5] above, wherein at least the surface of the sheet-like piece made of a polymer material further contains an extracellular matrix. [7] The method according to any one of [1] to [6] above, further comprising, after step (s2), a step (s3) of expanding the induced pluripotent stem cells established in step (s2). [8] The method according to [7] above, wherein step (s3) comprises the steps of detaching the induced pluripotent stem cells from the sheet-like piece by stirring in a liquid medium in which the induced pluripotent stem cells adhered to the sheet-like piece are present, and expanding and culturing the detached induced pluripotent stem cells. [9] The method according to [8], wherein the step of peeling by stirring is carried out by rotating a stirring blade and the impact when the sheet-like piece collides with the stirring blade, or by the flow of the liquid medium caused by the stirring blade.

[10] The method according to any one of [7] to [9] above, wherein the expansion culture in step (s3) is carried out by introducing a new sheet-like piece into the liquid medium in which the induced pluripotent stem cells adhering to the sheet-like piece are present, allowing the sheet-like piece (a1) to adhere to the induced pluripotent stem cells and the newly introduced sheet-like piece (a2) to settle and come into contact with each other, transferring the induced pluripotent stem cells proliferated on the sheet-like piece (a1) to the sheet-like piece (a2), and proliferating the induced pluripotent stem cells on the sheet-like piece (a2) as well.

[11] A method for producing differentiated cells, comprising the step (s4) of inducing differentiation of induced pluripotent stem cells produced by the method according to any one of [1] to

[10] above. [Effects of the Invention]

[0013] In the production method of the present invention, iPS cells are established using a polymer sheet as a scaffold. The use of this sheet provides a sufficient scaffold surface for iPS cell establishment, even in a standard-sized culture vessel with a simple structure. This allows for a more compact design of the production equipment capable of mass-producing iPS cells, improving space efficiency and production efficiency.

[0014] The sheet-like piece used as a scaffold for establishing iPS cells in the present invention, the composition of which is described in detail below, is a small piece having a sheet-like structure of a polymer material. That is, the sheet-like piece is not a porous sheet (such as a nonwoven fabric, woven sheet, knitted sheet, net-like sheet (including mesh sheet and punched sheet), or porous foam sheet). Therefore, by selecting an appropriate polymer material, the sheet-like piece can be made into a highly transparent film piece, allowing for closer observation of iPS cells adhered to the sheet-like piece under a microscope or the like.

[0015] The sheet-like pieces are not composed of a collection of fibers (such as nonwoven fabric) and do not break easily like conventional beads, thus eliminating the problems of disintegration into fine constituent fibers or shattering in liquid culture media.

[0016] The surfaces of the sheet-like piece (front and back surfaces, peripheral side surfaces) are made of a polymer material (i.e., non-porous surfaces) and can be smooth or even glossy. Therefore, while the surface is preferable as a scaffold for establishing iPS cells, it has the advantage that iPS cells do not penetrate deeply and are easily detached compared to various porous sheets (particularly nonwoven fabrics and mesh-like materials). This allows iPS cells to be detached from the sheet-like piece by simply using the flow of liquid medium caused by pipetting or stirring, and / or by the impact of rotating stirring blades colliding with the sheet-like piece, without the need for a cell dissociation reagent to detach the iPS cells from the scaffold material. This can alleviate the problem of iPS cells being damaged by the use of cell dissociation reagents. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 is a partially enlarged view showing the steps of the production method according to the present invention. In Figure 1(a), the sheet-like piece, somatic cells, and reprogramming factors are depicted at an appropriate enlarged scale, regardless of their relative size ratio, to clearly show them. Also, in this figure, somatic cells are represented by white circles, and reprogramming factors are represented by black circles. Figure 1(b) is a perspective view showing the cross section of the disc-shaped sheet-like piece and the cross section of the iPS cell layer to clearly show the state of iPS cells adhering to the surface of the sheet-like piece. In Figure 1(b), the cross section of the sheet-like piece is hatched, and the cross section of the iPS cell layer is shaded. [Figure 2] Fig. 2 is a perspective view illustrating the overall shape of a sheet piece used in the present invention. Fig. 2(a) depicts the thickness of the sheet piece and shows a cross section, while Figs. 2(b) to 2(d) omit depicting the thickness of the sheet piece. [Figure 3] FIG. 3 is a cross section illustrating the internal structure of a sheet piece used in the present invention. [Figure 4] FIG. 4 is a diagram illustrating the outer shape of a sheet piece used in the present invention, showing one main surface of the sheet piece as viewed from the front. [Figure 5] FIG. 5 is a block diagram illustrating the steps of a manufacturing method according to the present invention. [Figure 6] FIG. 6 is a diagram illustrating an example of a culture vessel used in the production method of the present invention, showing how the production method is carried out in the culture vessel. [Figure 7] FIG. 7 is a perspective view (FIG. 7(a)) showing an example of a culture apparatus having a preferred culture vessel used in the production method of the present invention, and a diagram (FIG. 7(b)) showing the action of the stirring blades in the culture vessel. [Figure 8] Figure 8 is a photograph (Figure 8(a)) showing the production of iPS cells using the culture device of Figure 7(a) in an example of the present invention, and a photograph (Figure 8(b)) showing the top surface of the lid of the culture vessel included in the culture device. [Figure 9] FIG. 9 is a photomicrograph showing the results of a single cell adhesion test in an example of the present invention. [Figure 10] FIG. 10 is a micrograph showing the surface of a sheet piece when subcultured in an example of the present invention. [Figure 11] FIG. 11 is a micrograph showing the surface of a sheet piece when subcultured in an example of the present invention. [Figure 12] FIG. 12 shows micrographs showing the results of iPS cell establishment using a control (2D) and microcarriers in an example of the present invention. [Figure 13] FIG. 13 shows micrographs of iPS cells established on each microcarrier in an example of the present invention. [Figure 14] FIG. 14 is a micrograph of iPS cells established on a sheet-like piece in an example of the present invention. [Figure 15] FIG. 15 is a micrograph of iPS cells established on a sheet-like piece in an example of the present invention. [Figure 16] FIG. 16 shows the results of analyzing the ratio of CD14-positive cells by flow cytometry in an example of the present invention (Example 1). [Figure 17] FIG. 17 is a micrograph taken 20 days after the start of differentiation when a sheet-like piece (Example 6: PET disc, diameter 0.8 mm) was used in an example of the present invention. [Figure 18] FIG. 18 shows the results of analyzing the ratio of CD14-positive cells by flow cytometry in an example of the present invention (Example 6). [Figure 19] FIG. 19 shows the measurement results of the troponin T positive rate (22.1%) when the microcarrier of Example 1 was used in an example of the present invention. [Figure 20] FIG. 20 shows the measurement results of the troponin T positive rate (50.7%) when the sheet piece of Example 6 was used in an example of the present invention. [Figure 21] FIG. 21 shows the results of an FSC / SSC plot and analysis of the proportion of SSEA4 and TRA-1-60 positive cells through flow cytometry when the sheet piece of Example 12 was used in an example of the present invention. [Figure 22] Figure 22 shows the results of an analysis of the ratio of SSEA4 and TRA-1-60 positive cells through FSC / SSC plots and flow cytometry when iPS cells were established using the sheet piece of Example 11 without using a PBS reactor in an example of the present invention. [Figure 23] FIG. 23 shows an FSC / SSC plot of Example 18 (Comparative Example) in an example of the present invention, and the results of analyzing the proportion of SSEA4 and TRA-1-60 positive cells through flow cytometry. [Figure 24] FIG. 24 shows the results of analyzing the ratio of SSEA4 and TRA-1-60 positive cells through FSC / SSC plots and flow cytometry when the microcarrier of Example 4 (Comparative Example) was used in an example of the present invention. [Figure 25] FIG. 25 is a micrograph showing the surface of a sheet-like piece when adhesion of iPS cells to the sheet-like piece was confirmed before the start of maintenance culture (maintenance culture day 0) in an example of the present invention. [Figure 26]FIG. 26 is a micrograph showing the surface of a sheet piece when maintenance culture was carried out in an example of the present invention. [Figure 27] FIG. 27 is a micrograph showing the surface of a sheet piece when a reprogramming factor introduction vector was contacted with somatic cells in an example of the present invention. [Figure 28] FIG. 28 is a micrograph of iPS cells established on a sheet in an example of the present invention. [Figure 29] 29 shows the results of flow cytometry performed on cells stained with TRA-1-BV421 and SSEA4-PreCP-Cy5.5 in an example of the present invention. As a result, expression of undifferentiated markers related to reprogramming (TRA-1-60 and SSEA-4) was confirmed. [Figure 30] Figure 30 shows the results of flow cytometry performed on cells stained with TRA-1-BV421 and SSEA4-PreCP-Cy5.5 in an example of the present invention. As a result, the expression of undifferentiated markers related to reprogramming (TRA-1-60 and SSEA-4) was confirmed. DETAILED DESCRIPTION OF THE INVENTION

[0018] The method for producing iPS cells according to the present invention will be described in detail below. As shown in FIG. 1(a), this method comprises a step (s1) of contacting somatic cells 20 with reprogramming factors 30 in a liquid medium 10, and a step (s2) of establishing iPS cells 40 from the somatic cells 20 that have been subjected to step (s1) in the presence of a sheet-like piece 100, as shown in FIG. 1(b). In the example of FIG. 1(b), the established iPS cells 40 are illustrated as a cell layer that has proliferated two-dimensionally and surrounds the entire surface of the sheet-like piece 100. The sheet-like piece 100 is a small sheet-like (film-like) piece made of a polymer material. The above-mentioned effects can be achieved by using the sheet-like piece 100 as a scaffold during the establishment of iPS cells.

[0019] (Step (s1): contacting somatic cells with reprogramming factors) In the example shown in FIG. 1(a), somatic cells 20, reprogramming factors 30, and sheet-like pieces 100 are supplied to a liquid medium 10. However, as described below, the sheet-like pieces 100 may be supplied to the liquid medium 10 after the reprogramming factors 30 have come into contact with the somatic cells 20. That is, step (s1) may be performed as a separate step in which the sheet pieces are not present. In step (s1), the liquid medium 10 is preferably stirred sufficiently so that the somatic cells 20 and the reprogramming factors 30 come into contact with each other. The stirring method will be described later together with the culture vessel. Once stirring is complete, the liquid medium is replaced and the reprogramming factors are removed. Removal of the reprogramming factors also includes reduction of the reprogramming factors (reduction of the concentration of the reprogramming factors). A small amount of the reprogramming factors that have not been removed may remain in the liquid medium, and a small amount of somatic cells that have not come into contact with the reprogramming factors may be present.

[0020] (Step (s2): Establishing iPS cells) In step (s2), as shown in Figure 1(b), the somatic cells contacted with the reprogramming factors in step (s1) are further cultured in the presence of a sheet piece (i.e., in a liquid medium in which the sheet piece serving as a scaffold is suspended), thereby establishing iPS cells adhered to the surface of the sheet piece. In this step, the iPS cells may adhere not only to the surface of the sheet piece but also to the inner surface of the culture vessel.

[0021] The period for performing step (s2) is not particularly limited as long as iPS cells are established, but is typically, for example, 10 days or more, preferably 14 days or more. The upper limit of the period for performing step (s2) is not particularly limited, but is typically 30 days or less, preferably 40 days or less.

[0022] Whether iPS cells have been established can be confirmed appropriately by publicly known methods, such as by checking the expression of the introduced reprogramming factors (e.g., Oct3 / 4, SOX2, Nanog, TRA-1-60, TRA-1-81, SSEA3, SSEA4, alkaline phosphatase, etc.). However, while the concept of Quality By Test (QbT) requires confirmation of iPS cell establishment, the concept of Quality By Design (QbD) does not necessarily require confirmation of iPS cell establishment, as long as a process designed to produce iPS cells and differentiated cells is followed, with the aim of obtaining differentiated cells for use in regenerative medicine from patient-derived somatic cells.

[0023] The order of material introduction, the amount of introduction, and the supply route from the container containing each material to the culture container (e.g., an example of a closed system configuration) in carrying out steps (s1) and (s2) will be described later. In this specification, the terms "introduction" and "addition" may be used interchangeably.

[0024] (induced pluripotent stem cells) As used herein, "induced pluripotent stem cells (iPS cells)" are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells through the introduction of reprogramming factors. iPS cells can differentiate into various tissues and cells with different morphologies and functions in the body, and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm).

[0025] 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.

[0026] This method can be used to produce any existing iPS cells.

[0027] As used herein, iPS cells may be cells derived from a patient with a genetic disease. Cells induced to differentiate from iPS 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 the CRISPR-Cas system or the like, and then differentiated into target cells, making it possible to use the cells as a therapeutic agent for the disease.

[0028] (somatic cells) In the present invention, "somatic cells" refer to raw material cells (original cells) to be processed to produce iPS cells. As used herein, "somatic cells" refers to cells that constitute an animal, other than germ cells. Somatic cells are not particularly limited and include both mature, healthy and diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells may be, for example, floating cells (e.g., blood cells) or adherent cells, with floating cells being preferred. Examples of somatic cells used in the production method of the present invention include, but are not limited to, mesenchymal stem cells derived from skin fibroblasts, etc., skin cells, visual cells, brain cells, hair cells, oral mucosa, dental pulp cells, lung cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells, kidney cells, neural stem cells, wisdom teeth, etc., tissue stem cells, tissue progenitor cells, blood (blood cell) cells (e.g., hematopoietic stem cells, peripheral blood mononuclear cells (PBMCs) (including T cells and non-T cells), leukocytes (e.g., lymphocytes), umbilical cord blood cells, etc.), epithelial cells, endothelial cells (e.g., vascular endothelial cells), muscle cells, etc.

[0029] In one embodiment, when blood cells (for example, peripheral blood mononuclear cells) are used as somatic cells, the cells can be obtained by centrifuging whole blood (density gradient centrifugation, specific gravity centrifugation, etc.), separating the cells using a filter (leukocyte removal filter, etc.), or by using an antibody, a magnetic substance (magnetic beads, etc.), or a hydrophilic polysaccharide (Ficoll TM etc.) can be obtained by separation.

[0030] (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).

[0031] In the present specification, the species from which the somatic cells are derived is not particularly limited, and the species from which the somatic cells are derived is preferably human.

[0032] (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.

[0033] 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.

[0034] 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.

[0035] (initialization factor) 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.

[0036] 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.

[0037] 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).

[0038] 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.

[0039] (liquid medium) Liquid media can be used not only for culturing cells but also for various purposes such as washing cells, diluting chemical solutions, etc. Examples of liquid media that can be used in the present invention include the following:

[0040] The liquid medium is not particularly limited, but may be, for example, Essential 8 medium (CTS TM Essential 8 TM Medium, Essential 8 TM Medium, Essential 8 TM Flex Medium, Essential 6 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.

[0041] If necessary, physiologically active substances and nutritional factors necessary for cell survival or proliferation can be added to the liquid medium.

[0042] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the liquid medium as needed.

[0043] In this specification, known serum can be used in the liquid medium.

[0044] As used herein, the liquid medium may or may not contain serum substitutes as well as serum.

[0045] (sheet-like pieces) In the present invention, a sheet-like piece is a small sheet-like member that serves as a scaffold for adherent cells in a liquid medium. As illustrated in Figures 1(b) and 2(a), a sheet-like piece 100 has one main surface 101 and the other main surface 102 located on the reverse side. This main surface is the so-called sheet surface (or film surface). In the present invention, the terms "sheet," "film," and "plate" are terms that indicate the same form, regardless of thickness or material, and all have one main surface and the other main surface located on the reverse side.

[0046] (Overall shape of sheet piece) The overall shape of the sheet-like piece may be a flat plate (e.g., a flat disk) as shown in FIG. 2(a), a shape with an entirely curved main surface as shown in FIG. 2(b), a shape with a main surface bent in two along an arbitrary straight line as shown in FIG. 2(c), or a shape with a hemispherically recessed main surface as shown in FIG. 2(d). In the example of FIG. 2(b), the main surface 101 of the disk-shaped sheet is entirely concavely curved, and the other main surface 102 is correspondingly convexly curved. In the example of FIG. 2(c), the main surface 101 of the disk-shaped sheet is concavely bent in two along the diameter of the circle, and the other main surface 102 is correspondingly convexly bent. In the example of FIG. 2(d), the main surface 101 of the disk-shaped sheet is entirely concavely recessed, and the other main surface 102 is correspondingly convexly bulged.

[0047] Furthermore, the overall shape of the sheet-like piece 100 may be plate-like, having front and back main surfaces (101, 102) and an outer peripheral side surface 103, as in the example of Figure 2(a), or it may have a shape in which the thickness gradually decreases as you move from the center of the main surface to the outer peripheral edge, resulting in an edge-like shape (not shown), or a shape in which the outer peripheral edge is bulged (not shown), and the boundary between the main surface and the side surface may not be distinguishable.

[0048] (Layer structure of sheet-like pieces) The layer structure of the sheet-like piece 100 is preferably a single layer made entirely of one type of polymer material, as this is low cost, but in order to adjust the specific gravity, mechanical strength, rigidity, elasticity, etc. of the entire sheet-like piece, it may be a two-layer structure (not shown), a simple three-layer structure (outer layer 100a, middle layer 100b, outer layer 100c) as shown in Figure 3(a), a multi-layer structure (not shown), a three-layer structure as shown in Figure 3(b) (a structure in which the inner layer 100b is sandwiched between two outer layers (100a, 100c) and sealed), or a structure in which the entire core layer 100d is covered with an outer layer 100e as shown in Figure 3(c).

[0049] (Outline of one main surface of the sheet piece) The outer shape of one main surface of the sheet-like piece (hereinafter referred to as the outer shape of the sheet-like piece) is not particularly limited, but is preferably a simple shape in which the surface areas are concentrated in one place (i.e., a shape closer to a circle), such as a triangle (especially an equilateral triangle), a rectangle (especially a square), a polygon (especially a regular polygon), a circle ( FIG. 4(c)), an ellipse, or an irregular shape, because these simple shapes are less likely to tangle with each other and are easy to manufacture. Furthermore, because sharp corners in triangles, rectangles, and the like may damage iPS cells in liquid culture, it is preferable to provide sufficient rounding of the corners, as shown in the example of a square in FIG. 4(a), or sufficient chamfering of the corners, as shown in the example of a square in FIG. 4(b). Among these outer shapes, the circle shown in FIG. 4(c) (i.e., a disk-shaped sheet-like piece) is preferred because it can be manufactured relatively easily and inexpensively and has no corners. On the other hand, even if the external shape has the same area, shapes such as a long, thin, strip-like rectangle as shown in Figure 4(d), a complex, meandering shape of a long, thin strip as shown in Figure 4(e), or a spiral shape (not shown) are not excluded from the present invention, but are undesirable in that they tend to become tangled with each other or entangled on the stirring blades, etc., and make it difficult to detach iPS cells.

[0050] (area of ​​one main surface of the sheet piece) The area (E) of one main surface of the sheet-like piece is not particularly limited, but is preferably 0.007 to 80 mm 2 The preferred thickness is 0.5 to 80 mm. 2 The preferred range is 0.7 to 20 mm.2 is more preferable. When the outer shape of the sheet-like piece is circular, the diameter of the circle, which roughly corresponds to the area (E) of the main surface, is preferably about 0.1 to 10 mm, more preferably about 0.8 to 10 mm, and even more preferably about 1 to 5 mm. When the outer shape of the sheet-like piece is a shape other than a circle (triangle, square, polygon, ellipse, irregular shape, etc.), the main dimensions such as the length of the sides of each shape may be appropriately determined so as to have the area (E) of the main surface. When the area (E) of one main surface of the sheet-like piece is within the above-mentioned suitable range, it is easy to handle and can be easily suspended appropriately in a culture vessel of normal capacity with only normal gentle stirring.

[0051] (sheet thickness) The thickness (T) of the sheet-like piece is not particularly limited, but is preferably about 0.005 to 3 mm, and more preferably about 0.012 to 0.5 mm. Regarding the thickness (T) of the sheet-like piece, it is preferable to select the thickness (T) so as to obtain a preferable sheet-like piece depending on the area (E) and diameter (or circle-equivalent diameter) of the main surface of the sheet-like piece. For example, if the overall shape of the sheet-like piece is disk-shaped and its diameter is the lower limit (0.1 mm), selecting the upper limit (3 mm) for the thickness will result in an overall shape closer to a thin cylinder (rod) rather than a disk. Rather than using such thin cylinder-shaped pieces, selecting a smaller thickness (T) to obtain an overall shape closer to a disk and using a larger number of sheet-like pieces is preferable, as this will increase the surface area. On the other hand, if the diameter of the disk is the upper limit (10 mm), selecting the lower limit (0.005 mm) for the thickness will result in a sheet-like piece that is very thin compared to the width of one main surface. Such wide and thin sheet-like pieces will easily bend when subjected to resistance from the liquid medium, etc., and will have difficulty maintaining their disk shape, so it is preferable to select a larger thickness (T) to provide appropriate rigidity. Furthermore, thicker sheet pieces tend to settle more quickly in a liquid medium, while thinner pieces tend to take longer to settle. Therefore, it is preferable to appropriately select the area (E) and thickness (T) of the main surface of the sheet piece to impart favorable buoyancy to the sheet piece.

[0052] For the above reasons, when the outer shape of the sheet-like piece is circular, the ratio (T / D) of the thickness (T) of the sheet-like piece to the diameter (D) of the circle is preferably about 0.0009 to 0.5, more preferably about 0.001 to 0.6, and even more preferably about 0.002 to 0.5. Combinations of thickness (T) and diameter (D) outside these ratio ranges are not excluded from the present invention, and the thicknesses (T) and diameters (D) may be freely combined according to various circumstances. As a result of such combinations, pieces that have an overall shape similar to the elongated cylindrical shape described above also fall within the scope of the sheet-like piece of the present invention.

[0053] When the outer shape of the sheet-like piece is not circular (triangle, square, polygon, ellipse, irregular shape, etc.), the circle-equivalent diameter of the non-circular shape can be used as the diameter (D) to calculate the ratio (T / D). The "circle-equivalent diameter" is the diameter of a circle having the same area as the area of ​​the non-circular shape.

[0054] The main surface of the sheet-like piece may have localized protrusions or depressions, in which case the thickness (T) of the sheet-like piece can be the thickness of the portion other than the localized protrusions or depressions. It is preferable that the thickness of the sheet-like piece is uniform, but if the thickness varies, the average value of the thickness at multiple points can be used as the thickness (T) of the sheet-like piece.

[0055] (Sheet material) The sheet piece 100 may have a composite structure combining various materials such as metals and polymer materials, but at least one of its front and back main surfaces 101, 102 contains a polymer material (i.e., a surface that is partially or entirely made of a polymer material). Here, the "surface made of a polymer material" refers to a surface formed by spreading a polymer material, and is different from the surface of a porous sheet (a porous surface such as a nonwoven fabric surface or a mesh surface). The surface made of a polymer material may be smooth or roughened. In a preferred embodiment, both main surfaces of the sheet piece contain a polymer material, and further, all of both main surfaces of the sheet piece are made of a polymer material. Furthermore, in a shape having side surfaces, such as the examples of Figures 1(b) and 2(a), it is preferred that all of the side surfaces are made of a polymer material.

[0056] Polymer materials (resin materials) used for the sheet-like pieces include those that can serve as scaffolds for iPS cells, such as polystyrene, polyolefins (polyethylene, polypropylene, etc.), polyesters (particularly polyethylene terephthalate), polyethers, polyvinyl alcohol, polyvinyl acetals (e.g., polyvinyl butyral), poly(meth)acrylic acid esters (poly(meth)acrylates), epoxy resins, polyamides, polyimides, polyurethanes, polycarbonates, cellulose, dextran, and polypeptides (e.g., gelatin). Any material selected from these may also be mixed. Among these polymer materials, thermoplastic resins are preferred due to their excellent processability. Polyethylene terephthalate (PET) is also preferred as a material for the sheet-like pieces because it has high mechanical strength and transparency, moderate rigidity and elasticity, high chemical resistance, is stable in liquid medium, and has little effect on the iPS cells to be cultured.

[0057] (Weight per unit volume of sheet piece) The weight per unit volume of the sheet-like piece (unit volume weight) is preferably such that it remains stationary (floats) or settles slowly in the liquid medium used. Slow settling here refers to settling at a rate such that the time required for the sheet-like piece to fall 100 mm in the liquid medium left stationary is at least about 1 second, preferably at least about 3 seconds, and more preferably at least about 5 seconds. There is no upper limit to the time required for this fall. If the unit volume weight of the sheet-like piece is such that it floats on the surface of the liquid medium, it becomes difficult for iPS cells and the like to use it as a scaffold. Furthermore, if the sheet-like piece settles excessively quickly in the liquid medium, it will not float sufficiently in the liquid medium even with stirring, which is undesirable because it reduces the probability of contact between the reprogramming factors, cells, and the sheet-like piece. If the material of the sheet piece is one of the above-mentioned polymer materials with a specific gravity equal to or greater than that of the liquid medium to be used (especially PET (specific gravity of approximately 1.29 to 1.4)), it will remain stationary or slowly settle in a normal liquid medium, although this will depend on the overall shape of the sheet piece. Note that when a polymer material with a specific gravity lower than that of the liquid medium to be used is used as the main surface material, a layer made of a material with a higher specific gravity can be added to a part of the sheet piece (such as the interior) to adjust the unit volume weight of the sheet piece.

[0058] (Method of manufacturing sheet pieces) The method for producing the sheet-like pieces is not particularly limited, and conventional resin processing methods can be used, such as various resin molding methods using a molding die, methods of punching a stretched polymer material sheet using a press or the like (which may be bent as needed), methods of obtaining the sheet-like pieces by removing the periphery of the desired sheet-like piece from a stretched polymer material sheet using etching or the like (which may be bent as needed), laser processing methods, and forming methods using a 3D printer. Among these processing methods, methods of punching a polymer material sheet obtained by unstretching, uniaxial stretching, or biaxial stretching using a press or the like are preferred because they allow for inexpensive mass production of sheet-like pieces with relatively high dimensional accuracy. If burrs or sharp edges resulting from punching using a press or the like are problematic, conventional deburring processes can be added as appropriate.

[0059] (extracellular matrix) In one embodiment, at least the polymer surface of the sheet-like piece may further contain an extracellular matrix. For example, the polymer surface may be pre-coated with an extracellular matrix, or the sheet-like piece and the extracellular matrix may be placed in a liquid medium to allow the extracellular matrix to adhere to the polymer surface.

[0060] Examples of extracellular matrices include basement membrane preparations (e.g., Matrigel (manufactured by Corning) and Geltrex matrix (manufactured by Thermo Fisher Scientific)), fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, vitronectin, and the like, or combinations thereof. The above-listed extracellular matrices may be natural products, artificially synthesized using recombinant DNA technology, fragments obtained by cleavage with restriction enzymes, or synthetic proteins or peptides based on these biological substances.

[0061] (Other materials to be supplied) In addition to the materials mentioned above, substances necessary for cell processing may be added as appropriate, such as liquids (e.g., saline, buffer solutions, etc.), powders, additives, release agents, cryoprotectants, CO2 gas, etc.

[0062] (Time to insert sheet pieces) Fig. 5 is a block diagram showing a typical example of the order in which materials are introduced into a culture vessel when carrying out the method, and is also a block diagram showing an example of the supply path of materials in a manufacturing apparatus for carrying out the method.

[0063] In the example of Figure 5(a), the culture vessel 200 is configured to be supplied with liquid medium 10 from vessel 310, somatic cells 20 from vessel 320, reprogramming factors 30 from vessel 330, sheet-like pieces 100 from vessel 340, and necessary incidental materials such as extracellular matrix from vessel 350. Depending on the size of the sheet-like pieces 100, it may be difficult to transport them using a normal tube and peristaltic pump. In such cases, the sheet-like pieces 100 may be separately introduced into the culture vessel 200 manually or using a dedicated dispenser. Container 360 is a container for containing waste, and container 370 is a container for removing the produced iPS cells. Each vessel may be a sealed vessel. Each vessel may be connected to a culture vessel by a tube or the like. Furthermore, the vessel may be configured to move materials or the like using a peristaltic pump, or the entire vessel may be a closed system.

[0064] In the example of Figure 5(a), liquid medium 10, somatic cells 20, reprogramming factors 30, sheet-like pieces 100, etc. may be added to culture vessel 200 at the same time, and sheet-like pieces 100 may be present in liquid medium 10 when reprogramming factors 30 come into contact with somatic cells 20. In the example of Figure 5(a), the liquid medium 10, somatic cells 20, and reprogramming factors 30 may be introduced into the culture vessel 200 at the same time, and the sheet-like piece 100 may be introduced into the culture vessel 200 after the reprogramming factors have come into contact with the somatic cells and the liquid medium has been replaced (i.e., after the reprogramming factors have been washed away).

[0065] In the example of FIG. 5(b), a cell processing container 200A is added for contacting somatic cells 20 with reprogramming factors 30. The cell processing container 200A is configured so that liquid medium 10 is supplied from container 310A, somatic cells 20 are supplied from container 320, and reprogramming factors 30 are supplied from container 330. After the contact step between the somatic cells 20 and the reprogramming factors 30 in the cell processing container 200A is completed and the liquid medium is replaced (i.e., after the reprogramming factors are washed away), the somatic cells 20 are transferred to the culture container 200B. The liquid medium 10 is supplied from container 310B, and the sheet-like piece 100 is supplied from container 340 to the culture container 200B. Container 360 is a container for containing waste, and container 370 is a container for removing the produced iPS cells. The tubes, feed pump, and sheet-like piece introduction means connecting each container may be the same as those in FIG. 5(a).

[0066] In the example of Figure 5(b), the cell processing container 200A for contacting the somatic cells 20 with the reprogramming factors 30 may be a container similar to the culture container 200, or may be a rotatable sealed chamber in a cell processing device having a centrifugal function (e.g., a counterflow centrifugal function) for efficiently contacting the somatic cells with the reprogramming factors by spinoculation or the like.

[0067] (Amount of each material and sheet piece added to the liquid medium) The amounts of somatic cells, reprogramming factors, sheet-like pieces, etc. added per liquid medium are not particularly limited, but an example is as follows. (1) The culture vessel is a well plate (per well: bottom area 9.6 cm 2 , depth 2cm) Somatic cells (PBMC): 0.5×10 4 ~5×10 5 cell / well Reprogramming factor introduction vector (SeV): MOI = 0.3 to 3 (specified amount of reagent) Sheets: 3 to 15 sheets / well Liquid medium: 2-5 ml (2) When the culture vessel is a PBS Bioreactor (PBS0.1) manufactured by PBS Biotech (width 6.4 cm, depth 4.6 cm, height 12.8 cm) Somatic cells (PBMC): 1×10 5 cells Reprogramming factor introduction vector (SeV): MOI = 0.3 to 3 (specified amount of reagent) Sheet pieces: 10 to 60 pieces Liquid medium: 2-5 ml at the time of establishment, 60-80 ml from the 7th day The present invention can be carried out even if the input amounts are outside the ranges of the above-mentioned examples. Furthermore, when scaling up the capacity of the culture vessel, the above-mentioned input amounts can be appropriately increased or decreased so as to maintain agitation properties, etc.

[0068] (Culture container) The culture vessel used to carry out this method is not particularly limited, and any vessel generally used for cell culture can be used. In particular, a vessel that is closed or airtight (sealed vessel) that prevents microorganisms and viruses from entering from the outside world is preferred.

[0069] The term "closed" or "hermetically sealed" refers not only to a state in which the interior is kept airtight or liquidtight, but also to a state in which the interior is isolated from the outside world to such an extent that microorganisms or viruses cannot enter from the outside world, i.e., to such an extent that sterility of the interior is maintained. For example, a sealed container provided with a porous filter that does not allow bacteria or viruses to pass through but allows fluids (particularly gases) to pass through will allow outside air to pass through the porous filter and into the sealed container, but will not allow bacteria or viruses to enter, and sterility within the closed system will be maintained, so the container has closed or hermetically sealed properties.

[0070] FIG. 6 is a diagram illustrating an example of a culture vessel used in this method. In the example of FIG. 6(a), the culture vessel 200 is a sealable vessel having a vessel body 210 with a U-shaped bottom and a lid 220 that seals the opening at the top. As described below, a filtered air vent (not shown) is provided on the top surface of the lid 220, allowing gas exchange between the inside of the culture vessel and the outside world through the air vent, and maintaining the internal pressure of the culture vessel at atmospheric pressure. In the example of FIG. 6(a), appropriate amounts of liquid medium 10, somatic cells 20, reprogramming factors 30, and sheet-like pieces 100 are each added to the culture vessel 200 so that they are simultaneously present, and step (s1) is performed while the liquid medium 10, somatic cells 20, reprogramming factors 30, and sheet-like pieces 100 are stirred by a stirrer 230.

[0071] In the example of Figure 6(b), as in the example of Figure 6(a), the culture vessel 400 has a general vessel body 410 and an agitator 420, and appropriate amounts of liquid medium 10, somatic cells 20, reprogramming factors 30, and sheet-like pieces 100 are each added so that they are simultaneously present in the culture vessel 400, and step (s1) is carried out while being agitated by the agitator 420.

[0072] The agitator 230 in the example of Fig. 6(a) is a device configured such that an agitator blade, called a vertical wheel, rotates around a horizontal shaft 232 like a water wheel. On the other hand, the agitator 420 in the example of Fig. 6(b) has a very general configuration, in which an agitator blade 244 attached to the tip of a vertical shaft 242 is driven by the vertical shaft 242 and rotates in the direction of the arrow in the figure.

[0073] The stirring principle and device are not particularly limited, and a stirrer, the stirring device shown in Figure 6(b), shaking the container, fluid injection, etc. can be used, but the stirring impeller shown in Figure 6(a) is preferred because it rotates gently to minimize damage to iPS cells while still sufficiently stirring the liquid medium and suspended matter in the culture container. Next, this stirring impeller will be described.

[0074] (Vertical Wheel) As shown in FIG. 7(a), a vertical wheel is rotatably mounted inside a culture vessel (also called a culture vessel) of a PBS bioreactor (e.g., PBS MINI (product number PBS0.1 MAG) manufactured by PBS Biotech). As shown in FIG. 6(a), a horizontal shaft 232 is fixed inside the culture vessel. A boss is rotatably mounted around the boss. A pair of fan-shaped vanes (screw propeller-like blades) 236 extend radially from the boss. The fan-shaped vanes 236 support an annular rim 234 like spokes. The annular rim 234 is provided with multiple outer impellers 238 spaced at equal intervals around its circumference. As shown in FIG. 7(a), the culture vessel 200 is attached to a drive unit 240. The magnetic rotational driving force from the drive unit 240 rotates the fan-shaped vanes around the horizontal shaft 232 within the sealed culture vessel, as shown in FIG. 7(b).

[0075] An important feature of the vertical wheel is that mixing is further promoted by the cooperative action of the central sectorial vane 236 and the peripheral impeller 238. The peripheral impeller 238 generates a circumferential flow (the direction of arrow y1 in FIG. 7(b)) in the gap between the impeller and the U-shaped bottom of the vessel body 210. Meanwhile, the pair of central sectorial vanes 236 generate two horizontal flows in opposite directions, as indicated by the thick arrows in FIG. 7(b). These flows in a total of three directions effectively mix the liquid medium 10 (containing the somatic cells 20, reprogramming factors 30, and sheet-like pieces 100) within the vessel body 210, thereby promoting contact between the somatic cells 20 and the reprogramming factors 30 and the establishment of iPS cells.

[0076] (Volume of the container body and stirring speed) Figure 8(a) is a photograph showing the operating state of the PBS bioreactor (PBS 0.1 MAG) used in the examples of the present invention. Figure 8(b) is a photograph showing the top surface of the lid 220. As shown in Figure 8(b), an air vent 222 is provided on the top surface of the lid 220, and this air vent has a structure in which a through-hole provided in the lid is closed with a gas-permeable filter that prevents bacteria and viruses from passing through. Gas exchange between the inside of the culture vessel and the outside world is performed through this filter, and the internal pressure of the culture vessel is maintained at atmospheric pressure. The capacity of the container body shown in Figure 8(a) is approximately 60 to 100 ml, and the diameter of the vertical wheel is approximately 30 mm. In this case, the stirring speed (the speed of the vertical wheel's circumference) for carrying out steps (s1) and (s2) may be any speed that causes the sheet-like pieces to float in the liquid medium due to stirring, and may be, for example, approximately 0.47 to 4.7 m / min (rotation speed approximately 5 to 50 rpm). PBS bioreactors are available in several varieties, each with a different culture vessel capacity and a different outer diameter for the vertical wheel. Therefore, when scaling up the culture vessel, the stirring speed of the vertical wheel can be adjusted appropriately to achieve the same stirring effect as in the previous example. Even when using impellers other than vertical wheels, the type of impeller and stirring speed can be selected appropriately to achieve the same stirring effect as in the previous example.

[0077] (Step (s3): Step of expanding iPS cells) From the viewpoint of the yield of iPS cells, this method may further include a step (s3) of expanding the iPS cells after the step (s2) of establishing the iPS cells. The step (s3) may be performed using the culture vessel 100 used in the steps (s1) and (s2), or the iPS cells established in the step (s2) may be transferred to a different culture vessel. In this case, the iPS cells established in the step (s2) while adhered to the sheet-like piece may be expanded while still adhered to the sheet-like piece, without being detached from the sheet-like piece. This reduces damage to the iPS cells caused by detachment.

[0078] (Agitation for peeling) In step (s3), the iPS cells can be easily detached from the sheet by agitating the liquid medium containing the iPS cells adhered to the sheet. This agitation can then be used to detach the iPS cells from the sheet, and the detached iPS cells can then be expanded. A separate culture vessel can also be used for the expansion of the detached iPS cells. The culture vessel and agitator blades used for this detachment may be a PBS Bioreactor (larger model) manufactured by PBS Biotech, Inc., as shown in Figure 6(a) and its vertical wheel. The vertical wheel is preferred because it allows gentle agitation while generating a strong agitation flow when rotated at a higher speed. In this case, the peripheral speed of the vertical wheel, which has a diameter of approximately 30 mm, is preferably approximately 8 to 10 m / min (rotation speed: approximately 80 to 100 rpm).

[0079] In step (s3), the agitation step of detaching iPS cells from the sheet piece may involve rotating the impeller in the culture vessel at a higher speed than in steps (s1) and (s2), and detaching iPS cells from the sheet piece by the impact of the sheet piece colliding with the impeller or by the flow of liquid medium caused by the impeller. The culture vessel and impeller used for detachment may be a PBS Bioreactor (larger model) manufactured by PBS Biotech, Inc., as shown in Figure 6(a) and its vertical wheel. Rotating the vertical wheel at a higher speed generates a strong agitation flow and adequately impacts the sheet piece, resulting in a moderate impact on the iPS cells, resulting in a moderate detachment of the iPS cells. In this case, the peripheral speed of the vertical wheel, approximately 30 mm in diameter, is preferably approximately 8 to 10 m / min (rotation speed: approximately 80 to 100 rpm), as described above. When scaling up the culture vessel and vertical wheel, the stirring speed of the vertical wheel can be adjusted appropriately to obtain the same stirring effect as in the above example. Also, when using a stirring impeller other than a vertical wheel, the type of the stirring impeller and stirring speed can be selected appropriately to obtain the same stirring effect as in the above example.

[0080] (The process of transferring iPS cells from one sheet to another and expanding them) In step (s3), a new sheet piece (referred to as sheet piece (a2)) is placed into the liquid medium containing iPS cells established in a state adhered to a sheet piece (referred to as sheet piece (a1)), and the sheet pieces (a1) and (a2) are allowed to settle together and come into contact with each other at the bottom of the culture vessel. This is expected to cause some of the iPS cells established on sheet piece (a1) to transfer and adhere to sheet piece (a2), leading to proliferation of the iPS cells on sheet piece (a2). It is also expected that iPS cells will detach from sheet piece (a1) by agitation, float in the liquid medium, and then adhere to other floating sheet pieces (a2) and proliferate thereon.

[0081] More specifically, when the sheet pieces (a1) and (a2) are allowed to settle to the bottom of the culture vessel, the sheet pieces may partially or completely overlap at the bottom. In this case, the contact state between the overlapping sheet pieces is likely to be surface contact, unlike the contact state between beads (point contact). Therefore, it is expected that some of the iPS cells proliferated on the previous sheet piece (a1) will efficiently migrate to the new sheet piece (a2) and continue to proliferate on the new sheet piece (a2). This proliferation method for transferring iPS cells from the sheet piece (a1) to the sheet piece (a2) by utilizing surface contact due to sedimentation does not fundamentally involve the step of detaching iPS cells, and is therefore advantageous in that the detachment step and detachment agent can be omitted and damage to the iPS cells is minimized.

[0082] The above-described method of transferring iPS cells from sheet piece (a1) to sheet piece (a2) may be performed immediately after iPS cells are established on sheet piece (a1), or after detaching the iPS cells from the sheet piece by stirring as described above. In this way, it is expected that iPS cells that are not completely detached by stirring and remain on sheet piece (a1) will migrate to a new sheet piece (a2).

[0083] Those skilled in the art can appropriately select the medium and other ingredients used in step (s3) from the liquid media described above. In one embodiment, when it is intended to expand the established iPS cells, a factor for maintaining undifferentiated states can be added. As used herein, the term "factor for maintaining undifferentiated states" refers to a substance that has the effect of suppressing the differentiation of induced pluripotent stem cells, and is not particularly limited as long as it is such a substance. Examples of factors for maintaining undifferentiated states commonly used by those skilled in the art include bFGF, FGF2, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, and TGFβ2. The factors for maintaining undifferentiated states used in the present invention are preferably isolated. "Isolated" means that the target components and factors other than cells have been removed, and the factor has escaped from its naturally occurring state.

[0084] In step (s3), the number of times expansion culture is performed is not particularly limited as long as the desired number of iPS cells is obtained, but in typical processing procedures, it is preferably performed about 1 to 5 times, more preferably about 2 to 5 times. The period for performing step (s3) is not particularly limited as long as the desired number of iPS cells is obtained, but is typically, for example, 1 to 40 days, 3 to 20 days, or 5 to 10 days. If the Sendai virus vector used for reprogramming contains a GFP marker, it is desirable to perform expansion culture until the marker is lost.

[0085] (Quality assessment of iPS cells) After step (s2) or step (s3) of the production method of the present invention, the quality of the obtained iPS cells may be evaluated by a known method.

[0086] (Method for producing differentiated cells) The method for producing differentiated cells according to the present invention is a method comprising the step (s4) of inducing differentiation of iPS cells produced by the method for producing iPS cells according to the present invention described above. Furthermore, the method for producing differentiated cells may be a method that essentially includes steps (s1) and (s2) of the method for producing iPS cells according to the present invention described above, and further includes steps (s1) to (s3) as steps preceding step (s4). Furthermore, the method for producing differentiated cells may involve inducing differentiation of iPS cells that have been produced by the method for producing iPS cells according to the present invention described above and that are attached to a sheet-like piece, while the sheet-like piece remains attached. Furthermore, the method for producing differentiated cells may involve inducing differentiation of iPS cells that have been produced by the method for producing iPS cells according to the present invention described above and that are attached to a sheet-like piece, by detaching the iPS cells from the sheet-like piece by stirring as described above, and then leaving the iPS cells free from the sheet-like piece. The method for producing differentiated cells may also be a method using iPS cells produced by the method for producing iPS cells according to the present invention described above, placing the iPS cells and a sheet-like piece (a3) ​​in a liquid medium, and inducing differentiation of the iPS cells in the presence of the sheet-like piece (a3).

[0087] The step (s4) may be performed using the culture vessel used in the above-described method for producing iPS cells according to the present invention, or may be performed in a vessel in which the produced iPS cells are transferred for differentiation induction.

[0088] As containers for differentiation induction, the culture containers used in the above steps (s1) and (s2), the containers used in the above expansion culture, sealed containers included in various reactors for differentiation induction, G-Rex (Wilson Wolf), and sealed containers equipped with tubes for air venting, tubes for medium exchange, and tubes for CO2 gas, etc., can be used as appropriate.

[0089] The step (s4) may be performed after the establishment step (s2) in the method for producing iPS cells according to the present invention described above, without performing the step (s3) of expanding the iPS cells, or may be performed after the establishment step (s2) and the step (s3) of expanding the iPS cells.

[0090] After the step (s2) of establishing iPS cells and before the step (s4) of inducing differentiation of iPS cells, one or both of a cell sorting step and a gene transfer step may be further performed. The cell sorting step can be performed by connecting a known cell sorting device to the closed system portion. The gene transfer step can use electroporation, lipofection, or a viral vector. For electroporation, an electroporator can be used. For methods using lipofection, liposomes, or viral vectors, a cell processing device capable of counterflow centrifugation can be used. The gene transfer method can be designed appropriately depending on the gene to be transferred.

[0091] (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.

[0092] 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.

[0093] 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.

[0094] (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.

[0095] 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.

[0096] (differentiation induction method) In the method for producing differentiated cells according to 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), followed by adhesion culture (suspension culture using a scaffold material) in a medium containing a TGFβ inhibitor and a GSK3β inhibitor, thereby differentiating them into neural crest cells.

[0097] 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.

[0098] Differentiation into mesenchymal stromal cells can be induced, for example, by the following method: Neural crest cells are seeded in a culture vessel and cultured for one day in CDM medium containing SB431542 and CHIR99021. After one day, the medium is replaced with αMEM containing FBS. Mesenchymal stromal cells can be obtained approximately 14 days after the start of differentiation induction.

[0099] 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.

[0100] Examples of methods for inducing differentiation of pluripotent stem cells into cardiomyocytes include those described in WO2015 / 141827, Laflamme MA and Murry CE, Nature. 473(7347):326-35 (2011), S Funakoshi et al., Sci Rep. 2016 Jan 8:6:19111. doi: 10.1038 / srep19111, etc. Other methods include, for example, a method for producing cardiomyocytes by forming embryoid bodies through suspension culture of induced pluripotent stem cells, a method for producing cardiomyocytes in the presence of a substance that suppresses BMP signaling (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), and a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical (classical) Wnt signaling pathway (WO2007 / 126077). Typically, for example, marker proteins for cardiomyocytes include NKX2.5 (a cardiac muscle-specific transcription factor) and TNNT2 (troponin T), while marker proteins for cardiac progenitor cells include KDR (a receptor for vascular endothelial growth factor (VEGF)) and ISL1 (a LIM homeodomain transcription factor).

[0101] 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.

[0102] 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.

[0103] The method for producing differentiated cells according to the present invention may involve culturing under feeder-free and / or xeno-free conditions for all or part of the period. From the perspective of clinical use, it is preferable that the differentiation induction method of the present invention is carried out under feeder-free and xeno-free conditions for the entire period.

[0104] The method for producing differentiated cells according to 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 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, or the like.

[0105] In the method for producing differentiated cells according to 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 method for producing differentiated cells according to the present invention, and can be performed by adding a known agent for removing undifferentiated cells to the medium (e.g., Di Mao., et al., Angewandte Chemie International Edition; 9 January 2017; Ben-David, U., et al., Cell Stem Cell, 12, 167 (2013); WO2019 / 187918; JP 2016-93178 A; Yoshiki Nakashima, et al., Molecular Therapy Vol. 26 No. 7 July 2018, etc.).

[0106] Quality testing may also be performed as appropriate to determine whether the cells, organoids, etc. obtained by the method for producing differentiated cells according to the present invention are desirable. 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 appropriate for each item can be used.

[0107] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0108] In the examples shown below, induced pluripotent stem cells and differentiated cells derived from said cells were produced by the production method of the present invention using the following reagents, equipment, and materials, and various evaluations were performed. In addition, as a comparative example, experiments using known microcarriers were also performed.

[0109] (1) Reagents and equipment [Microcarriers]: BioNOC II TM Cell Culture Carriers, Esco VacciXcell Cytodex 3, cytiva Corning Microcarrier (Synthemax® coated), Catalog Number: 3781 Corning Microcarrier (no coating), Catalog Number: 3772 Atelocollagen: Takaken Co., Ltd., MIC-00 Sheet-like piece in the present invention: a circular plate formed by punching a polyethylene terephthalate plate with a press device [iMatrix-511]:MATRIXOME [12-well plate]: Multidishes Thermo Fisher Scientific 150200 [TC 6-well plate]: MS-80060 Adherent Cell Culture Plate 6F Independent Well Type, Sumitomo Bakelite [Culture vessel equipped with agitator blades (vertical wheels)]: PBS Bioreactor (PBS0.1), manufactured by PBS Biotech [PBS(-)]: Nacalai Tesque [TrypLE]: TrypLE TM Select(1X) Gibco, 12563-029 [ROCK inhibitor (Y)]: CultureSure 10 mmol / L Y-27632 solution, animal-derived-free, Wako Pure Chemical Industries, Ltd., 035-24593 [StemSpan-AOF]:Stemcell Technologies, British Columbia, Canada [UL 6well plate]:Ultra-Low 6well plate 3471 Corning [6 types of cytokines used in 6CytoMix 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) [PBMC]: Peripheral blood from healthy volunteer donors was collected using CliniMACS Prodigy (Miltenyi Biotec) or BD Vacutainer® CPT TM Separated by [SeV]:SRV4 SRV TM iPSC-4, Tokiwa Bio Co., Ltd., equipped with GFP. [Seesaw shaker]: Wave-SI2 (Taitec) [Microscope]: Olympus Corporation, inverted routine microscope, trinocular tube phase contrast set (pre-centered) CKX41 [Automated cell counter]: Countess II, Thermo Fisher Scientific [Flow cytometry]: SA3800 Spectral Cell Analyzer, Sony [5% CO2 incubator]: Panasonic Healthcare, MCO-170AICUVH-PJ

[0110] (2) Coating of iMatrix on the sheet-like piece in the present invention 1) In all of the examples described below (Examples 6 to 17), up to 10 sheets were placed per well in a non-treated plate 12wp. 1 mL of PBS(-) and 22.8 μL of iMatrix 511 (recommended amount per well x 3 x 2 (for both sides)) were added per well. The sheet was suspended by pipetting to coat the entire surface of the sheet with iMatrix. 2) It was left standing for more than 1 hour. 3) The sheet was washed twice with PBS(-). 4) 1 mL of StemSpan was placed in each well of the well plate. 5) 1.5 mL of StemSpan and 6CytoMix was placed per well in a UL 6-well plate, and the sheet was transferred to the UL 6-well plate. 6) The sheet was left to stand in an incubator at 37°C.

[0111] (2') Coating of iMatrix onto a 6-well control plate For control purposes, we also performed an experiment in which iPS cells were established in 2D without using microcarriers. TC 6-well plates were coated with iMatrix using one of the following methods. 1) A TC 6-well plate was precoated with 9.6 μL of iMatrix511 / 1.5 mL of PBS(-) per well. After coating, the solution was replaced with StemSpan+6CytoMix at 1.2 mL / well and the plate was left to stand at 37°C. 2) 9.6 μL of iMatrix511 and 1.2 mL of StemSpan+6CytoMix were added to a TC 6-well plate and allowed to stand at 37°C.

[0112] (3) Single cell adhesion test 1. Remove the medium from the iPS cells established on the plate that reached 80% confluence and wash with 1 mL of PBS(-). 2.1 mL of 0.5x TrypLE Select was added, and the mixture was left to stand at 37°C for 10 minutes. 3. The supernatant was removed, 1 mL of PBS(-) was added, and the cells were recovered by pipetting. 4. After cell counting, single cells were seeded onto microcarriers using a pipette (recommended seeding volume: 1 cm). 2 5x10 per 3 cells). 5. Culture was performed at 37°C and 5% CO2. 6. Observed under a microscope. 7. The next day, the specimens were observed under a microscope to determine whether they had adhered. The image of Example 13 (diameter 2 mm, 500 μm) observed with the microscope one day after the start of the adhesion test is shown in Figure 9. As shown in Figure 9, the iPS cells were not detached on the day after the start of the adhesion test and were observed to have a leg-like morphology, indicating that they were adhered.

[0113] (4) Passage test (mechanical passage) Following the single cell adhesion test, the following subculture test was performed. For the microcarriers in Example 1, we tried detaching them using TrypLE and adding new iMatrix-coated microcarriers, but we were unable to subculture them. So I tried the following mechanical passage. 1) 4 mL of AK03N+Y was placed in a UL 6-well plate. 2) After confirming that the cells had reached 80% confluence, the medium was replaced with 4 mL of AK03N+Y. 3) Using a Pipetman P1000, the iPS cells were detached by pipetting. 4) Microcarriers (coated if necessary) were placed in the UL 6-well plate prepared in 1). 5) We confirmed under a microscope whether iPS cells had adhered to the newly inserted microcarriers and had been subcultured.

[0114] 〔result〕 FIG. 10 shows the results for the sheet-like piece of Example 13 (diameter 2 mm), showing micrographs of the sheet-like piece on Day 5 after subculture (the disc on the right in the figure) and a newly inserted sheet-like piece (the disc on the left in the figure). Furthermore, Figure 11 shows photographs of the sheet-like piece of Example 13 (diameter 2 mm), showing the sheet-like piece on Day 6 after subculture (disc on the left in the figure) and the sheet-like piece one day after being newly placed (disc on the right in the figure). As is clear from the images in Figures 10 and 11, iPS cell colonies were observed on the newly placed sheet one day after the cells were cultured, indicating that the passage was successful.

[0115] (5) Establishment of iPS cells (SeV infection) 1) A UL 6-well plate containing microcarriers (including the sheet-like pieces of the present invention) was prepared. Only in Example 11, a PBS bioreactor (PBS 0.1) was used instead of a UL 6-well plate. 2) The required amounts of PBMC and SeV (SRV4) were determined as follows and added to the wells in 1). PBMC: 5x10 5 cell / well (1x10 5 Some cells / well were also established.) SeV: MOI=3 (It was also possible to establish SeV at MOI=1 and 0.3.) 3) After adding 2), the cells were placed on a seesaw at 37°C for 30 minutes and then cultured statically at 37°C. In Example 11 only, a PBS bioreactor (PBS 0.1) was used, and the cells were cultured at 37°C while stirring with a vertical wheel (rotation speed 10 rpm). 4) From the next day (day 1), medium was added. Day 1, 3, 5: Medium addition Day 7 and after: The medium was changed every two days. 5) After establishment, the cells on the microcarriers were observed under a microscope. The ease of confirming iPS cell establishment for each microcarrier was evaluated according to the following criteria.

[0116] <Evaluation criteria for ease of cell observation> A: iPS cell colonies were observed on the microcarriers, and it was easy to determine that iPS cells had been established on the microcarriers. B: Cells were observed adhering to the microcarrier, but it was difficult to determine from their shape whether they were differentiated cells or iPS cells. C: It was possible to observe something adhering to the microcarrier, but it was difficult to determine from its shape whether it was an impurity or a cell. D: The adhesion of iPS cells to the microcarriers could not be observed under a microscope, and the adhesion was determined by the change in color of the medium caused by cell metabolites.

[0117] 6) After cell counting, undifferentiated markers were confirmed.

[0118] 〔result〕 The state of iPS cell establishment in the control (2D) and in the case of using microcarriers was observed under a microscope. The micrographs are shown in Figure 12. Micrographs of iPS cells established on each microcarrier are shown in Figures 13 to 15.

[0119] As is clear from Figures 12 to 15, when using bead microcarriers, it takes several days to determine whether iPS cells have been established, whereas when the sheet-like piece of the present invention is used as the microcarrier, establishment can be confirmed as early as one or two days after establishment. Furthermore, when the microcarrier is a transparent and flat sheet like the sheet-like piece of the present invention (a PET disk), iPS cell colonies are easily formed, just like in 2D culture on a 6-well plate, making microscopic observation easier. These results (Examples 1 to 18) are shown in Tables 1 and 2 below. In Table 1, the FACS results for Example 4 are the results of measurements on cells after one passage, and "*" indicates a comparative example.

[0120] [Table 1]

[0121] [Table 2]

[0122] As shown by the flow cytometry results, the sheet-like piece of the present invention tended to have stronger expression of undifferentiated markers at P0 than commercially available microbeads (Tables 1 and 2, Figures 21 to 24). This suggests that cells can more easily initiate reprogramming when attached to a flat surface (sheet-like surface) than to a convex surface such as a microbead.

[0123] (6) Monocyte differentiation In Examples 1, 3, 4, 6, 8 to 10, 13, 14, and 17, iPS cells were induced to differentiate into monocytes without detaching them from each microcarrier using the protocol described in Noriko Shimasaki, et al., Cytotherapy 25 (2023) 1338-1348. In Example 11, differentiation was performed using a PBS bioreactor (PBS 0.1) with stirring using a vertical wheel (rotation speed 10 rpm). As a result, monocytes were observed under a microscope 10 days after the start of differentiation induction. On day 20 after the start of differentiation, the cells were detached from the microcarriers 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, which are markers of monocytic differentiation, was examined.

[0124] BioNOC TM The analysis results of II Cell Culture Carriers (Example 1) are shown in FIG. Furthermore, Figure 17 shows a micrograph of the sheet-like piece of the present invention (Example 6: PET disc, diameter 0.8 mm) taken 20 days after the start of differentiation, and Figure 18 shows the results of the flow cytometry analysis described above. As can be seen from Figures 16 to 18, the sheet-like piece of the present invention was capable of monocyte differentiation, similar to commercially available microcarriers.

[0125] (7) Differentiation into cardiomyocytes In Examples 1 and 6, the iPS cells on each microcarrier were differentiated into cardiomyocytes without detachment using the cardiac differentiation method described on the Kyoto University iPS Cell Research Foundation webpage (https: / / www.cira-foundation.or.jp / 2023 / 04 / cardiomyocytes_protocol.pdf). In Example 1, the troponin T positivity rate on day 15 from the start of differentiation was measured to be 22.1% (Figure 19), and in Example 6, the troponin T positivity rate on day 15 from the start of differentiation was measured to be 50.7% (Figure 20). These results demonstrate that the cell production method of the present invention is a significantly superior method compared to conventional methods.

[0126] Example 18 (1) Adhesion test In the same manner as in the method described above in "(3) Single cell adhesion test," adhesion of the iPS cells to the discs was confirmed using the iPS cells before seeding (maintenance culture day 0).

[0127] 〔result〕 Adhesion of iPS cells to the discs was confirmed by microscopic observation (Figure 25). The cells were then maintained in the culture medium, and adhesion of the iPS cells to the discs was confirmed by microscopic observation (Figure 26).

[0128] (2) Establishment of iPS cells (SeV infection) The experiment was carried out in the same manner as described in "(5) Establishment of iPS cells (SeV infection)" above.

[0129] 〔result〕 ·Establishment day0 SeV infection The granular objects in Figure 27 are PBMCs. It was confirmed that when a disc was placed from the time of establishment, iPS cells could be established while adhering to the disc (Figure 27). ·Establishment day13 As a result of staining with TRA-1-BV421 and SSEA4-PreCP-Cy5.5 and performing flow cytometry, the expression of reprogramming (undifferentiated) markers was confirmed as shown in Figures 29 and 30 . ·Establishment day15 As shown in Figure 28, the iPS cells proliferated. The results of Example 18 are shown in Table 3 below.

[0130] (3) Differentiation into cardiomyocytes When differentiated into cardiomyocytes using the same method as in Example 1, pulsation was observed on day 12 from the start of differentiation.

[0131] [Table 3] [Industrial Applicability]

[0132] The present invention provides a method for producing iPS cells using a new scaffold material that can alleviate the problems associated with conventional microcarriers, and makes it possible to preferably produce differentiated cells using iPS cells obtained by this production method. [Explanation of symbols]

[0133] 10 Liquid medium 20 somatic cells 30 Initialization factor 100 sheets 101 One main surface of the sheet piece (one side of the sheet surface) 102 The other main surface of the sheet piece (rear sheet surface)

Claims

1. A method for producing induced pluripotent stem cells, comprising: a step (s1) of contacting a somatic cell with a reprogramming factor; a step (s2) of establishing induced pluripotent stem cells from the somatic cells in the presence of a sheet-like piece having a surface containing a polymer material as at least one main surface; The method comprising:

2. The area of ​​one main surface of the sheet-like piece is 0.007 to 80 mm ２ The method according to claim 1, wherein the thickness of the sheet-like piece is 0.005 to 3 mm.

3. 3. The method according to claim 1 or 2, wherein the overall shape of the sheet-like piece is a disk, one main surface of the disk is entirely curved, or one main surface of the disk is bent in two at the diameter portion.

4. The method according to claim 1 or 2, wherein the sheet-like piece has a single layer structure made of the polymer material.

5. 3. The method of claim 1 or 2, wherein the polymeric material is polyethylene terephthalate.

6. The method according to claim 1 or 2, wherein at least the surface of the sheet-like piece made of a polymer material further comprises an extracellular matrix.

7. The method according to claim 1 or 2, further comprising, after step (s2), a step (s3) of expanding the induced pluripotent stem cells established in step (s2).

8. The method of claim 7, wherein step (s3) comprises the steps of detaching the induced pluripotent stem cells from the sheet-like piece by stirring in a liquid medium in which the induced pluripotent stem cells adhered to the sheet-like piece are present, and expanding and culturing the detached induced pluripotent stem cells.

9. The method according to claim 8, wherein the step of peeling by stirring is carried out by rotating a stirring blade and by the impact when the sheet-like piece collides with the stirring blade, or by the flow of the liquid medium caused by the stirring blade.

10. The method of claim 7, wherein the expansion culture in step (s3) is carried out by introducing a new sheet piece into a liquid medium containing induced pluripotent stem cells adhered to the sheet piece, allowing the sheet piece (a1) to contact the newly introduced sheet piece (a2) by settling, transferring the induced pluripotent stem cells proliferated on the sheet piece (a1) to the sheet piece (a2), and proliferating the induced pluripotent stem cells on the sheet piece (a2).

11. A method for producing differentiated cells, comprising a step (s4) of inducing differentiation of induced pluripotent stem cells produced by the method of claim 1 or 2.

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