Method for producing induced pluripotent stem cells

Abstract

Provided is a method for producing induced pluripotent stem cells with which it is possible to easily obtain induced pluripotent stem cells having good quality in a short time. A method for producing induced pluripotent stem cells according to the present invention comprises, in the following order: an inoculation step for inoculating somatic cells on a cell culture substrate provided with a cell scaffold material; a removal step for removing the suspended somatic cells that do not adhere to the cell scaffold material; and an introduction step for introducing a reprogramming factor into the somatic cells adhered to the cell scaffold material.

Description

Technical Field

[0001] The present invention relates to a method for producing induced pluripotent stem cells. Background Art

[0002] Induced pluripotent stem cells (iPS cells) are attracting attention in research fields such as academia, drug development, and regenerative medicine. iPS cells can be generated by introducing reprogramming factors (e.g., OCT3 / 4, SOX2, KLF4, and c-MYC) into somatic cells (e.g., Patent Document 1).

[0003] As a method for producing iPS cells, a method of culturing somatic cells into which reprogramming factors have been introduced on a plate coated with laminin is widely used (for example, Non-Patent Document 1). Laminin functions as a cell scaffold.

[0004] However, Patent Document 2 below describes a cell culture scaffold material comprising a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. Furthermore, the Examples in Patent Document 2 describe the use of this scaffold material for culturing iPS cells. However, Patent Document 2 does not describe the establishment of iPS cells from somatic cells.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2019-162054

[0008] Patent Document 2: WO2020 / 230885A1

[0009] Non-patent literature

[0010] Non-Patent Document 1: Protocol for Establishing and Maintaining Human iPS Cells in a Feeder-Free Environment, CiRA_Ff-iPSC_protocol_JP_v140310 (iPS Cell Research Institute, Kyoto University, https: / / www.cira.kyoto-u.ac.jp / j / research / img / protocol / hipsprotocolFf_140311.pdf, English version URL: https: / / www.cira.kyoto-u.ac.jp / j / research / img / protocol / Ff-iPSC-culture_protocol_E_v140311.pdf) Summary of the Invention

[0011] Technical problem to be solved by the invention

[0012] In the past, somatic cells were inoculated into a cell culture vessel and cultured, then the suspended somatic cells were recovered in another vessel and reprogramming factors were introduced into the recovered somatic cells to produce induced pluripotent stem cells (iPS cells). However, this previous method required the step of recovering the suspended somatic cells in another vessel, making the operation complicated.

[0013] Furthermore, conventional methods have made it difficult to obtain high-quality induced pluripotent stem cells in a short period of time. For example, conventional methods have sometimes failed to sufficiently increase the positive rate of TRA-1-60, an undifferentiation marker.

[0014] An object of the present invention is to provide a method for producing induced pluripotent stem cells, which can easily and quickly obtain high-quality induced pluripotent stem cells.

[0015] Technical means to solve the problem

[0016] This specification discloses the following method for producing induced pluripotent stem cells.

[0017] Item 1. A method for producing induced pluripotent stem cells, comprising:

[0018] a seeding step of seeding somatic cells on a cell culture substrate having a cell scaffold material;

[0019] a removal step of removing somatic cells that are not attached to the cell scaffold material and are suspended; and

[0020] The introduction step is to introduce reprogramming factors into the somatic cells adhered to the cell scaffold material.

[0021] Item 2. The method for producing induced pluripotent stem cells according to Item 1, wherein:

[0022] The somatic cells inoculated in the inoculation step include peripheral blood mononuclear cells.

[0023] Item 3. The method for producing induced pluripotent stem cells according to Item 1 or 2, wherein:

[0024] The somatic cells adhered to the cell scaffold material in the introduction step include cells having phagocytic function.

[0025] Item 4. The method for producing induced pluripotent stem cells according to any one of Items 1 to 3, wherein

[0026] The cell scaffold material has an RGD sequence.

[0027] Item 5. The method for producing induced pluripotent stem cells according to any one of Items 1 to 4, wherein:

[0028] The cell scaffold material comprises a peptide-containing resin having a synthetic resin portion and a peptide portion.

[0029] Item 6. The method for producing induced pluripotent stem cells according to Item 5, wherein:

[0030] The peptide-containing resin has a polyvinyl acetal resin portion and a peptide portion.

[0031] Item 7. The method for producing induced pluripotent stem cells according to any one of Items 1 to 6, wherein

[0032] In the introduction step, reprogramming factors are introduced into the somatic cells adhered to the cell scaffold material using a Sendai virus vector.

[0033] Effects of the Invention

[0034] The method for producing induced pluripotent stem cells according to the present invention comprises the following steps:

[0035] a seeding step of seeding somatic cells on a cell culture substrate having a cell scaffold material;

[0036] a removal step of removing somatic cells that are not attached to the cell scaffold material and are suspended; and

[0037] The introduction step is to introduce reprogramming factors into the somatic cells adhered to the cell scaffold material. In the method for producing induced pluripotent stem cells according to the present invention, since it has the above-mentioned structure, it is possible to obtain high-quality induced pluripotent stem cells simply and in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] [ Figure 1 ] Figure 1 This is a dot plot of FSC-SSC when the somatic cells removed in the removal step (suspended somatic cells) and the somatic cells adhered to the cell scaffold material after the removal step were measured by flow cytometry in Example 1.

[0039] [ Figure 2 ] Figure 2 This is a microscopic photograph obtained when the phagocytic ability of somatic cells adhered to the cell scaffold material after the removal step in Example 1 was evaluated.

[0040] [ Figure 3 ] Figure 3 These are microscopic photographs of somatic cells into which reprogramming factors were introduced in Example 1 and Comparative Example 2, taken one day after culture.

[0041] [ Figure 4 ] Figure 4 This is a graph showing the relationship between the number of passages after culturing somatic cells into which reprogramming factors have been introduced and the virus survival rate.

[0042] [ Figure 5 ] Figure 5 This is a graph showing the relationship between the number of passages after culturing somatic cells into which reprogramming factors have been introduced and the expression level of TRA-1-60.

[0043] [ Figure 6 ] Figure 6 This is a microscopic photograph of the iPS cell colony established from monocytes in Example 3. DETAILED DESCRIPTION

[0044] Hereinafter, the present invention will be described in detail.

[0045] The method for producing induced pluripotent stem cells according to the present invention comprises the following steps:

[0046] a seeding step of seeding somatic cells on a cell culture substrate having a cell scaffold material;

[0047] a removal step of removing somatic cells that are not attached to the cell scaffold material and are suspended; and

[0048] The introduction step is to introduce reprogramming factors into the somatic cells adhered to the cell scaffold material.

[0049] In the method for producing induced pluripotent stem cells according to the present invention, since it includes the above-mentioned configuration, high-quality induced pluripotent stem cells can be obtained simply and in a short time.

[0050] Hereinafter, in this specification, “induced pluripotent stem cells” may be referred to as “iPS cells”.

[0051] In the past, iPS cells were produced by seeding somatic cells into a cell culture vessel and culturing them. The suspended somatic cells were then recovered in another vessel, and reprogramming factors were introduced into the recovered somatic cells to produce iPS cells. The present inventors unexpectedly discovered that, instead of introducing reprogramming factors into the suspended somatic cells, the reprogramming factors were introduced into the somatic cells adhered to a cell scaffold material after the suspended somatic cells were removed. This allowed for the simple and rapid production of high-quality iPS cells. The iPS cell production method of the present invention eliminates the need for the complex and careful process of recovering the suspended somatic cells. The same cell culture substrate can be used for the seeding and introduction steps, allowing for the simple and convenient production of iPS cells. Furthermore, the iPS cell production method of the present invention can produce high-quality iPS cells in a short period of time. For example, the iPS cell production method of the present invention can produce iPS cells with high expression levels of TRA-1-60, a marker of undifferentiation, at an early stage. Therefore, the iPS cell production method of the present invention allows for the simple and rapid establishment of iPS cells with high undifferentiated properties. Furthermore, in the method for producing iPS cells of the present invention, when a viral vector is used as a reprogramming factor, the time until the viral vector disappears is shortened, and iPS cells with a low amount of residual viral vector can be obtained.

[0052] Furthermore, the iPS cell production method of the present invention can reduce the amount of reprogramming factors required to produce iPS cells compared to conventional methods, thereby reducing the production cost of iPS cells.

[0053] First, somatic cells and cell culture substrates that can be used in the present invention are described.

[0054] (somatic cells)

[0055] As the somatic cells, conventionally known somatic cells used for establishing iPS cells can be used. The somatic cells are preferably animal cells, more preferably mammalian cells, and even more preferably human cells.

[0056] Examples of the somatic cells include blood cells, fibroblasts, hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, and dental pulp cells. Sources of the somatic cells include peripheral blood, umbilical cord blood, skin tissue, and teeth.

[0057] Examples of the blood cells include nucleated cells such as mononuclear cells, neutrophils, eosinophils, basophils, and lymphocytes. The blood cells may be endothelial progenitor cells, blood stem / progenitor cells, T cells, or B cells.

[0058] The somatic cells inoculated in the inoculation step are preferably blood cells, more preferably mononuclear cells, and even more preferably peripheral blood mononuclear cells (PMBCs). In the inoculation step, the cell population comprising peripheral blood mononuclear cells (PMBCs) is preferably inoculated onto a cell culture substrate having a cell scaffold material. Peripheral blood mononuclear cells (PMBCs) are particularly preferably used because they have low invasiveness to the patient when collected, have a low probability of genomic mutation, and are easy to sterile.

[0059] (Cell Culture Substrate)

[0060] The cell culture substrate comprises a cell scaffold material. The cell culture substrate may be a cell culture container, a cell culture microcarrier, a cell culture fiber, or other materials.

[0061] The cell culture substrate preferably includes a base material (cell culture substrate main body) and a cell scaffold material.

[0062] <Substrate (Cell Culture Substrate Main Body)>

[0063] The material, size, and shape of the substrate are not particularly limited. As the substrate, a conventionally known substrate can be used.

[0064] Examples of the substrate include containers, fibers, nonwoven fabrics, hollow fibers, particles, membranes, and porous membranes.

[0065] Examples of the material of the substrate include synthetic resins, metals, and glass. Examples of the synthetic resin include polystyrene, polyethylene, polypropylene, polyethersulfone, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamideimide, (meth)acrylic resin, epoxy resin, and polysiloxane.

[0066] The substrate is preferably a container. The cell culture substrate is preferably a cell culture container. The cell culture substrate is preferably a cell culture container comprising a container (cell culture container body) and a cell scaffold material disposed on the first surface of the container. In this case, the cell scaffold material may be disposed on the entire first surface of the container or on a portion of the first surface of the container.

[0067] The shape and size of the container (cell culture container body) are not particularly limited. Examples of the container (cell culture container body) include 2-384 well plates, single-layer culture flasks, multi-layer culture flasks, multi-faceted culture flasks, culture dishes, roller bottles, bags, insert cups, and microfluidic chips. The container can be a closed system container. The first surface of the container is preferably the bottom surface of the container.

[0068] The substrate is preferably a particle (substrate particle). The cell culture substrate is preferably a cell culture microcarrier. The cell culture substrate is preferably a cell culture microcarrier having a cell scaffold material of a substrate particle and a cell coating the outer surface of the substrate particle. In this case, the cell scaffold material can coat the entire outer surface of the substrate particle or a part of the outer surface of the substrate particle.

[0069] <Cell Scaffold Materials>

[0070] The cell culture substrate comprises a cell scaffold material, which is used as a scaffold for cells when culturing the cells.

[0071] Examples of the components contained in the cell scaffold material include cell adhesive proteins and synthetic resins, etc. These components may be used alone or in combination of two or more.

[0072] The cell scaffold material preferably has an RGD sequence. More specifically, the cell scaffold material preferably includes a component having an RGD sequence. In this case, the effects of the present invention can be more effectively exerted.

[0073] Examples of the cell adhesive protein include laminin, vitronectin, and collagen. The cell scaffold material may include a cell adhesive protein such as matrigel. Examples of the matrigel include matrigel derived from mouse sarcoma.

[0074] As described below, the synthetic resin may include a synthetic resin having a synthetic resin portion and a peptide portion (synthetic resin bound to a peptide). The synthetic resin may include a synthetic resin not having a peptide portion (synthetic resin not bound to a peptide). Examples of the synthetic resin include polyvinyl alcohol derivatives, peptide-containing polyvinyl alcohol derivatives having a polyvinyl alcohol derivative portion and a peptide portion, (meth) acrylic copolymers, and peptide-containing (meth) acrylic copolymers having a poly(meth)acrylate portion and a peptide portion. Examples of the polyvinyl alcohol derivative include polyvinyl acetal resins.

[0075] Hereinafter, in this specification, the “synthetic resin having a synthetic resin portion and a peptide portion” may be referred to as a “peptide-containing resin”.

[0076] The cell scaffold material preferably comprises a cell adhesive protein or a synthetic resin, more preferably comprises laminin, vitronectin, or a peptide-containing resin, and even more preferably comprises a peptide-containing resin. In this case, the effects of the present invention can be more effectively exerted.

[0077] <Peptide-conjugated resin>

[0078] From the perspective of further effectively exerting the effects of the present invention and improving the manufacturing efficiency of the cell culture substrate, the cell scaffold material preferably comprises a peptide-containing resin having a synthetic resin portion and a peptide portion. The peptide-containing resin is a synthetic resin bound to a peptide. The peptide-containing resin comprises a synthetic resin portion and a peptide portion. The peptide-containing resins may be used alone or in combination of two or more.

[0079] The synthetic resin portion preferably has a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion, more preferably a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion. The peptide-containing resin preferably has a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion and a peptide portion. In this case, the effects of the present invention can be more effectively exerted. The peptide-containing resin may have both a polyvinyl alcohol derivative portion and a poly(meth)acrylate portion.

[0080] In the peptide-containing resin having the polyvinyl alcohol derivative moiety, the polyvinyl alcohol derivative moiety is preferably bound to the peptide moiety via a linker moiety. Therefore, the peptide-containing resin having the polyvinyl alcohol derivative moiety preferably comprises a polyvinyl alcohol derivative moiety, a peptide moiety, and a linker moiety.

[0081] In the peptide-containing resin having the poly(meth)acrylate moiety, the poly(meth)acrylate moiety and the peptide moiety may be bound via a linker moiety or directly bound without a linker moiety. The peptide-containing resin having the poly(meth)acrylate moiety may comprise a poly(meth)acrylate moiety, a peptide moiety, and a linker moiety.

[0082] In this specification, “(meth)acrylic” refers to one or both of “acrylic” and “methacrylic”, and “(meth)acrylate” refers to one or both of “acrylate” and “methacrylate”.

[0083] <<Polyvinyl alcohol derivatives section>>

[0084] The polyvinyl alcohol derivative portion (polyvinyl alcohol derivative skeleton) is a structural portion derived from a polyvinyl alcohol derivative. The polyvinyl alcohol derivative is a compound derived from polyvinyl alcohol.

[0085] The peptide-containing resin is preferably a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. The cell scaffold material preferably comprises a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. By using the peptide-containing polyvinyl alcohol derivative, the effects of the present invention can be further effectively exerted, and cell proliferation can be further improved.

[0086] From the perspective of further effectively exerting the effects of the present invention and further enhancing cell proliferation, the polyvinyl alcohol derivative is preferably a polyvinyl acetal resin, and the polyvinyl alcohol derivative portion is preferably a polyvinyl acetal resin portion. That is, the peptide-containing resin preferably comprises a polyvinyl acetal resin portion and a peptide portion. The cell scaffold material preferably comprises a peptide-containing polyvinyl acetal resin portion having a polyvinyl acetal resin portion and a peptide portion. The polyvinyl alcohol derivative and the polyvinyl acetal resin may be used singly or in combination of two or more.

[0087] The polyvinyl alcohol derivative portion and the polyvinyl acetal resin portion preferably have acetal groups, hydroxyl groups, and acetyl groups in their side chains. However, the polyvinyl alcohol derivative portion and the polyvinyl acetal resin portion may not have acetyl groups, for example. For example, by binding all acetyl groups in the polyvinyl acetal resin portion to the linker, the polyvinyl alcohol derivative portion and the polyvinyl acetal resin portion may not have acetyl groups.

[0088] The polyvinyl acetal resin can be synthesized by acetalizing polyvinyl alcohol with aldehyde.

[0089] The aldehyde used in the acetalization of polyvinyl alcohol is not particularly limited. Examples of the aldehyde include aldehydes having 1 to 10 carbon atoms. The aldehyde may or may not have a chain aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a chain aldehyde or a cyclic aldehyde. The aldehyde may be used alone or in combination of two or more.

[0090] From the perspective of further improving the adhesion of the cell scaffold material to the cells and further effectively exerting the effects of the present invention, the aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde or valeraldehyde, more preferably butyraldehyde. Therefore, the polyvinyl acetal resin is more preferably a polyvinyl butyral resin. From the perspective of further improving the adhesion of the cell scaffold material to the cells and further effectively exerting the effects of the present invention, the polyvinyl acetal resin portion is preferably a polyvinyl butyral resin portion. The peptide-containing resin preferably has a polyvinyl butyral resin portion and a peptide portion. The cell scaffold material preferably comprises a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion and a peptide portion.

[0091] In the peptide-containing polyvinyl acetal resin, the degree of acetalization of the polyvinyl acetal resin portion (in the case of the polyvinyl butyral resin portion, the degree of butyralization) is preferably 40 mol% or more, more preferably 50 mol% or more, preferably 90 mol% or less, and more preferably 85 mol% or less. When the degree of acetalization is above the lower limit, the peptide-containing polyvinyl acetal resin is less likely to swell in the culture medium. When the degree of acetalization is below the upper limit, the solubility in the solvent can be improved.

[0092] In the peptide-containing polyvinyl acetal resin, the hydroxyl content (hydroxyl amount) of the polyvinyl acetal resin portion is preferably 15 mol% or more, more preferably 20 mol% or more, preferably 45 mol% or less, more preferably 30 mol% or less, and further preferably 25 mol% or less.

[0093] In the peptide-containing polyvinyl acetal resin, the degree of acetylation (amount of acetyl groups) of the polyvinyl acetal resin portion is preferably 1 mol% or more, more preferably 2 mol% or more, preferably 5 mol% or less, more preferably 4 mol% or less. When the degree of acetylation is above the lower limit and below the upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be improved.

[0094] The degree of acetalization, degree of acetylation and amount of hydroxyl groups of the polyvinyl acetal resin can be 1 The results were measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0095] <<Poly(meth)acrylate portion>>

[0096] The poly(meth)acrylate portion (poly(meth)acrylate backbone) is a structural portion derived from poly(meth)acrylate.

[0097] The peptide-containing resin preferably has a poly(meth)acrylate portion, more preferably a peptide-containing (meth)acrylic acid copolymer having a poly(meth)acrylate portion and a peptide portion. The cell scaffold material preferably comprises a peptide-containing (meth)acrylic acid copolymer having a poly(meth)acrylate portion and a peptide portion. By using the peptide-containing (meth)acrylic acid copolymer, the effects of the present invention can be further effectively exerted, and the proliferation of cells can be further improved.

[0098] The poly(meth)acrylate portion has a skeleton derived from (meth)acrylate. The poly(meth)acrylate is obtained by polymerizing (meth)acrylate. The poly(meth)acrylate may be used alone or in combination of two or more.

[0099] Examples of the (meth)acrylate include alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aryl (meth)acrylates, polyethylene glycol (meth)acrylates, phosphorylcholine (meth)acrylates, etc. The (meth)acrylates may be used alone or in combination of two or more.

[0100] Examples of the alkyl (meth)acrylate include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, tert-butyl (meth)acrylate, n-octyl (meth)acrylate, isooctyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, isononyl (meth)acrylate, decyl (meth)acrylate, isodecyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, and isotetradecyl (meth)acrylate.

[0101] The alkyl (meth)acrylate may be substituted with a substituent such as an alkoxy group having 1 to 3 carbon atoms or a tetrahydrofurfuryl group. Examples of such alkyl (meth)acrylate include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0102] Examples of the cyclic alkyl (meth)acrylate include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0103] Examples of the aryl (meth)acrylate include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0104] Examples of the (meth)acrylic polyethylene glycols include methoxy-polyethylene glycol (meth)acrylate, ethoxy-polyethylene glycol (meth)acrylate, hydroxy-polyethylene glycol (meth)acrylate, methoxy-diethylene glycol (meth)acrylate, ethoxy-diethylene glycol (meth)acrylate, hydroxy-diethylene glycol (meth)acrylate, methoxy-triethylene glycol (meth)acrylate, ethoxy-triethylene glycol (meth)acrylate, and hydroxy-triethylene glycol (meth)acrylate.

[0105] Examples of the (meth)acryloylphosphocholine include 2-(meth)acryloyloxyethylphosphocholine.

[0106] The peptide-containing resin having a poly(meth)acrylate portion may have a skeleton derived from a monomer other than (meth)acrylate.

[0107] Examples of monomers other than the (meth)acrylate include (meth)acrylamides and vinyl compounds. The monomers other than the (meth)acrylate may be used alone or in combination of two or more.

[0108] Examples of the (meth)acrylamides include (meth)acrylamide, N-isopropyl (meth)acrylamide, N-tert-butyl (meth)acrylamide, N,N'-dimethyl (meth)acrylamide, (3-(meth)acrylamidopropyl)trimethylammonium chloride, 4-(meth)acryloylmorpholine, 3-(meth)acryloyl-2- Oxazolidinone, N-[3-(dimethylamino)propyl](meth)acrylamide, N-(2-hydroxyethyl)(meth)acrylamide, N-hydroxymethyl(meth)acrylamide, and 6-(meth)acrylamidohexanoic acid, etc.

[0109] Examples of the vinyl compound include ethylene, allylamine, vinyl pyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, and vinylamine.

[0110] <<Peptide Section>>

[0111] The peptide portion (peptide backbone) is a structural portion derived from a peptide. The peptide portion has an amino acid sequence. The peptide constituting the peptide portion may be an oligopeptide or a polypeptide. The peptide may be used alone or in combination of two or more.

[0112] The number of amino acid residues in the peptide portion is preferably 3 or more, more preferably 4 or more, further preferably 5 or more, preferably 10 or less, more preferably 8 or less, and further preferably 6 or less. When the number of amino acid residues is above the lower limit and below the upper limit, the adhesion between the cell scaffold material and the inoculated cells can be further improved, and the cell proliferation rate can be further increased. The number of amino acid residues in the peptide portion can exceed 10 or exceed 15.

[0113] The peptide portion preferably has an amino acid sequence with cell adhesion. It should be noted that the amino acid sequence with cell adhesion refers to an amino acid sequence whose cell adhesion activity has been confirmed by phage display, agarose bead method, or plate coating method. As the phage display method, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 1189-1196" can be used. As the agarose bead method, for example, the method described in "Protein Nuclease Vol. 45 No. 15 (2000) 2477" can be used. As the plate coating method, for example, the method described in "Protein Nuclease Vol. 45 No. 15 (2000) 2477" can be used.

[0114] Examples of the cell adhesive amino acid sequence include the RGD sequence (Arg-Gly-Asp), the YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), the PDSGR sequence (Pro-Asp-Ser-Gly-Arg), the HAV sequence (His-Ala-Val), the ADT sequence (Ala-Asp-Thr), the QAV sequence (Gln-Ala-Val), the LDV sequence (Leu-Asp-Val), the IDS sequence (Ile-Asp-Ser), the REDV sequence (Arg-Glu-Asp-Val), the IDAPS sequence (Ile-Asp-Ala-Pro-Ser), the KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val), and the TDE sequence (Thr-Asp-Glu). Examples of the cell-adhesive amino acid sequence include those described in "Patiophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Journal of the Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, pp. 58-66, 1992." The peptide portion may have only one of the cell-adhesive amino acid sequences or two or more of them.

[0115] The peptide portion preferably comprises at least one of the cell-adhesive amino acid sequences, more preferably comprises at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, further preferably comprises an RGD sequence, and particularly preferably comprises at least an RGD sequence represented by the following formula (1). In this case, the adhesion between the cell scaffold material and the seeded cells can be further improved, and the cell proliferation rate can be further increased.

[0116] Arg-Gly-Asp-X ···Formula (1)

[0117] In the formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro or Asn.

[0118] The peptide portion may be linear or may have a cyclic peptide backbone. The cyclic peptide backbone refers to a cyclic backbone composed of multiple amino acids. From the perspective of more effectively exerting the effects of the present invention, the cyclic peptide backbone is preferably composed of 4 or more amino acids, more preferably composed of 5 or more amino acids, and preferably composed of 10 or less amino acids.

[0119] In the described peptide-containing resin, the containing rate of the peptide portion is preferably more than 0.05 % by mole, more preferably more than 0.1 % by mole, more preferably more than 0.15 % by mole, particularly preferably more than 0.2 % by mole, preferably below 25 % by mole, more preferably below 20 % by mole, more preferably below 15 % by mole, particularly preferably below 10 % by mole. When the containing rate of the peptide portion is above the lower limit, the adhesion of the cell scaffold material and the inoculated cell can be further improved, the proliferation rate of the cell can be further improved. In addition, if the containing rate of the peptide portion is below the upper limit, manufacturing cost can be suppressed.

[0120] The content rate (mol %) of the peptide portion is the amount of the peptide portion relative to the total amount of each structural unit constituting the peptide-containing resin.

[0121] The content of the peptide portion can be measured by, for example, NMR (nuclear magnetic resonance).

[0122] <<Joint Section>>

[0123] The linker portion is a structural portion derived from a joint. The linker portion is generally located between the polyvinyl alcohol derivative portion or the poly(meth)acrylate portion and the peptide portion. The polyvinyl alcohol derivative portion or the poly(meth)acrylate portion and the peptide portion are combined via the linker portion. The linker portion is formed by a linker (cross-linking agent). The linker may be used alone or in combination of two or more.

[0124] The linker is preferably a compound having a functional group capable of binding to the peptide, more preferably a compound having a functional group capable of condensing with a carboxyl group of the peptide or a functional group capable of condensing with an amino group.

[0125] Examples of the functional group capable of condensing with the carboxyl group of the peptide or the functional group capable of condensing with the amino group include a carboxyl group, a thiol group, an amino group, a hydroxyl group, and a cyano group.

[0126] From the viewpoint of reacting well with a peptide, the linker is preferably a compound having a carboxyl group or an amino group, and more preferably a compound having a carboxyl group.

[0127] When obtaining a peptide-containing resin having a polyvinyl alcohol derivative portion, examples of the linker having a carboxyl group include (meth)acrylic acid and carboxyl-containing acrylamide. By using a carboxylic acid (carboxylic acid monomer) having a polymerizable unsaturated group as the linker having a carboxyl group, the carboxylic acid monomer can be polymerized by graft polymerization during the introduction of the linker, thereby increasing the number of carboxyl groups reactive with the peptide.

[0128] From the viewpoint of achieving good binding between the polyvinyl alcohol derivative and the peptide, the linker is preferably (meth)acrylic acid, and more preferably acrylic acid.

[0129] When obtaining a peptide-containing resin having a poly(meth)acrylate moiety, the linker preferably has a functional group capable of binding to (meth)acrylate. Examples of the functional group capable of binding to (meth)acrylate include vinyl, (meth)acryloyl, and allyl groups. The linker more preferably has a (meth)acryloyl group as the functional group capable of binding to (meth)acrylate, and is preferably a compound having a carboxyl group or an amino group and a (meth)acryloyl group.

[0130] Examples of the linker when obtaining a peptide-containing resin having a poly(meth)acrylate moiety include (meth)acrylic acid, itaconic acid, and acrylamide.

[0131] From the viewpoint of achieving good binding of poly(meth)acrylate to the peptide, the linker is preferably (meth)acrylic acid or itaconic acid, and more preferably (meth)acrylic acid.

[0132] <Additional details of cell scaffold materials>

[0133] The number average molecular weight of the peptide-containing resin is preferably 10,000 or more, more preferably 50,000 or more, further preferably 100,000 or more, preferably 5,000,000 or less, more preferably 2,500,000 or less, and further preferably 1,000,000 or less. When the number average molecular weight is above the lower limit, the cell scaffold material is not easily eluted into the liquid culture medium during cell culture, and the cell scaffold material is not easily peeled off from the substrate during cell culture. When the number average molecular weight is below the upper limit, the solubility in alcohol solvents can be improved.

[0134] It should be noted that the number average molecular weight of the peptide-containing resin can be measured, for example, by the following method. The peptide-containing resin is dissolved in tetrahydrofuran (THF) to prepare a 0.2% by weight solution of the peptide-containing resin. Next, the peptide-containing resin is evaluated using a gel permeation chromatography (GPC) measuring apparatus (APC system, manufactured by Waters) under the following measurement conditions.

[0135] Column: HSPgel HR MB-M 6.0×150mm

[0136] Flow rate: 0.5 mL / min

[0137] Column temperature: 40°C

[0138] Injection volume: 10 μL

[0139] Detector: RI, PDA

[0140] Standard sample: polystyrene

[0141] The cell scaffold material may include a synthetic resin without a peptide portion (a synthetic resin not bound to a peptide) and a peptide-containing resin. The cell scaffold material may include: a synthetic resin having a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion and not having a peptide portion, and a peptide-containing resin. The cell scaffold material may include other components such as a resin that does not have both the polyvinyl alcohol derivative portion and the poly(meth)acrylate portion. As the other components, polyolefin resins, polyether resins, polyesters, epoxy resins, polyamide resins, polyimide resins, polyurethane resins, polycarbonate resins, cellulose, and polypeptides may be cited. Only one of the other components may be used, or two or more may be used in combination.

[0142] The cell scaffold material preferably comprises the peptide-containing resin and does not contain or comprises a synthetic resin to which no peptide is bound.

[0143] The cell scaffold material preferably comprises a layer comprising the peptide-containing resin. The cell scaffold material may comprise only a layer comprising the peptide-containing resin, or may comprise a layer comprising the peptide-containing resin and a layer comprising a synthetic resin to which no peptide is bound. From the perspective of further effectively exerting the effects of the present invention and further enhancing cell proliferation, the first surface comprising the peptide-containing resin layer is preferably the upper surface of the cell scaffold material. The peptide-containing resin is preferably present at least on the surface that adheres to somatic cells (the surface that adheres to somatic cells).

[0144] In 100% by weight of the cell scaffold material, the content of the peptide-containing resin is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the content of the peptide-containing resin is above the lower limit, the effect of the present invention can be more effectively exerted. In 100% by weight of the cell scaffold material, the content of the peptide-containing resin can be 100% by weight or less, or less than 100% by weight.

[0145] In 100% by weight of the cell scaffold material, the total content of the peptide-containing resin and the content of the synthetic resin not bound to the peptide is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the total is above the lower limit, the effect of the present invention can be more effectively exerted. In 100% by weight of the cell scaffold material, the total content of the peptide-containing resin and the content of the synthetic resin not bound to the peptide can be 100% by weight or less, or less than 100% by weight.

[0146] The cell scaffold material preferably comprises a layer comprising the cell adhesive protein. The cell scaffold material may comprise only a layer comprising the cell adhesive protein, or may comprise a layer comprising the cell adhesive protein and a layer not comprising the cell adhesive protein. From the perspective of further effectively exerting the effects of the present invention and further improving cell proliferation, the first surface of the layer comprising the cell adhesive protein is preferably the upper surface of the cell scaffold material. The cell adhesive protein is preferably present at least on the surface that adheres to somatic cells (the surface that adheres to somatic cells).

[0147] In 100% by weight of the cell scaffold material, the content of the cell adhesive protein is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the content of the cell adhesive protein is above the lower limit, the effect of the present invention can be more effectively exerted. In 100% by weight of the cell scaffold material, the content of the cell adhesive protein can be 100% by weight or less, or less than 100% by weight.

[0148] A cell culture substrate comprising a cell scaffold material containing a peptide-containing resin can be prepared, for example, by the following method (A) or (B). (A) is an example of a method for preparing a cell culture substrate using a method for synthesizing a peptide-containing resin by a liquid phase method, and (B) is an example of a method for preparing a cell culture substrate using a method for synthesizing a peptide-containing resin by a solid phase method.

[0149] (A) A solution containing a synthetic resin having a functional group reactive with an amino group or a functional group reactive with a carboxyl group (hereinafter sometimes referred to as "synthetic resin X") and a peptide is prepared. Synthetic resin X and the peptide are reacted to obtain a peptide-containing resin. The solution containing the peptide-containing resin and a solvent is applied to the surface of a substrate, and the solvent is evaporated.

[0150] (B) A solution containing a synthetic resin (synthetic resin X) having a functional group reactive with an amino group or a functional group reactive with a carboxyl group is prepared. The solution containing synthetic resin X is applied to the surface of a substrate. A liquid containing a peptide is added to the surface of the substrate to allow the synthetic resin X to react with the peptide.

[0151] Examples of the synthetic resin X include (meth)acrylate polymers and linker-bonded polyvinyl acetal derivatives.

[0152] A cell culture substrate comprising a cell scaffold material containing a cell adhesive protein can be prepared, for example, by applying a solution containing a cell adhesive protein onto the surface of the substrate and then volatilizing the solvent.

[0153] Hereinafter, the method for producing iPS cells of the present invention will be described in further detail.

[0154] The iPS cell production method sequentially comprises: a seeding step of seeding somatic cells on a cell culture substrate comprising a cell scaffold material; a removal step of removing suspended somatic cells that are not adhered to the cell scaffold material; and an introduction step of introducing reprogramming factors into the somatic cells adhered to the cell scaffold material. In the iPS cell production method, the seeding step through the introduction step can be performed using the same cell culture substrate.

[0155] The method for producing iPS cells preferably comprises a culturing step (A) of culturing the inoculated somatic cells between the inoculation step and the removal step. The method for producing iPS cells preferably comprises the inoculation step, the culturing step (A), the removal step, and the introduction step in sequence.

[0156] The manufacturing method of the iPS cell preferably has a culturing step (B) of culturing somatic cells having the reprogramming factors introduced therein on the surface of the cell scaffold material. The culturing step (B) is a step of culturing the somatic cells using the cell culture substrate used in the introduction step. The manufacturing method of the iPS cell preferably has the inoculation step, the removal step, the introduction step and the culturing step (B) in sequence. The manufacturing method of the iPS cell more preferably has the inoculation step, the culturing step (A), the removal step, the introduction step and the culturing step (B) in sequence. In the manufacturing method of the iPS cell, the same cell culture substrate can be used to carry out the inoculation step to the culturing step (B).

[0157] (Inoculation process)

[0158] The method for producing iPS cells includes a step of seeding somatic cells on a cell culture substrate including a cell scaffold material (seeding step).

[0159] When the cell culture substrate is a cell culture container, the area of ​​the upper surface of the cell scaffold material is 1 cm 2 The seeding density of somatic cells in the seeding step is preferably 2×10 3 cells or more, more preferably 5×10 3 cells or more, more preferably 1×10 4 cells or more, preferably 1×10 8 cells or less, more preferably 1×10 7 cells or less, more preferably 1×10 6cells or less.

[0160] In the case where the cell culture substrate is a cell culture microcarrier or a cell culture fiber, the area of ​​1 cm relative to the outer surface of the cell scaffold material is 2 The seeding density of somatic cells in the seeding step is preferably 1×10 3 cells or more, more preferably 2×10 3 cells or more, more preferably 5×10 3 cells or more, preferably 1×10 7 cells or less, more preferably 1×10 6 cells or less, more preferably 1×10 5 cells or less.

[0161] (Cultivation Step (A))

[0162] The method for producing iPS cells preferably includes a step of culturing the inoculated somatic cells (culturing step (A)). In the culturing step (A), the somatic cells inoculated in the inoculation step are cultured. The culturing step (A) is preferably a step of pre-culturing the somatic cells.

[0163] As the culture medium for culturing somatic cells in the culturing step (A), a conventionally known culture medium can be used. The culture medium is preferably a liquid culture medium. As the culture medium, a commercially available culture medium can be used, or a culture medium prepared by oneself can be used. As the culture medium, for example, the culture medium described in the non-patent document 1 can be used.

[0164] Commercially available culture media include "Stem Fit" manufactured by Ajinomoto, "StemSpan-ACF," "TeSR E8," and "mTeSR1" manufactured by STEMCELL Technologies, "X-VIVO 10" manufactured by Lonza, and "StemFlex" and "Essential 8" manufactured by Thermo Fisher Scientific. Furthermore, the culture media may be supplemented with various cytokines from the commercially available products.

[0165] When peripheral blood mononuclear cells (PMBCs) are used as the somatic cells, a culture medium containing 45-55 ng / mL IL-6, 45-55 ng / mL SCF, 9-11 ng / mL TPO, 18-22 ng / mL Flt-3L, 18-22 ng / mL IL-3, and 9-11 ng / mL G-CSF is preferably used as the final concentration of the cytokines. The culture medium having the above cytokine concentrations can be obtained, for example, by adding these cytokines to the commercially available products so that the final concentrations are the above.

[0166] The culture temperature in the culture step (A) is preferably 35°C or higher, preferably 38°C or lower. The CO2 concentration during the culture step (A) is preferably 4% or higher, preferably 6% or lower. For example, the culture conditions in the culture step (A) are 37°C and a CO2 concentration of 5%.

[0167] The culture time of the culture step (A) is preferably 3 days or more, more preferably 4 days or more, further preferably 5 days or more, preferably 8 days or less, more preferably 7 days or less, and further preferably 6 days or less. The culture time of the culture step (A) is the number of days after the somatic cells are inoculated in the inoculation step. More specifically, the culture time of the culture step (A) is the number of days from the inoculation of somatic cells in the inoculation step to the removal of somatic cells that are not adhered to the cell scaffold material and are suspended in the removal step. The date of inoculation of somatic cells in the inoculation step is set as day 0, and the day after inoculation is set as day 1.

[0168] During the culture step (A), the culture medium may or may not be replaced. If the culture medium is replaced, it is preferably performed after the somatic cells inoculated in the inoculation step have adhered to the cell scaffold material. Specifically, for example, if the culture medium is replaced during the culture step (A), it is preferably performed after the third day from the start of the culture step (A).

[0169] When the culture medium is replaced in the culturing step (A), preferably 20% by volume or more of the culture medium is replaced, more preferably 40% by volume or more of the culture medium is replaced, relative to 100% by volume of the culture medium before the culture medium replacement.

[0170] (Removal process)

[0171] The method for producing iPS cells includes a step of removing somatic cells that are not adhered to the cell scaffold material and are suspended (removal step). Before the removal step, there are somatic cells that are not adhered to the cell scaffold material and are suspended, and somatic cells that are adhered to the cell scaffold material. For example, if the culturing step (A) is performed, there are somatic cells that are not adhered to the cell scaffold material and are suspended, and somatic cells that are adhered to the cell scaffold material. In the removal step, the somatic cells that are not adhered to the cell scaffold material and are suspended are removed.

[0172] In the removal step, the suspended somatic cells may be removed by removing the suspended somatic cells together with the culture medium. Examples of methods for removing the suspended somatic cells together with the culture medium include methods using a pipette. It should be noted that the suspended somatic cells may be washed with a buffer solution such as PBS or a liquid such as a culture medium before or after removal of the suspended somatic cells together with the culture medium.

[0173] From the perspective of easily removing the suspended somatic cells, in the removal step, the method for removing the suspended somatic cells is preferably a method of removing the suspended somatic cells together with the culture medium. In the removal step, the suspended somatic cells are preferably removed by removing the culture medium containing the suspended somatic cells.

[0174] In the removal step, preferably 50% or more of the suspended somatic cells, based on the number of cells, are removed, more preferably 70% or more of the somatic cells are removed, even more preferably 90% or more of the somatic cells are removed, particularly preferably 95% or more of the somatic cells are removed, and most preferably 99% or more of the somatic cells are removed. In this case, the effects of the present invention can be more effectively exerted. In the removal step, less than 100% of the somatic cells, less than 100%, or less than 99% of the somatic cells, based on the number of cells, can be removed from the suspended somatic cells.

[0175] In the case of removing the culture medium in the removal process, relative to 100 volume % of the culture medium before the culture medium is removed, preferably 10 volume % or more of the culture medium is removed, more preferably 20 volume % or more of the culture medium is removed, further preferably 40 volume % or more of the culture medium is removed, further preferably 60 volume % or more of the culture medium is removed, particularly preferably 80 volume % or more of the culture medium is removed, and most preferably 90 volume % or more of the culture medium is removed. If the ratio of the culture medium removed is above the lower limit, more suspended somatic cells can be removed, and thus the effect of the present invention can be further effectively exerted. In the case of removing the culture medium in the removal process, relative to 100 volume % of the culture medium before the culture medium is removed, less than 100 volume % of the culture medium can be removed, less than 95 volume % of the culture medium can be removed, and less than 90 volume % of the culture medium can be removed.

[0176] The time from inoculation of somatic cells in the inoculation step to removal of the suspended somatic cells in the removal step is preferably 1 day or longer, more preferably 2 days or longer, even more preferably 3 days or longer, even more preferably 4 days or longer, and particularly preferably 5 days or longer, preferably 15 days or shorter, more preferably 10 days or shorter, even more preferably 8 days or shorter, even more preferably 7 days or shorter, and particularly preferably 6 days or shorter. When the time is above the lower limit and below the upper limit, the effects of the present invention can be more effectively exerted. The time is the number of days after inoculation of somatic cells in the inoculation step. The date of inoculation of somatic cells in the inoculation step is designated as Day 0, and the day following inoculation is designated as Day 1.

[0177] (Introduction process)

[0178] The manufacturing method of the iPS cell is equipped with a process (introduction process) of introducing reprogramming factors into the somatic cells adhered to the cell scaffold material. In the introduction process, reprogramming factors are introduced into the somatic cells (somatic cell group) adhered to the cell scaffold material after the removal process. That is, in the introduction process, reprogramming factors are introduced into the somatic cells (somatic cell group) remaining on the surface of the cell scaffold material after the removal process.

[0179] In the introduction step, the somatic cells adhered to the cell scaffold material can be introduced with reprogramming factors without collecting the somatic cells adhered to the cell scaffold material. Therefore, iPS cells can be produced easily and conveniently with a simple process.

[0180] The somatic cells adhered to the cell scaffold material during the introduction step preferably include cells with phagocytic activity, more preferably monocytes, macrophages, or dendritic cells, and even more preferably monocytes or macrophages. In this case, high-quality iPS cells can be obtained in a short period of time.

[0181] As the reprogramming factor (nuclear reprogramming factor), conventionally known reprogramming factors can be used.

[0182] Examples of genes included in the 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. The reprogramming factors may be used alone or in combination of two or more.

[0183] The reprogramming factors preferably include Oct family genes, Sox family genes, Klf family genes, Myc family genes, Lin28 family genes or Nanog family genes.

[0184] The reprogramming factors more preferably include Oct family genes, Klf family genes and Sox family genes, further preferably include Oct3 / 4, Sox2 and Klf4, more preferably include Oct3 / 4, Sox2, Klf4 and c-Myc, and more preferably include Oct3 / 4, Sox2, Klf4, c-Myc, Lin28 and Nanog.

[0185] As a method for introducing the reprogramming factors into somatic cells, conventionally known introduction methods can be used.

[0186] Methods for introducing the reprogramming factors into somatic cells include methods using vectors and methods using mRNA. Examples of the vectors include viral vectors such as Sendai virus vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus vectors, as well as non-viral vectors such as episomal vectors and plasmid vectors.

[0187] In the introduction step, it is more preferred to use a vector, more preferably a viral vector, and even more preferably a Sendai virus vector. Specifically, in the introduction step, it is more preferred to use a Sendai virus vector to introduce reprogramming factors into the somatic cells adhered to the cell scaffold material. In this case, safety can be improved.

[0188] Commercially available products of the Sendai virus vector include the "CytoTune-iPS" series manufactured by ID PHARMA and the "SRV iPSC Vector" series manufactured by TOKIWA-BIO.

[0189] In the method for manufacturing the iPS cells, in the introduction step, iPS cells can be established with a small amount of viral vectors compared to previous methods. In the introduction step, the viral vector (particularly the Sendai virus vector) is preferably infected with an MOI (Multiplicity of Infection) of less than 3, more preferably infected with an MOI of less than 2, further preferably infected with an MOI of less than 1, preferably infected with an MOI of more than 0.001, and more preferably infected with an MOI of more than 0.01. In the introduction step, the viral vector (particularly the Sendai virus vector) is preferably infected with an MOI of more than 0.001 and less than 3, more preferably infected with an MOI of more than 0.2 and less than 3, and further preferably infected with an MOI of more than 0.5 and less than 2. In addition, the infection time is preferably more than 15 minutes and less than 4 hours, more preferably more than 30 minutes and less than 4 hours. It should be noted that the MOI in this specification refers to the amount of the viral vector relative to the number of somatic cells inoculated in the inoculation step.

[0190] In the introduction process, mRNA is also preferably used as the reprogramming factor. The reprogramming factor is preferably mRNA. That is, in the introduction process, mRNA can also be used to introduce the reprogramming factor into the somatic cells adhered to the cell scaffold material. In this case, even a smaller amount of mRNA can also be used to produce iPS cells. In addition, when using mRNA, safety can be improved compared to using viral vectors as reprogramming factors.

[0191] When mRNA is used as the reprogramming factor in the introduction step, examples of methods for introducing the reprogramming factor (mRNA) into somatic cells include plasma gene transfer, electroporation, and lipofection.

[0192] The introduction process is preferably carried out within 2 minutes after the suspended somatic cells are removed in the removal process, more preferably within 1 minute, and further preferably within 0.5 minutes. In this case, iPS cells of good quality can be obtained in a shorter period of time. It should be noted that "the introduction process is carried out within X minutes after the suspended somatic cells are removed in the removal process" means that after the suspended somatic cells are removed in the removal process, the time from the somatic cells adhered to the cell scaffold material to the contact with the components (vectors, etc.) containing the reprogramming factors is within X minutes.

[0193] (Cultivation Step (B))

[0194] The method for producing iPS cells preferably includes a step of culturing somatic cells (somatic cell population) into which the reprogramming factors have been introduced on the surface of the cell scaffold material (culturing step (B)). In the culturing step (B), the somatic cells (somatic cell population) obtained in the introduction step are cultured. In the culturing step (B), adherent culture is preferably performed.

[0195] As the culture medium for culturing somatic cells in the culturing step (B), a conventionally known culture medium can be used. The culture medium is preferably a liquid culture medium. As the culture medium, a commercially available culture medium can be used, or a culture medium prepared by oneself can be used. As the culture medium, for example, the culture medium described in the non-patent document 1 can be used.

[0196] Commercially available culture media include "Stem Fit" manufactured by Ajinomoto, "StemSpan-ACF", "TeSR-E8" and "mTeSR1" manufactured by Stemcell Technologies, "X-VIVO 10" manufactured by Lonza, and "StemFlex" and "Essential 8" manufactured by Thermo Fisher Scientific. Furthermore, the culture media may be supplemented with various cytokines from the commercially available products.

[0197] The culture temperature of the culture step (B) is preferably 36°C or higher, preferably 38°C or lower. The CO2 concentration during the culture step (B) is preferably 4% or higher, preferably 6% or lower. For example, the culture conditions of the culture step (B) are 37°C and a CO2 concentration of 5%. In addition, during the culture step (B), the culture medium may or may not be replaced. When the culture medium is replaced, for example, it may be replaced with a fresh culture medium at a frequency of once every two days.

[0198] iPS cells may or may not be established in the culture step (B). Furthermore, it is not necessary to confirm whether iPS cells have been established at the end of the culture step (B). In the method for producing iPS cells of the present invention, iPS cells can be induced, for example, within about 14 days from the start of culture in the culture step (B), to obtain a cell population containing iPS cells.

[0199] (Subculture process)

[0200] From the viewpoint of obtaining a cell population containing iPS cells of higher quality, the method for producing iPS cells preferably comprises a subculturing step of subculturing the cells (cell population) obtained in the culturing step (B).

[0201] In the subculture step, the same type of cell culture substrate as that used in the inoculation step to the culturing step (B) may be used, or a different type of cell culture substrate may be used.

[0202] Examples of the subculturing method in the subculturing step include a colony picking method and a method using a cell detachment agent.

[0203] From the perspective of simplifying cell passage, the subculture method in the subculture step preferably uses a cell detachment agent. Conventional iPS cell production methods require colony selection, particularly during the first subculture. However, the iPS cell production method of the present invention includes both the removal step and the introduction step, allowing the cells to be subcultured using a cell detachment agent even during the first subculture.

[0204] Examples of commercially available cell detachment agents include "TrypLE Select" and "TrypLE Express" manufactured by Thermo Fisher Scientific.

[0205] When culturing cells in the subculture step, adherent culture is preferably performed.

[0206] As the culture medium that can be used in the subculture step, a culture medium known in the past can be used. The culture medium is preferably a liquid culture medium. As the culture medium, a commercially available culture medium can be used, or a culture medium prepared by oneself can be used. As the culture medium, for example, the culture medium described in the non-patent document 1 can be used. As the culture medium, the culture medium described in the culture step (B) column can be used.

[0207] The culture temperature in the subculture step is preferably 36°C or higher, preferably 38°C or lower. The CO2 concentration during the subculture step is preferably 4% or higher, preferably 6% or lower. For example, the culture conditions in the subculture step are 37°C and a CO2 concentration of 5%. Furthermore, the culture medium may or may not be replaced during the subculture step. If the culture medium is replaced, it may be replaced with fresh culture medium every two days, for example.

[0208] The number of cell passages in the subculture process can be 1 time, 2 times, more than 2 times, 3 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, less than 10 times, less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, or less than 4 times.

[0209] Conventional iPS cell production methods require repeated cell passages to obtain high-quality iPS cells. However, the iPS cell production method of the present invention allows high-quality iPS cells to be obtained even with a reduced number of cell passages. Therefore, the iPS cell production method of the present invention allows the production of high-quality iPS cells in a short period of time.

[0210] Whether somatic cells are induced into iPS cells can be confirmed, for example, by observing the morphology of the cells and analyzing by flow cytometry whether undifferentiation markers are expressed in the cells. As such undifferentiation markers, TRA-1-60 and SSEA4 are known. In particular, TRA-1-60 is a specific antigen for iPS cells or ES cells and cannot be detected in somatic cells. Therefore, it is a suitable marker for confirming whether they have been induced into iPS cells (for example, WO2018 / 155595A1, Japanese Patent Application Laid-Open No. 2019-162054, Elayne M Chan1 et al. (Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells, Nature Biotechnology Volume 27 Number 11 November 2009)).

[0211] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

[0212] As cell culture substrates, the following cell culture vessels and cell culture microcarriers were prepared.

[0213] Culture vessel (P) (equipped with a plate containing a cell scaffold material comprising a peptide-containing polyvinyl acetal resin, prepared by the following "Method for preparing a culture vessel (P)")

[0214] Microcarrier (P) (a microcarrier comprising a cell scaffold material comprising a peptide-containing polyvinyl acetal resin, prepared by the following "Microcarrier (P) Preparation Method")

[0215] Culture container (L) (a plate coated with "Laminin 511E8 fragment (iMatrix-511)" manufactured by NIPPI, prepared by the following "Method for preparing culture container (L)")

[0216] It should be noted that in the table below, the "peptide-containing polyvinyl acetal resin" serving as the cell scaffold material possessed by the culture vessel (P) and the microcarrier (P) is recorded as "PVB-AA", and the "laminin 511E8 fragment (iMatrix-511)" serving as the cell scaffold material possessed by the culture vessel (L) is recorded as "laminin".

[0217] [Method for preparing culture container (P)]

[0218] Preparation of coating solution containing peptide-containing polyvinyl acetal resin:

[0219] As a polyvinyl acetal resin, a polyvinyl butyral resin having an acetalization degree (butyralization degree) of 65 mol%, a hydroxyl amount of 32 mol%, and an acetyl amount of 3 mol% was prepared. 30 parts by weight of acrylic acid and 70 parts by weight of polyvinyl acetal resin were dissolved in 255 parts by weight of tetrahydrofuran to obtain a polymer mixed solution. 0.015 parts by weight of PERBUTYLO (manufactured by NOF Corporation) was dissolved in the obtained polymer mixed solution and reacted at 90°C for 6 hours. Subsequently, the reacted solution was mixed with 30,000 parts by weight of water. The obtained precipitate was vacuum dried at 80°C for 3 hours to prepare a polyvinyl acetal resin having a joint (acrylic acid) (polyvinyl butyral resin having a joint). Hereinafter, "polyvinyl acetal resin having a joint" may sometimes be described as "polyvinyl acetal resin (X)".

[0220] Prepare dimethylformamide (DMF) as the first solvent. Prepare dimethylformamide (DMF) as the second solvent. As a peptide, prepare a cyclic peptide having the amino acid sequence of Arg-Gly-Asp-Phe-Lys (5 amino acid residues, a cyclic skeleton formed by the combination of Arg and Lys, and Phe is D-type). Prepare 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a condensing agent. Mix 50 parts by weight of polyvinyl acetal resin (X) and 2 parts by weight of peptide in 1000 parts by weight of the first solvent to prepare a first solution. In addition, mix 1 part by weight of the condensing agent in 1000 parts by weight of the second solvent to prepare a second solution. Mix the first solution and the second solution to prepare a solution containing polyvinyl acetal resin (X), peptide and condensing agent.

[0221] The obtained solution was reacted at 40°C for 2 hours to cause dehydration condensation between the carboxyl group in the acrylic acid-derived structural unit of the polyvinyl acetal resin (X) and the amino group of Lys in the peptide, thereby obtaining a solution containing the peptide-containing polyvinyl acetal resin.

[0222] The obtained solution containing the peptide-containing polyvinyl acetal resin was diluted 100-fold with DMF and added dropwise to a column filled with an ion exchange resin (manufactured by Organo) at a rate of 0.3 mL / min for washing. The washed solution was vacuum-dried at 60°C for 3 hours to obtain a dried solid, which was dissolved in butanol (alcohol solvent). 5 parts by weight of acetic acid (pH adjuster) was added to 100 parts by weight of butanol (alcohol solvent) to obtain a coating solution containing the peptide-containing polyvinyl acetal resin and butanol. It should be noted that the content of the peptide-containing polyvinyl acetal resin in the coating solution was 0.1% by weight.

[0223] Preparation of culture vessel (P):

[0224] 20 μL of the obtained coating solution was applied to a 96-well plate (bottom area of ​​each well: 0.32 cm) by cast coating. 2 ) wells, and then the alcohol solvent was removed by vacuum drying at 60°C for 3 hours. This yielded a culture vessel (P) in which a cell scaffold material (resin film serving as a dried layer of the coating solution) comprising a peptide-containing polyvinyl acetal resin was placed on the bottom surface of each well. The peptide-containing polyvinyl acetal resin had a peptide moiety content of 1 mol %. Furthermore, the number average molecular weight of the peptide-containing polyvinyl acetal resin was 50,000.

[0225] [Method for preparing microcarriers (P)]

[0226] As substrate particles, divinylbenzene particles with an average particle size of 300 μm and a CV value of 1% of the particle size were prepared. 1 part by weight of the polyvinyl acetal resin (X) was dissolved in 19 parts by weight of butanol. After adding 1 part by weight of the substrate particles to the obtained solution and stirring, the solution was filtered and washed with pure water, and vacuum-dried at 60°C for 5 hours to obtain polyvinyl acetal resin-coated particles with a joint portion. In addition, the peptide (a cyclic peptide having an amino acid sequence of Arg-Gly-Asp-Phe-Lys (5 amino acid residues, a cyclic skeleton formed by the combination of Arg and Lys, and Phe is D-type) was prepared. 1 part by weight of the peptide and 1 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to a phosphate-buffered saline solution containing neither calcium nor magnesium so that the final concentration of the peptide was 1 mM to prepare a peptide-containing solution. In the obtained 20 parts by weight of the peptide-containing solution, 1 part by weight of polyvinyl acetal resin coated particles having a linker portion was added to dehydrate and condense the carboxyl group of the linker portion with the amino group of Lys of the peptide. The resulting suspension was filtered and washed with pure water, and vacuum dried at 60°C for 5 hours. In this way, a microcarrier (P) having a cell scaffold material comprising a peptide-containing polyvinyl acetal resin configured on the outer surface of the substrate particles was obtained. It should be noted that the content of the peptide portion in the peptide-containing polyvinyl acetal resin is 1 mol%. In addition, the number average molecular weight of the peptide-containing polyvinyl acetal resin is 50,000.

[0227] [Method for preparing culture container (L)]

[0228] "Laminin 511E8 fragment (iMatrix-511, 0.5 mg / mL)" manufactured by NIPPI was diluted 300-fold with phosphate-buffered saline (PBS (Ca- / Mg-)) to obtain a solution of 0.5 μg / cm 2 Add to 96-well plate (bottom area of ​​each well: 0.32cm 2 After the 96-well plate was allowed to stand in an incubator at 37°C and a CO2 concentration of 5% for 1 hour, the supernatant was removed to obtain a culture container (L) in which the laminin 511E8 fragment was disposed on the bottom surface of each well.

[0229] As a culture medium, the following culture medium was prepared.

[0230] Culture medium (1) (prepared by the following "Method for preparing culture medium (1)", equivalent to the mononuclear cell culture medium for non-T cells described in Non-Patent Document 1)

[0231] Culture medium (2) (prepared by the following "Method for preparing culture medium (2)", equivalent to the maintenance culture medium StemFit described in Non-Patent Document 1)

[0232] [Method for preparing culture medium (1)]

[0233] 400 mL of "Solution A for Stem Fit AK02N" manufactured by AJINOMOTO Co., Ltd. and 100 mL of "Solution B for Stem Fit AK02N" manufactured by AJINOMOTO Co., Ltd. were mixed. To the resulting mixed solution, the following cytokines were added so that the final concentrations were 50 ng / mL of IL-6, 50 ng / mL of SCF, 10 ng / mL of TPO, 20 ng / mL of Flt-3L, 20 ng / mL of IL-3, and 10 ng / mL of G-CSF. Thus, a culture medium (1) was obtained.

[0234] [Method for preparing culture medium (2)]

[0235] 400 mL of "Solution A for Stem Fit AK02N" manufactured by AJINOMOTO, 100 mL of "Solution B for Stem Fit AK02N" manufactured by AJINOMOTO, and 2 mL of "Solution C for Stem Fit AK02N" manufactured by AJINOMOTO were mixed to obtain a culture medium (2).

[0236] In the following examples, cell culture and introduction of reprogramming factors were performed according to the protocol described in Non-Patent Document 1, except for the parts specifically described below.

[0237] (Example 1)

[0238] <Inoculation process>

[0239] Peripheral blood mononuclear cells ("normal human PBMC-Japanese donor purified" manufactured by Precision for Medicine) were prepared as somatic cells. 150 μL of culture medium (1) was added per well to the culture container (P). 8.0×10 4 The inoculum volume of cells / well was inoculated with peripheral blood mononuclear cells.

[0240] <Cultivation Step (A)>

[0241] The cells seeded into the culture vessel (P) were cultured in an incubator at 37°C and a CO2 concentration of 5% for 5 days. On the third day from the start of culture, 75 μL of the liquid medium in the well was collected from a position near the liquid surface using a pipette to minimize the removal of suspended somatic cells and then replaced with 75 μL of fresh liquid medium.

[0242] <Removal Process>

[0243] On the fifth day from the start of culture (the fifth day after the somatic cells were seeded in the seeding step), all somatic cells that were not attached to the cell scaffold material and were suspended were removed together with the liquid culture medium in the culture vessel.

[0244] <Introduction process>

[0245] Sendai virus vector ("SRV TM iPSC-4 Vector”) and culture medium (1) are mixed to obtain a virus vector-containing liquid. The virus vector-containing liquid is added to each well of the culture container (P) after the removal process in a manner that the entire well is immersed. It should be noted that the addition of the virus vector-containing liquid to each well of the culture container (P) is carried out within 0.5 minutes after the suspended somatic cells are removed in the removal process. The culture container (P) to which the virus vector-containing liquid is added is allowed to stand in an incubator at 37°C and a CO2 concentration of 5% for 2 hours. Then, 150 μL of fresh culture medium (1) is added to each well, and a washing operation of removing 150 μL of culture medium is performed a total of 3 times. After the washing operation, 100 μL of fresh culture medium (1) is added to each well. In this way, reprogramming factors are introduced into the somatic cells.

[0246] <Cultivation Step (B)>

[0247] 70 μL of fresh culture medium (2) is added to each well of the culture container (P), and the somatic cells into which the reprogramming factors have been introduced are cultured adherently in an incubator at 37°C and a CO2 concentration of 5%. It should be noted that on the third day from the start of the culture in the culture step (B), 70 μL of fresh culture medium (2) is added to each well of the culture container (P). On the fifth and seventh days from the start of the culture in the culture step (B), 70 μL of culture medium is removed from each well of the culture container (P) and 70 μL of fresh culture medium (2) is added. After the ninth day from the start of the culture in the culture step (B), the liquid in the well is replaced with 70 μL of fresh culture medium (2) at a frequency of once or twice every two days.

[0248] <Subculture Step>

[0249] The cell population on day 14 of the culture step (B) was separated into single cells using a stripping agent (TrypLE Select manufactured by Thermo Fisher Scientific). The total number of cells present in each well of the cell culture container was counted using an automated cell counter. Based on the total number of cells calculated, 1×10 4 cells~50×10 4The cells of cells were inoculated into a new culture container (P) to perform cell passage. It should be noted that the cell passage was performed once or twice a week, and the culture medium used was culture medium (2). During the passage, a ROCK inhibitor (Y-27632) was added to the culture medium (2) to a final concentration of 10 μmol / L. In addition, after the second day of the passage, the culture medium was replaced at a frequency of once every three days or more.

[0250] (Example 2)

[0251] The inoculation step to the subculture step were carried out in the same manner as in Example 1, except that the culture container (L) was used instead of the culture container (P).

[0252] (Example 3)

[0253] Monocytes ("human peripheral blood-derived CD14-positive monocytes" manufactured by KAC) were used as somatic cells. The inoculation step to the subculture step were performed in the same manner as in Example 1 except that the inoculation amount in the inoculation step was changed as described in the table below.

[0254] (Example 4)

[0255] As a pretreatment, the microcarriers (P) were immersed in PBS for 30 minutes. 2 ), the pretreated microcarriers (P) were added to each well of the 96-well plate so that the microcarriers (P) formed two to three layers in the well. Adhesive culture was performed on the surface of the microcarriers (P) using this 96-well plate. The inoculation step to the culturing step (B) were performed in the same manner as in Example 1, except that the inoculation amount in the inoculation step was changed as described in the table below.

[0256] The subculture step was carried out in the same manner as in Example 1 except for the following.

[0257] On the 21st day after the start of the culture step (B), 100 μL of a cell detachment agent ("TrypLE Express" manufactured by Thermo Fisher Scientific) was added to each well and allowed to stand for 10 minutes. Next, 20 to 30 pipettings were performed, and the microcarriers (P) were separated using a cell strainer (manufactured by Corning, 40 μm pore size) provided in the tube, and only the cells were recovered in the tube. New microcarriers (P) were prepared, and after pretreatment in the same manner as above, the pretreated microcarriers (P) were added to each well of a 48-well plate, and the microcarriers (P) were formed into 2 to 3 layers in the well. The recovered cells were plated at 1.25×10 4 cells / well were seeded in 48-well plates for cell passage.

[0258] (Comparative Example 1)

[0259] In Comparative Example 1, somatic cells adhered to the cell scaffold material were recovered, and reprogramming factors were introduced into the recovered somatic cells. Specifically, iPS cells were established according to the following procedure.

[0260] <Inoculation Step and Cultivation Step (A)>

[0261] The inoculation step to the culturing step (A) were performed in the same manner as in Example 1, except that the inoculation amount in the inoculation step was changed as described in the following table.

[0262] <Recycling Process>

[0263] After all the culture supernatant was removed, each well was washed twice with 1 mL of PBS. Then, somatic cells adhering to the cell scaffold material were detached using a cell detachment agent ("TrypLE Select" manufactured by Thermo Fisher Scientific) and collected in a 15 mL tube.

[0264] <Introduction process>

[0265] The somatic cells recovered in a 15 mL tube were plated at 3 × 10 5 The number of cells was placed in a 1.5 mL tube and the 1.5 mL tube was centrifuged at 300 × g for 5 minutes. After centrifugation, the supernatant was removed and an appropriate amount of culture medium (1) was added to the 1.5 mL tube. Then, Sendai virus vector ("SRV" manufactured by TOKIWA-BIO) was added to the 1.5 mL tube so that MOI = 3. TM iPSC-4 Vector"). Next, the 1.5 mL tube was placed in a 37°C incubator for 2 hours. Next, 1 mL of culture medium (1) was added to the 1.5 mL tube and centrifuged at 300 × g for 5 minutes. After centrifugation, the supernatant was removed and tapped. The steps of adding the culture medium (1), centrifuging, and removing the supernatant were repeated twice. Next, 0.1 mL of culture medium (1) was added to the 1.5 mL tube and mixed thoroughly. In this way, the reprogramming factors were introduced into the somatic cells.

[0266] <Cultivation Step (B)>

[0267] 0.75 mL of culture medium (2) was added per well of the culture container (P). The somatic cells introduced with the reprogramming factors obtained in the introduction step were cultured at a rate of 1.5 × 10 5Cells at a number of cells / well were seeded on a cell scaffold material in a culture container (P) and cultured in an incubator at 37°C and a CO2 concentration of 5%. It should be noted that 0.5 mL of fresh culture medium (2) was added to the culture container (P) on the 1st, 3rd, 5th, and 7th day after the start of the culture in the culture step (B). In addition, after the 9th day after the start of the culture in the culture step (B), the liquid in the culture container (P) was replaced with 0.75 mL of fresh culture medium (2) at a frequency of once every two days or more.

[0268] <Subculture Step>

[0269] The cell population obtained in the culture step (B) was subjected to the subculture step in the same manner as in Example 1.

[0270] (Comparative Example 2)

[0271] After the culture step (A), the same procedures as in Comparative Example 1 from the seeding step to the subculture step were carried out except that the somatic cells that were not attached to the cell scaffold material and were suspended were recovered and used to carry out the introduction step.

[0272] In Examples 1 to 4 and Comparative Examples 1 and 2, colony morphology was confirmed on the 14th day from the start of culture in the culture step (B). As a result, the colony morphology of iPS cells was confirmed.

[0273] (evaluate)

[0274] In the following description and figures, the numbers used with P refer to the number of passages from the subculture step. P0 is the 0th passage and refers to cells after the culture step (B) (cells provided before the subculture step).

[0275] (1) Flow cytometry analysis of cells before virus infection

[0276] In Example 1, the somatic cells removed by the removal step (suspended somatic cells) and the somatic cells adhered to the cell support material after the removal step were measured by flow cytometry ("Attune NxTFlow Cytometer" manufactured by Thermo Fisher Scientific). It should be noted that the sample of somatic cells adhered to the cell support material for flow cytometry was obtained by washing the wells twice with 1 mL of PBS and then peeling them with a cell peeling agent ("TrypLE Select" manufactured by ThermoFisher Scientific). The results of the FSC-SSC dot plot are shown in FIG. Figure 1 As shown. Figure 1As shown, compared with the suspended somatic cells, the somatic cells adhered to the cell scaffold material contained significantly more cells with larger FSC and SSC.

[0277] (2) Phagocytic ability of somatic cells adhered to cell scaffold materials

[0278] In Example 1, the phagocytic ability of somatic cells adhered to the cell scaffold material after the removal process was evaluated in the following order. pHrodo Green S. aureus BioParticles conjugates (manufactured by Life Technologies) were added to the culture medium (1) in a manner that the final concentration was 0.1 mg / mL, and 750 μL was added to the wells of the culture container after the removal process. Then, the culture medium was allowed to stand for 90 minutes at 37°C and a CO2 concentration of 5%. Then, the wells were observed at a magnification of 200 times in the fluorescent field and bright field using a fluorescence microscope ("BZ-X800" manufactured by KEYENCE). It should be noted that as a negative control, the evaluation was also carried out according to the following steps. First, cytochalasin D was added to the culture medium (1) in a manner that the final concentration was 10 μM, and 750 μL was added to the wells of the culture container after the removal process. Then, the culture medium was allowed to stand for 30 minutes at 37°C and a CO2 concentration of 5%. Next, pHrodo Green S. aureus BioParticles conjugates were added to the wells at a final concentration of 0.1 mg / mL and allowed to stand in an incubator at 37°C and 5% CO2 for 90 minutes. The wells were then observed under the same observation conditions as above. Figure 2 As shown. Figure 2 As shown, under the fluorescence field, cells adhered to the cell support material were observed only in the absence of cytochalasin D. pHrodo Green S. aureus Bioparticles conjugates do not fluoresce outside the phagosome, but do fluoresce inside the phagosome. Furthermore, cytochalasin D acts as an inhibitor of phagocytosis. Therefore, it was confirmed that the cells adhered to the cell support material after the removal step were phagocytic cells.

[0279] (3) Morphological observation of cells after virus infection

[0280] In Example 1 and Comparative Example 2, the morphology of the cells on the first day after the somatic cells into which the reprogramming factors were introduced (the first day after the virus infection) were cultured was observed in the following order. The wells of the culture container on the first day from the start of the culture step (B) were washed once with 1×DPBS. The wells were observed and photographed at a magnification of 100 times under a bright field using a fluorescence microscope ("BZ-X800" manufactured by KEYENCE). The results are as follows. Figure 3 As shown. Figure 3 As shown, in Example 1, compared with Comparative Example 2, many cells with large size and elongated shape were observed.

[0281] (4) Virus survival rate

[0282] In Examples 1 to 3 and Comparative Examples 1 and 2, the virus survival rate was evaluated in the following order. During cell passage, a portion of the single cells was recovered into a 1.5 mL tube. The recovered single cells were measured by flow cytometry ("Attune NxT Flow Cytometer" manufactured by Thermo Fisher Scientific). The proportion of GFP-positive cells contained in the main cell population developed on the FSC-SSC dot plot was analyzed. The results are shown in Tables 1 to 3 and Figure 4 It should be noted that Figure 4 The results of Examples 1 and 2 and Comparative Examples 1 and 2 and the number N are shown in FIG. Figure 4 In the figure, from the left are the data of P2 and P3. Figure 4 The smaller the GFP positive rate on the vertical axis, the lower the virus survival rate. Figure 4 As shown, in Examples 1 to 3, the results of the virus survival rate were particularly excellent, and the time until the virus disappeared was short.

[0283] (5) Undifferentiated (TRA-1-60 positive rate)

[0284] An antibody targeting TRA-1-60, labeled with the fluorescent dye PE ("TRA-1-60 PE Antibody" manufactured by Thermo Fisher Scientific), was prepared. P2 and P4 cells were each exposed to the antibody. Next, the TRA-1-60 positive rate was determined using a flow cytometer ("Attune NxT Flow Cytometer" manufactured by Thermo Fisher Scientific). Specifically, in Examples 1 to 3 and Comparative Examples 1 and 2, evaluation was performed according to the following procedure.

[0285] The cells of P2 and P4 were separated into single cells using a stripping agent (TrypLE Select manufactured by Thermo Fisher Scientific). The obtained single cells were fed to an automatic cell counter to calculate the cell concentration. 5 The cells of cells were collected into 1.5mL tubes and centrifuged at 300×g and 5 minutes. After centrifugation, the supernatant was removed and 1×DPBS was added to the 1.5mL tube. Then, centrifugation and supernatant removal were performed again under the conditions described above. Then, blocking was implemented with DPBS containing 1% by weight BSA. Then, an appropriate amount of antibody solution containing the antibody (“TRA-1-60 PE Antibody” manufactured by Thermo Fisher Scientific) was added to the 1.5mL tube and incubated for 1 hour in the dark and at room temperature. Then, after centrifuging the 1.5mL tube at 300×g and 5 minutes, the supernatant was removed and washed twice with 1×DPBS. Then, 0.4mL of PBS containing 1% by weight BSA was added to the 1.5mL tube and mixed, and measured by flow cytometry. The proportion of TRA-1-60 positive cells contained in the main cell population expanded on the FSC-SSC dot plot was analyzed (TRA-1-60 positive rate). The results are shown in Tables 1 to 3 and Figure 5 It should be noted that Figure 5 The results of Examples 1 and 2 and Comparative Examples 1 and 2 and the number N are shown in FIG. Figure 5 From the left, the data are P2 and P4. Figure 5 As shown, in Examples 1 to 3, a cell population (cell population including iPS cells) with a high TRA-1-60 positive rate was obtained in a short period of time.

[0286] The details and results are shown in the following Tables 1 to 3 and the figures. In Example 4, the establishment of iPS cells was visually confirmed.

[0287] [Table 1]

[0288]

[0289] [Table 2]

[0290]

[0291] [Table 3]

[0292]

[0293] Figure 6 This is a micrograph of the iPS cell colony established from monocytes in Example 3. More specifically, Figure 6 This is a microscopic photograph (magnification: 100x) of P2 cells on the 5th day after culture.

Claims

1. A method for producing induced pluripotent stem cells, comprising: a seeding step of seeding somatic cells on a cell culture substrate having a cell scaffold material; a removal step of removing somatic cells that are not attached to the cell scaffold material and are suspended; and The introduction step is to introduce reprogramming factors into the somatic cells adhered to the cell scaffold material.

2. The method for producing induced pluripotent stem cells according to claim 1, wherein The somatic cells inoculated in the inoculation step include peripheral blood mononuclear cells.

3. The method for producing induced pluripotent stem cells according to claim 1 or 2, wherein: The somatic cells adhered to the cell scaffold material in the introduction step include cells having phagocytic function.

4. The method for producing induced pluripotent stem cells according to any one of claims 1 to 3, wherein The cell scaffold material has an RGD sequence.

5. The method for producing induced pluripotent stem cells according to any one of claims 1 to 4, wherein The cell scaffold material comprises a peptide-containing resin having a synthetic resin portion and a peptide portion.

6. The method for producing induced pluripotent stem cells according to claim 5, wherein: The peptide-containing resin has a polyvinyl acetal resin portion and a peptide portion.

7. The method for producing induced pluripotent stem cells according to any one of claims 1 to 6, wherein In the introduction step, reprogramming factors are introduced into the somatic cells adhered to the cell scaffold material using a Sendai virus vector.

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