Method for producing induced pluripotent stem cells

Abstract

Provided is a method for producing induced pluripotent stem cells by which induced pluripotent stem cells having high undifferentiated and differentiated capabilities can be easily obtained. This method for producing induced pluripotent stem cells comprises: a step for introducing a reprogramming factor into somatic cells; and a step for culturing the somatic cells into which the reprogramming factor is introduced in the presence of a cytoskeleton material containing a peptide-containing polyvinyl acetal resin.

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) have attracted attention in research fields such as academia, drug development, and regenerative medicine. iPS cells can be prepared by introducing reprogramming factors (e.g., OCT3 / 4, SOX2, KLF4, 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 Documents 1 and 2). Laminin functions as a cell scaffold material.

[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. In addition, Patent Document 2 describes the use of the scaffold material to culture iPS cells in an example. 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 Feeder-Free Establishment / Maintenance Culture of Human iPS Cells, 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)

[0011] Non-patent document 2: (8) iPS cell induction protocol from human peripheral blood mononuclear cells / monocytes using SRV(TM) iPSC-4 Vector (TKB_P-008-02) (TOKIWA-BIO Co., Ltd., http: / / tokiwa-bio.com / jp / wp / wp-content / uploads / 2022 / 02 / 8_%E3%83%92%E3%83%88%E6%9C%AB%E6%A2%A2%E8%A1%80%E5%8 D%98%E6%A0%B8%E7%90%83%E3%83%BB%E5%8D%98%E7%90%83%E3%81%8B%E3%82%89%E3%81%AEiPS%E7%B 4%B0%E8%83%9E%E8%AA%98%E5%B0%8E%E3%83%97%E3%83%AD%E3%83%88%E3%82%B3%E3%83%AB_02.pdf) Summary of the invention

[0012] Technical problem solved by the invention

[0013] Various attempts have been made to improve the efficiency of establishing undifferentiated and highly differentiated iPS cells. However, it is still difficult to efficiently obtain undifferentiated and highly differentiated iPS cells. When the undifferentiated nature of iPS cells is low, differentiation may occur unexpectedly during iPS cell culture.

[0014] An object of the present invention is to provide a method for producing induced pluripotent stem cells by which undifferentiated induced pluripotent stem cells and induced pluripotent stem cells with high differentiation ability can be easily obtained.

[0015] Technical means of solving problems

[0016] According to a broad aspect of the present invention, there is provided a method for producing induced pluripotent stem cells, comprising: introducing reprogramming factors into somatic cells; and culturing the somatic cells introduced with the reprogramming factors in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin.

[0017] In a specific aspect of the method for producing induced pluripotent stem cells of the present invention, the method for producing induced pluripotent stem cells comprises, before the step of introducing reprogramming factors into the somatic cells, a step of culturing the somatic cells introduced with the reprogramming factors in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin.

[0018] In a specific aspect of the method for producing induced pluripotent stem cells of the present invention, in the step of introducing the reprogramming factors into the somatic cells, the reprogramming factors are introduced into the somatic cells using a Sendai virus vector.

[0019] In a specific aspect of the method for producing induced pluripotent stem cells of the present invention, the reprogramming factor is mRNA.

[0020] In a specific aspect of the method for producing induced pluripotent stem cells of the present invention, the mRNA is introduced into the somatic cells by a plasma gene introduction method.

[0021] Effects of the Invention

[0022] According to the present invention, a method for producing induced pluripotent stem cells can be provided, which can easily obtain induced pluripotent stem cells that are undifferentiated and have high differentiation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [ Figure 1 ] Figure 1 This is a graph showing the relationship between the number of passages from the culture of somatic cells into which reprogramming factors have been introduced and the survival rate of viruses.

[0024] [ Figure 2 ] Figure 2 This is a graph showing the relationship between the number of culture days after culturing somatic cells into which reprogramming factors have been introduced and the cumulative number of cells.

[0025] [ Figure 3 ] Figure 3 This is a graph showing the relationship between the number of passages from the culture of somatic cells into which reprogramming factors have been introduced and the survival rate of the cells.

[0026] [ Figure 4 ] Figure 4 This is a microscopic photograph of cells (iPS cells) obtained by culturing somatic cells into which reprogramming factors have been introduced. Fluorescence indicates the expression of TRA-1-60.

[0027] [ Figure 5 ] Figure 5 This is a graph showing the relationship between the number of passages from the culture of somatic cells into which reprogramming factors have been introduced and the expression levels of Nanog and Oct3 / 4.

[0028] [ Figure 6 ] Figure 6 This is a microscopic photograph of cells on day 39 from the start of differentiation induction of iPS cells. In Examples 1 to 3, the formation of embryoid bodies was confirmed, but in Comparative Examples 1 to 3, the formation of embryoid bodies was not confirmed.

[0029] [ Figure 7 ] Figure 7 It is a graph showing the expression levels of endoderm markers (AFP, FOXA2, GATA4, GATA6, SOX17, SOX7).

[0030] [ Figure 8 ] Figure 8It is a graph showing the expression levels of mesoderm markers (KDR, T).

[0031] [ Fig. 9 ] Fig. 9 It is a graph showing the expression levels of ectoderm markers (NES, PAX6, SOX1).

[0032] [ Fig.10 ] Fig.10 It is a radar chart summarizing the differentiation scores into each germ layer.

[0033] [ Fig.11 ] Fig.11 It is a diagram showing the results of an experiment of introducing mRNA (reprogramming factor) into somatic cells.

[0034] [ Fig.12 ] Fig.12 This is a microscope photograph of cells under a fluorescent field of view 14 days after mRNA (reprogramming factor) was introduced into somatic cells by the plasma gene transfer method.

[0035] [ Fig.13 ] Fig.13 It is a graph showing the relationship between FITC fluorescence intensity and cell number.

[0036] [ Fig.14 ] Fig.14 It is a graph showing the expression levels (relative quantitative values) of NANOG, OCT3 / 4, and SOX2.

[0037] [ Fig.15 ] Fig.15 This is a microscope photo of immunostained cells under the fluorescent field.

[0038] [ Fig.16 ] Fig.16 It is a graph showing the expression levels (relative quantitative values) of endoderm markers (FOXA2, SOX17), mesoderm marker (T), and ectoderm marker (PAX6). DETAILED DESCRIPTION

[0039] Hereinafter, the present invention will be described in detail.

[0040] The method for producing induced pluripotent stem cells of the present invention comprises: a step of introducing reprogramming factors into somatic cells; and a step of culturing the somatic cells into which the reprogramming factors have been introduced in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin.

[0041] Since the method for producing induced pluripotent stem cells of the present invention has the above-mentioned structure, it is easy to obtain induced pluripotent stem cells that are undifferentiated and have high differentiation ability.

[0042] In the past, as a method for preparing induced pluripotent stem cells, a method of culturing somatic cells introduced with reprogramming factors on a plate coated with laminin (cell scaffold material) was widely carried out. The present inventors and others found that by culturing somatic cells introduced with the reprogramming factors in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin, it is easy to obtain induced pluripotent stem cells with high undifferentiation and differentiation ability. In particular, in the present invention, compared with the previous method of establishing induced pluripotent stem cells by culturing somatic cells introduced with reprogramming factors on laminin, induced pluripotent stem cells with a higher expression level of TRA-1-60 as an undifferentiation marker can be obtained. In addition, in the induced pluripotent stem cells obtained in the present invention, compared with the induced pluripotent stem cells obtained by culturing somatic cells introduced with reprogramming factors on laminin, embryoid bodies can be efficiently formed and differentiation ability can be maintained.

[0043] The method for producing induced pluripotent stem cells of the present invention preferably comprises a step of culturing somatic cells before the step of introducing reprogramming factors into the somatic cells.

[0044] In this specification, "induced pluripotent stem cells" are sometimes referred to as "iPS cells". In this specification, the "step of culturing somatic cells" performed before the step of introducing reprogramming factors into the somatic cells is sometimes referred to as "step (1)". In addition, in this specification, the "step of introducing reprogramming factors into somatic cells" is sometimes referred to as "step (2)", and the "step of culturing somatic cells introduced with reprogramming factors in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin" is sometimes referred to as "step (3)".

[0045] Therefore, the method for producing iPS cells of the present invention comprises step (2) and step (3). The method for producing iPS cells of the present invention preferably comprises step (1), step (2) and step (3).

[0046] First, the somatic cells and the peptide-containing polyvinyl acetal resin that can be used in the present invention are described.

[0047] (Somatic cells)

[0048] As the somatic cell, conventionally known somatic cells for establishing iPS cells can be used. The somatic cell is preferably an animal cell, more preferably a mammalian cell, and even more preferably a human cell.

[0049] Examples of the somatic cells include blood cells, fibroblasts, hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, and dental pulp cells. Examples of the sources of the somatic cells include peripheral blood, umbilical cord blood, skin tissue, and teeth.

[0050] Examples of the blood cells include nucleated cells such as monocytes, neutrophils, eosinophils, basophils, and lymphocytes. The blood cells may be endothelial progenitor cells, blood stem / progenitor cells, T cells, or B cells.

[0051] The somatic cells are preferably blood cells or fibroblasts, more preferably blood cells, and even more preferably monocytes.

[0052] (Polyvinyl acetal resin containing peptide)

[0053] The peptide-conjugated polyvinyl acetal resin has a polyvinyl acetal resin portion and a peptide portion. The peptide-conjugated polyvinyl acetal resin is a polyvinyl acetal resin combined with a peptide. In the peptide-conjugated polyvinyl acetal resin, the polyvinyl acetal resin portion and the peptide portion are preferably combined via a linker portion. Therefore, the peptide-conjugated polyvinyl acetal resin preferably has a polyvinyl acetal resin portion, a peptide portion and a linker portion.

[0054] In step (3), a cell scaffold material comprising the peptide-containing polyvinyl acetal resin is used. The cell scaffold material in step (3) comprises the peptide-containing polyvinyl acetal resin. In addition, as described below, step (1) is preferably a step of culturing somatic cells in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin. The cell scaffold material in step (1) preferably comprises the peptide-containing polyvinyl acetal resin.

[0055] In the present specification, the peptide-containing polyvinyl acetal resin used in step (1) may be described as "peptide-containing polyvinyl acetal resin (1)". In addition, the polyvinyl acetal resin part, peptide part and linker part of the peptide-containing polyvinyl acetal resin (1) may be described as "polyvinyl acetal resin part (1)", "peptide part (1)" and "linker part (1)", respectively.

[0056] In the present specification, the peptide-containing polyvinyl acetal resin used in step (3) is sometimes described as "peptide-containing polyvinyl acetal resin (3)". In addition, the polyvinyl acetal resin part, peptide part and linker part of the peptide-containing polyvinyl acetal resin (3) are sometimes described as "polyvinyl acetal resin part (3)", "peptide part (3)" and "linker part (3)", respectively.

[0057] In the following description, the common structure of the peptide-containing polyvinyl acetal resin used in step (1) and step (3) is described simply as "peptide-containing polyvinyl acetal resin". In addition, the common description of the polyvinyl acetal resin part, peptide part and linker part of the peptide-containing polyvinyl acetal resin used in step (1) and step (3) is described simply as "polyvinyl acetal resin part", "peptide part" and "linker part".

[0058] The peptide-containing polyvinyl acetal resin (1) and the peptide-containing polyvinyl acetal resin (3) may be the same or different. The polyvinyl acetal resin part (1) and the polyvinyl acetal resin part (3) may be the same or different. The peptide part (1) and the peptide part (3) may be the same or different. The linker part (1) and the linker part (3) may be the same or different.

[0059] <Polyvinyl acetal resin part>

[0060] The peptide-containing polyvinyl acetal resin has a polyvinyl acetal resin portion. The polyvinyl acetal resin portion is a structural portion derived from a polyvinyl acetal resin in the peptide-containing polyvinyl acetal resin. The polyvinyl acetal resin may be used alone or in combination of two or more.

[0061] The polyvinyl acetal resin part preferably has an acetal group, a hydroxyl group and an acetyl group in the side chain. The polyvinyl acetal resin part may not have an acetyl group, for example. For example, by combining all the acetyl groups of the polyvinyl acetal resin part with the linker, the polyvinyl acetal resin part may not have an acetyl group.

[0062] The polyvinyl acetal resin may be synthesized by acetalizing polyvinyl alcohol with aldehyde.

[0063] The aldehyde used in the acetalization of polyvinyl alcohol is not particularly limited. As the aldehyde, for example, an aldehyde having 1 to 10 carbon atoms can be cited. 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.

[0064] From the viewpoint of further improving the adhesion between the cell scaffold material and the cell and from the viewpoint of further effectively exerting the effect of the present invention, the aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde or valeraldehyde, and more preferably butyraldehyde. Therefore, the polyvinyl acetal resin is more preferably a polyvinyl butyral resin. The polyvinyl acetal resin portion (1) is preferably a polyvinyl butyral resin portion, and the polyvinyl acetal resin portion (3) is preferably a polyvinyl butyral resin portion. From the viewpoint of further improving the adhesion between the cell scaffold material and the cell and from the viewpoint of further effectively exerting the effect of the present invention, the peptide-containing polyvinyl acetal resin (1) is preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion and a peptide portion. From the viewpoint of further effectively exerting the effect of the present invention, the peptide-containing polyvinyl acetal resin (3) is preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion and a peptide portion.

[0065] In the peptide-containing polyvinyl acetal resin (1), the acetalization degree of the polyvinyl acetal resin part (1) (the butyralization degree in the case of the polyvinyl butyral resin part) is preferably 40 mol% or more, more preferably 50 mol% or more, preferably 90 mol% or less, more preferably 85 mol% or less. When the acetalization degree is above the lower limit, the peptide-containing polyvinyl acetal resin (1) becomes less likely to swell in the culture medium. When the acetalization degree is below the upper limit, the solubility in the solvent can be improved.

[0066] In the peptide-containing polyvinyl acetal resin (3), the acetalization degree of the polyvinyl acetal resin part (3) (the butyralization degree in the case of the polyvinyl butyral resin part) is preferably 40 mol% or more, more preferably 50 mol% or more, preferably 90 mol% or less, more preferably 85 mol% or less. When the acetalization degree is above the lower limit, the peptide-containing polyvinyl acetal resin (3) becomes less likely to swell in the culture medium. When the acetalization degree is below the upper limit, its solubility in the solvent can be improved.

[0067] In the peptide-containing polyvinyl acetal resin (1), the hydroxyl content (hydroxyl amount) of the polyvinyl acetal resin part (1) 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.

[0068] In the peptide-containing polyvinyl acetal resin (3), the hydroxyl content (hydroxyl amount) of the polyvinyl acetal resin part (3) 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.

[0069] In the peptide-containing polyvinyl acetal resin (1), the acetylation degree (acetyl group amount) of the polyvinyl acetal resin part (1) 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 acetylation degree is above the lower limit and below the upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be improved.

[0070] In the peptide-containing polyvinyl acetal resin (3), the acetylation degree (acetyl group amount) of the polyvinyl acetal resin part (3) 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 acetylation degree is above the lower limit and below the upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be improved.

[0071] The acetalization degree, acetylation degree and hydroxyl content of the polyvinyl acetal resin part can be 1 H-NMR (nuclear magnetic resonance spectroscopy) was used for measurement.

[0072] <Peptide section>

[0073] The peptide-containing polyvinyl acetal resin has a peptide portion. The peptide portion is a structural portion derived from a peptide in the peptide-containing polyvinyl acetal resin. 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.

[0074] The number of amino acid residues in the peptide portion (1) is preferably 3 or more, more preferably 4 or more, further preferably 5 or more, preferably 10 or less, more preferably 8 or less, further preferably 6 or less. When the number of amino acid residues is above the lower limit and below the upper limit, the adhesion to the inoculated cells can be further improved, and the proliferation rate of the cells can be further improved. However, the number of amino acid residues in the peptide portion (1) may exceed 10, or may exceed 15.

[0075] The number of amino acid residues in the peptide portion (3) is preferably 3 or more, more preferably 4 or more, further preferably 5 or more, preferably 10 or less, more preferably 8 or less, further preferably 6 or less. When the number of amino acid residues is above the lower limit and below the upper limit, the adhesion to the inoculated cells can be further improved, and the proliferation rate of the cells can be further improved. However, the number of amino acid residues in the peptide portion (3) can exceed 10, and can also exceed 15.

[0076] The peptide portion (1) preferably has an amino acid sequence with cell adhesion. The peptide portion (3) 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 a phage display method, an agarose bead method or a plate coating method. As the phage display method, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 19951189-1196" can be used. As the agarose bead method, for example, the method described in "Protein Nuclease Vol.45No.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.

[0077] As the cell adhesive amino acid sequence, for example, there can be mentioned RGD sequence (Arg-Gly-Asp), YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), PDSGR sequence (Pro-Asp-Ser-Gly-Arg), HAV sequence (His-Ala-Val), ADT sequence (Ala-Asp-Thr), QAV sequence (Gln-Ala-Val), LDV sequence (Leu-Asp-Val), IDS sequence (Ile-Asp-Ser), REDV sequence (Arg-Glu-Asp-Val), IDAPS sequence (Ile-Asp-Ala-Pro-Ser), KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val) and TDE sequence (Thr-Asp-Glu). In addition, examples of the cell adhesive amino acid sequence include sequences described in "Patient Physiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Journal of Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, pp. 58-66, 1992". The peptide portion may have only one cell adhesive amino acid sequence or may have two or more.

[0078] The cell adhesive amino acid sequence preferably has at least any one of the cell adhesive amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence or a PDSGR sequence, further preferably has an RGD sequence, and particularly preferably has at least an RGD sequence represented by the following formula (1). In this case, the adhesion to the inoculated cells can be further improved, and the cell proliferation rate can be further improved.

[0079] Arg-Gly-Asp-X ···Formula (1)

[0080] In the formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro or Asn.

[0081] The peptide portion (1) may be linear or have a cyclic peptide backbone. The peptide portion (3) may be linear or have a cyclic peptide backbone. The cyclic peptide backbone refers to a cyclic backbone composed of a plurality of amino acids. From the viewpoint 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.

[0082] In the peptide-containing polyvinyl alcohol acetal resin (1), the content of the peptide portion (1) is preferably 0.05 mol% or more, more preferably 0.1 mol% or more, further preferably 0.15 mol% or more, particularly preferably 0.2 mol% or more, preferably 25 mol% or less, more preferably 20 mol% or less, further preferably 15 mol% or less, particularly preferably 10 mol% or less. When the content of the peptide portion (1) is above the lower limit, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved. In addition, when the content of the peptide portion (1) is below the upper limit, the manufacturing cost can be suppressed.

[0083] In the peptide-containing polyvinyl acetal resin (3), the content of the peptide portion (3) is preferably 0.05 mol% or more, more preferably 0.1 mol% or more, further preferably 0.15 mol% or more, particularly preferably 0.2 mol% or more, preferably 25 mol% or less, more preferably 20 mol% or less, further preferably 15 mol% or less, particularly preferably 10 mol% or less. When the content of the peptide portion (3) is above the lower limit, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved. In addition, when the content of the peptide portion (3) is below the upper limit, the manufacturing cost can be suppressed.

[0084] The content rate (mol %) of the peptide part is the amount of the peptide part relative to the total amount of each structural unit constituting the peptide-containing polyvinyl acetal resin.

[0085] The content of the peptide portion can be measured by, for example, NMR (nuclear magnetic resonance).

[0086] <Joint>

[0087] The peptide-containing polyvinyl acetal resin preferably has a linker portion. The linker portion is a structural portion derived from a linker in the peptide-containing polyvinyl acetal resin. The linker portion is usually located between the polyvinyl acetal resin portion and the peptide portion. The polyvinyl acetal resin 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.

[0088] The linker is preferably a compound having a functional group capable of binding to the peptide, and more preferably a compound having a functional group capable of condensing with a carboxyl group or an amino group of the peptide.

[0089] Examples of the functional group capable of condensing with the carboxyl group or amino group of the peptide include a carboxyl group, a thiol group, an amino group, a hydroxyl group, and a cyano group.

[0090] 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.

[0091] As the linker having a carboxyl group, (meth) acrylic acid and carboxyl-containing acrylamide can be cited. 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 that can react with the peptide.

[0092] From the viewpoint of favorably bonding the polyvinyl acetal resin to the peptide, the linker is preferably (meth)acrylic acid, and more preferably acrylic acid.

[0093] <Other detailed descriptions of peptide-containing polyvinyl acetal resin>

[0094] The number average molecular weight of the peptide-containing polyvinyl acetal resin (1) 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, 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 container or the like during cell culture. When the number average molecular weight is below the upper limit, the solubility in alcohol solvents can be improved.

[0095] The number average molecular weight of the peptide-containing polyvinyl acetal resin (3) 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, 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 container or the like during cell culture. When the number average molecular weight is below the upper limit, the solubility in alcohol solvents can be improved.

[0096] It should be noted that the number average molecular weight of the peptide-containing polyvinyl acetal resin can be measured, for example, by the following method. The peptide-containing polyvinyl acetal resin is dissolved in tetrahydrofuran (THF) to prepare a 0.2 wt % solution of the peptide-containing polyvinyl acetal resin. Next, a gel permeation chromatography (GPC) measuring device (APC system, manufactured by Waters) is used to evaluate the following measuring conditions.

[0097] Column: HSPgel HR MB-M 6.0×150mm

[0098] Flow rate: 0.5mL / min

[0099] Column temperature: 40°C

[0100] Injection volume: 10 μL

[0101] Detector: RI, PDA

[0102] Standard sample: polystyrene

[0103] Examples of methods for obtaining the peptide-containing polyvinyl acetal resin include the following methods.

[0104] The polyvinyl acetal resin is reacted with the linker to obtain a reaction product in which the polyvinyl acetal resin and the linker are combined. The obtained reaction product is reacted with the peptide to obtain the peptide-containing polyvinyl acetal resin.

[0105] The culture container having the cell scaffold material containing the peptide-containing polyvinyl acetal resin can be prepared, for example, by the following method (A) or (B). (A) After applying a liquid containing the peptide-containing polyvinyl acetal resin and a solvent on the surface of the container (culture container body), the solvent is volatilized. (B) A layer containing a reactant formed by combining the polyvinyl acetal resin and a linker is arranged on the surface of the container (culture container body). A liquid containing a peptide is added to the surface of the layer containing the reactant to allow the reactant to react with the peptide.

[0106] Hereinafter, the method for producing iPS cells of the present invention will be described in further detail.

[0107] (Process (1))

[0108] The method for producing iPS cells preferably comprises a step of culturing somatic cells (step (1)). Step (1) is preferably a step of pre-culturing somatic cells. Step (1) may be a step of culturing somatic cells in the absence of a cell scaffold material, or a step of culturing somatic cells in the presence of a cell scaffold material. Step (1) may be a step of suspending and culturing somatic cells. From the viewpoint of effectively exerting the effects of the present invention, step (1) is preferably a step of culturing somatic cells in the presence of a cell scaffold material. The cell scaffold material is, for example, arranged on the surface of a culture substrate body. With regard to step (1), for example, it is a step of culturing somatic cells using a culture substrate comprising the culture substrate body and the cell scaffold material.

[0109] The shape and size of the culture substrate body are not particularly limited. Examples of the culture substrate body include containers, fibers, nonwoven fabrics, hollow fibers, particles, membranes, and porous membranes.

[0110] The culture substrate used in step (1) is preferably a culture container. In this case, the cell scaffold material is preferably arranged on the bottom surface of the culture container body. The culture container may be a culture dish or a plate having a plurality of holes.

[0111] Examples of cell scaffold materials used in step (1) include peptide-containing polyvinyl acetal resins, laminin, vitronectin, collagen, etc. Examples of commercially available cell scaffold materials used in step (1) include "iMatrix-511" manufactured by NIPPI and "Matrigel" manufactured by Corning Incorporated.

[0112] From the viewpoint of further effectively exerting the effect of the present invention, step (1) is preferably a step of culturing somatic cells in the presence of a cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin. When somatic cells are cultured in the presence of a cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin in step (1), there are somatic cells adhered to the cell scaffold material and somatic cells suspended without being adhered to the cell scaffold material. The reason why the effect of the present invention is further effectively exerted by using the cell scaffold material is still uncertain, and it is believed that useful cytokines may be secreted from somatic cells adhered to a cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin. However, the above is inferred, and the reason why the effect of the present invention is further effectively exerted by using a cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin in step (1) is not limited to this.

[0113] In the cell scaffold material used in step (1), the peptide-containing polyvinyl acetal resin is preferably present at least on the surface that adheres to somatic cells (the surface that adheres to somatic cells).

[0114] As the culture medium for culturing somatic cells in step (1), 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 at home can be used. As the culture medium, for example, a culture medium described in the non-patent document 1 can be used.

[0115] Commercially available products of the culture medium 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. In addition, as the culture medium, a culture medium to which various cytokines are added to the commercially available products can be used.

[0116] When monocytes are used as the somatic cells, a medium containing 45 to 55 ng / mL of IL-6, 45 to 55 ng / mL of SCF, 9 to 11 ng / mL of TPO, 18 to 22 ng / mL of Flt-3L, 18 to 22 ng / mL of IL-3, and 9 to 11 ng / mL of G-CSF is preferably used as the final concentration of the cytokines. The 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 obtained.

[0117] The culture temperature of step (1) is preferably above 35°C and preferably below 38°C. The CO2 concentration during the culture of step (1) is preferably above 4% and preferably below 6%. For example, the culture conditions of step (1) are 37°C and a CO2 concentration of 5%. The culture time of step (1) can be, for example, 4 to 6 days (preferably 5 days). During the culture of step (1), the culture medium may or may not be replaced. When the culture medium is replaced, for example, it can be replaced with fresh culture medium once a day.

[0118] (Process (2))

[0119] The method for producing iPS cells preferably comprises a step of introducing reprogramming factors into somatic cells (step (2)). In step (2), the reprogramming factors are preferably introduced into the somatic cells obtained in step (1).

[0120] From the viewpoint of more effectively exerting the effects of the present invention, step (2) preferably comprises the steps of collecting somatic cells after step (1) and introducing reprogramming factors into the collected somatic cells.

[0121] In the process of collecting the somatic cells, the somatic cells adhered to the cell scaffold material after step (1) can be collected, the somatic cells not adhered to the cell scaffold material after step (1) can be collected, and the somatic cells adhered to the cell scaffold material and the somatic cells not adhered after step (1) can be collected. In the process of collecting the somatic cells, it is preferred to pipette the solution (somatic cell-containing liquid) in the culture container and recover it to the tube. Then, the liquid obtained by adding DPBS (-) to the culture container can be recovered to the tube with the solution recovered, or it can not be recovered.

[0122] As the reprogramming factor (nuclear reprogramming factor), conventionally known reprogramming factors can be used.

[0123] As the genes contained in the reprogramming factors, 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, Glis1, etc. The reprogramming factors may be used alone or in combination of two or more.

[0124] The reprogramming factors preferably include Oct family genes, Sox family genes, Klf family genes, Myc family genes, Lin28 family genes or Nanog family genes.

[0125] 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.

[0126] As a method for introducing the reprogramming factors into somatic cells, a conventionally known introduction method can be used.

[0127] As a method for introducing the reprogramming factor into somatic cells, there can be mentioned a method using a vector and a method using mRNA, etc. As the vector, there can be mentioned viral vectors such as Sendai virus vectors, retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and non-viral vectors such as episomal vectors and plasmid vectors.

[0128] Methods of using carriers:

[0129] In the method of introducing the reprogramming factor into somatic cells, a vector is preferably used, and a Sendai virus vector is more preferably used. That is, in step (2), a Sendai virus vector is more preferably used to introduce the reprogramming factor into the somatic cell. When a Sendai virus vector is used, safety can be improved.

[0130] Examples of commercially available Sendai virus vectors include the "CytoTune-iPS" series manufactured by ID PHARMA and the "SRV iPSC Vector" series manufactured by TOKIWA-BIO.

[0131] In step (2), the virus vector (particularly Sendai virus vector) is preferably infected at an MOI (Multiplicity of Infection) of 1 to 6, and more preferably at an MOI of 3 to 4. Furthermore, the infection time is preferably 15 minutes to 4 hours, and more preferably 30 minutes to 4 hours.

[0132] In step (2) using a vector, after introducing reprogramming factors into somatic cells, the cells introduced with the reprogramming factors may be cultured without entering step (3), or after introducing reprogramming factors into somatic cells, the cells introduced with the reprogramming factors may be cultured before entering step (3). The step of culturing the somatic cells introduced with the reprogramming factors in step (2) is performed in the absence of a cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin. The culturing time of the step of culturing the somatic cells introduced with the reprogramming factors in step (2) is preferably more than 7 days, preferably less than 30 days.

[0133] Methods of using mRNA:

[0134] In the method of introducing the reprogramming factor into somatic cells, mRNA is preferably used. The reprogramming factor is also preferably mRNA. In this case, mRNA (reprogramming factor) can be introduced into somatic cells without using a virus, thereby improving safety.

[0135] Examples of methods for introducing mRNA (reprogramming factor) into somatic cells include plasma gene introduction, electroporation, and lipofection.

[0136] The method for introducing mRNA (reprogramming factor) into somatic cells is preferably a plasma gene introduction method. That is, mRNA (reprogramming factor) is preferably introduced into somatic cells by a plasma gene introduction method. In this case, the damage to the genome of the somatic cells can be suppressed, and mRNA (reprogramming factor) is introduced into the somatic cells. In addition, as the conditions for plasma irradiation in the plasma gene introduction method, previously known conditions can be adopted.

[0137] The introduction of mRNA (reprogramming factor) to somatic cells is preferably carried out in the presence of a cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin. Preferably, mRNA (reprogramming factor) is introduced into the somatic cells bonded to the cell scaffold material comprising a peptide-containing polyvinyl alcohol acetal resin. In this case, although the reason is still uncertain, compared with the situation carried out in the presence of a cell scaffold material comprising a protein, the introduction efficiency of mRNA to somatic cells can be further improved.

[0138] In step (2) using mRNA, step (1) and step (2) may be performed using the same culture substrate, step (2) and step (3) may be performed using the same culture substrate, and step (1), step (2), and step (3) may be performed using the same culture substrate.

[0139] (Process (3))

[0140] The method for producing iPS cells comprises: a step of culturing somatic cells into which reprogramming factors have been introduced in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin (step (3)). In step (3), the somatic cells obtained in step (2) are cultured in the presence of the cell scaffold material. The cell scaffold material is, for example, arranged on the surface of a culture substrate body. With respect to step (3), for example, it is a step of culturing somatic cells using a culture substrate having the culture substrate body and the cell scaffold material.

[0141] The shape and size of the culture substrate body are not particularly limited. Examples of the culture substrate body include containers, fibers, nonwoven fabrics, hollow fibers, particles, membranes, and porous membranes.

[0142] The culture substrate used in step (3) is preferably a culture container. In this case, the cell scaffold material is preferably arranged on the bottom surface of the culture container body. The culture container may be a culture dish or a plate having a plurality of holes.

[0143] The cell scaffold material used in step (3) comprises a peptide-containing polyvinyl acetal resin. In the cell scaffold material used in step (3), the peptide-containing polyvinyl acetal resin is preferably present at least on the surface that adheres to somatic cells (the surface that adheres to somatic cells).

[0144] In step (3), the somatic cells into which the reprogramming factors have been introduced are preferably cultured on the surface of the cell scaffold material. In step (3), adherent culture is preferably performed.

[0145] As the culture medium for culturing somatic cells in step (3), 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 at home can be used. As the culture medium, for example, a culture medium described in the non-patent document 1 can be used.

[0146] Commercially available products of the culture medium 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. In addition, as the culture medium, a culture medium to which various cytokines of the commercially available products are added can be used.

[0147] The culture temperature of step (3) is preferably 36°C or higher, and preferably 38°C or lower. The CO2 concentration during the culture of step (3) is preferably 4% or higher, and preferably 6% or lower. For example, the culture conditions of step (3) are 37°C and a CO2 concentration of 5%. In addition, during the culture of step (3), 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.

[0148] iPS cells can be induced in, for example, about 14 days from the start of the culture in step (3), and a cell population containing iPS cells can be obtained.

[0149] 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 the undifferentiation markers, TRA-1-60 and SSEA4 are known. In particular, TRA-1-60 is a specific antigen for iPS cells or ES cells, which cannot be detected in somatic cells, and is therefore a suitable marker for confirming whether they are induced into iPS cells (for example, WO2018 / 155595A1, Japanese Patent Publication 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)).

[0150] Hereinafter, the present invention will be specifically described with reference to Examples. The present invention is not limited to the following Examples.

[0151] As a culture container, the following container was prepared.

[0152] Culture container (S) (a plate having a cell scaffold material containing a peptide-containing polyvinyl acetal resin, prepared by the following preparation method (S))

[0153] Culture container (L) (a plate coated with "laminin 511E8 fragment (iMatrix-511)" manufactured by NIPPI, "mircroplate with lid, flat bottom, TC treated polystylene" manufactured by IWAKI)

[0154] Culture container (F) (suspension culture plate, "suspension culture plate" manufactured by Corning)

[0155] [Method for preparing culture container (S)]

[0156] Preparation of coating solution containing peptide-containing polyvinyl acetal resin:

[0157] 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% is prepared. 30 parts by weight of acrylic acid and 70 parts by weight of polyvinyl acetal resin are dissolved in 255 parts by weight of tetrahydrofuran to obtain a polymer mixed solution. 0.015 parts by weight of PERBUTYL O (manufactured by NOF Corporation) is dissolved in the obtained polymer mixed solution and reacted at 90°C for 6 hours. Then, the reacted solution is mixed with 30,000 parts by weight of water. The obtained precipitate is 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" is sometimes described as "polyvinyl acetal resin (X)".

[0158] Prepare dimethylformamide (DMF) as the first solvent. Prepare dimethylformamide (DMF) as the second solvent. As a peptide, prepare a cyclic peptide having an amino acid sequence of Arg-Gly-Asp-Phe-Lys (5 amino acid residues, a cyclic skeleton is 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.

[0159] The obtained solution was reacted at 40° C. for 2 hours to dehydrate and condense the carboxyl group in the acrylic acid-derived structural unit of the polyvinyl acetal resin (X) and the amino group of Lys of the peptide, thereby obtaining a solution containing a peptide-containing polyvinyl acetal resin.

[0160] The obtained solution containing the peptide-containing polyvinyl acetal resin was diluted 100 times 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 obtained by dissolving it in butanol (alcohol solvent), and 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 is 0.1% by weight.

[0161] Preparation of culture vessel(s):

[0162] 100 μL of the obtained coating solution was applied to a 6-well plate (bottom area of ​​each well: 9.5 cm) by cast coating. 2 ) in each well, and then the alcohol solvent was removed by vacuum drying at 60°C for 3 hours. In this way, a culture container (S) was obtained in which a cell scaffold material (resin film as a dried material layer of the coating liquid) containing a peptide-containing polyvinyl acetal resin was arranged on the bottom surface of each well. It should be noted that the content of the peptide part 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.

[0163] As a culture medium, the following culture medium was prepared.

[0164] Culture medium (1) (prepared by the following preparation method (1), equivalent to the monocyte culture medium for non-T cells described in Non-Patent Document 1)

[0165] Culture medium (2) (prepared by the following preparation method (1), equivalent to the monocyte culture medium for non-T cells described in Non-Patent Document 1, the same as culture medium (1))

[0166] Culture medium (3) (prepared by the following preparation method (3), equivalent to the maintenance culture medium StemFit described in Non-Patent Document 1)

[0167] [Method for preparing culture medium (1)]

[0168] 400 mL of "Stem Fit AK02N Solution A" manufactured by AJINOMOTO and 100 mL of "Stem Fit AK02N Solution B" manufactured by AJINOMOTO were mixed. To the resulting mixed solution, cytokines were added in such a manner that the final concentrations of IL-6, SCF, TPO, Flt-3L, IL-3, IL-3, and G-CSF were 50 ng / mL, 50 ng / mL, 10 ng / mL, 20 ng / mL, and 10 ng / mL, respectively. Thus, a culture medium (1) was obtained.

[0169] [Method for preparing culture medium (3)]

[0170] 400 mL of "Stem Fit AK02N Solution A" manufactured by AJINOMOTO, 100 mL of "Stem Fit AK02N Solution B" manufactured by AJINOMOTO, and 2 mL of "Stem Fit AK02N Solution C" manufactured by AJINOMOTO were mixed to obtain a culture medium (3).

[0171] In Examples 1 to 3 and Comparative Examples 1 to 3 described below, cell culture and introduction of reprogramming factors using Sendai virus vectors were performed according to the protocol described in Non-Patent Document 1, except for the parts specifically described below.

[0172] (Example 1)

[0173] Process (1):

[0174] As somatic cells, blood-derived mononuclear cells were prepared. In addition, 1.5 mL of the culture medium (1) was added per well to the culture container (S). Next, 1.0×10 6 The mononuclear cells were cultured at 37°C and 5% CO2 concentration in an incubator for 5 days. On the third day from the start of the culture, 0.75 mL of the liquid in the culture container was replaced with 0.75 mL of fresh liquid culture medium.

[0175] Step (2):

[0176] After culturing for 5 days in step (1), the somatic cells were collected into a 15 mL tube. 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 (2) 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 in such a manner 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 (2) 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 (2), centrifuging and removing the supernatant were further repeated twice. Next, 0.1 mL of culture medium (2) was added to the 1.5 mL tube and mixed thoroughly. In this way, reprogramming factors were introduced into the somatic cells.

[0177] After the reprogramming factors are introduced into the somatic cells, step (3) is performed.

[0178] Step (3):

[0179] 0.75 mL of the culture medium (2) was added per well of the culture container (S). The somatic cells introduced with the reprogramming factors obtained in step (2) were cultured at a rate of 1.5×10 5The number of cells / well was inoculated on the cell scaffold material in the culture container (S), and adherent culture was performed in an incubator at 37°C and a CO2 concentration of 5%. It should be noted that from the start of the culture in step (3), 0.5 mL of fresh culture medium (3) was added to the culture container (S) on the 1st, 3rd, 5th and 7th days. In addition, after the 9th day from the start of the culture in step (3), the liquid in the culture container (S) was replaced with 0.75 mL of fresh culture medium (3) at a frequency of more than once every 2 days. The colony morphology was confirmed on the 14th day from the start of the culture in step (3), and the result was a colony morphology of iPS cells.

[0180] (Examples 2, 3 and Comparative Examples 1 to 3)

[0181] Except that the type of culture container used in step (1) or (3) was changed as shown in the following table, steps (1) to (3) were performed in the same manner as in Example 1. In Examples 2 and 3 and Comparative Examples 1 to 3, the colony morphology was confirmed on the 14th day from the start of the culture in step (3), and the result was the colony morphology of iPS cells.

[0182] (evaluate)

[0183] In the following description and figures, the numbers used with P refer to the number of passages from the start of step (3). P0 is the 0th passage, specifically, the somatic cells obtained in step (2) (the somatic cells inoculated into the culture container at the start of step (3)). It should be noted that the cell passage is performed as follows.

[0184] Cell passaging:

[0185] The cell population on day 14 from step (3) 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 calculated using an automatic cell counter from the obtained single cells. In addition, based on the calculated total number of cells, 1×10 4 cells~50×10 4 Cells of cells were inoculated into the same type of culture container, thereby performing cell subculture. It should be noted that the cell subculture was performed once or twice a week, and the culture medium (3) was used as the culture medium. During subculture, ROCK inhibitor (Y-27632) was added to the culture medium (3) in such a manner that the final concentration was 10 μmol / L. In addition, after the second day of subculture, the culture medium was replaced at a frequency of once every three days or more.

[0186] In the following description and drawings, the first character of SS, LS, FS, LL, FL, and SL refers to the type of culture container used in step (1), and the second character refers to the type of culture container used in step (3). For example, LS means that a culture container (L) is used in step (1), and a culture container (S) is used in step (3).

[0187] (1) Virus survival rate

[0188] When cells were passaged, a portion of the single cells was collected into a 1.5 mL tube. The collected single cells were measured by flow cytometry (Attune NxT Flow Cytometer manufactured by ThermoFisher 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 Figure 2. Figure 1 shown. Figure 1 The smaller the GFP positive rate on the vertical axis, the lower the virus survival rate. Figure 1 As shown, the result of the virus survival rate in Example 1 was particularly excellent.

[0189] (2) Cell proliferation and cell survival rate

[0190] According to the number of days of culture after starting step (3), the cell proliferation and cell survival rate are evaluated according to the following steps. The cells recovered during the subculture are counted using a fully automatic cell counter. The number of cells in the well and the cell survival rate are calculated from the obtained results. In addition, the number of cells in the well is divided by the number of cells inoculated to calculate the cell proliferation rate during the period, and the theoretical cumulative number of cells obtained when the total number of cells is subcultured during the evaluation period is calculated. The results are as follows: Figure 2 and Figure 3 shown. Figure 2 This is a graph showing the relationship between the number of culture days from the time of culturing somatic cells into which reprogramming factors have been introduced (the number of culture days from the start of step (3)) and the cumulative number of cells. Figure 3 This is a graph showing the relationship between the number of passages from the time of culturing somatic cells into which reprogramming factors have been introduced (the number of passages from the start of step (3)) and the survival rate of cells. Figure 2 and Figure 3 As shown, the results of the cell proliferation of Example 1 are the same as those of the cell proliferation of Comparative Examples 1 to 3. In addition, the results of the cell survival rate of Examples 1 to 3 are the same as those of the cell survival rate of Comparative Examples 1 to 3.

[0191] (3) Doubling time

[0192] The doubling time of cells during cell recovery from P5 to P8 (the average time required for the number of cells to double) was calculated according to the following steps. First, the cumulative number of cells during cell recovery from P8 obtained by the "(2) Cell proliferation and cell survival rate" was divided by the cumulative number of cells during cell recovery from P5 to calculate the cell proliferation rate. The doubling time was calculated from the calculated cell proliferation rate and the culture time (set to 1 day = 24 hours). The results are shown in the table below. As shown in the table, the doubling time results of Example 1 were particularly excellent.

[0193] (4) Undifferentiated (cell surface markers)

[0194] (4-1) Microscope observation

[0195] Using the P3 cells in culture, the undifferentiation markers on the cell surface are stained according to the following steps to evaluate the undifferentiation. Prepare an antibody against TRA-1-60, that is, an antibody labeled with the NL557 fluorescent dye ("Glo LIVE anti TRA-1-60 NL557" manufactured by R&D Systems). In addition, prepare an antibody-containing culture medium (3) obtained by diluting the antibody culture medium (3) 50 times. The P3 cells in culture are inoculated in the culture container used in step (3), and on the 6th day from the inoculation, the culture medium (3) in the culture container is replaced with the antibody-containing culture medium (3). Then, culture is carried out for 1 hour in an incubator at 37°C and a CO2 concentration of 5%. After culturing for 1 hour, wash the wells of the culture container once with 1×DPBS. Use a fluorescence microscope ("BZ-X800" manufactured by KEYENCE) to observe the wells under the fluorescent field or bright field and take pictures. The results are as follows. Figure 4 shown. Figure 4 In the figure, the fluorescence field and bright field are the results of the images taken at a magnification of 20 times, and the fluorescence ×100 is the result of the images taken at a magnification of 100 times. Figure 4 The images of the fluorescent field and bright field in the well are obtained by automatically photographing the entire surface to obtain the full field of view in the well, and the whole well is combined into one image by the image software manufactured by KEYENCE. In Examples 1 to 3 and Comparative Examples 1 to 3, there is no significant difference in the area occupied by the cells in the well. On the other hand, in Examples 1 to 3, the number of cells labeled by the antibody is greater than that in Comparative Examples 1 to 3. In addition, in Examples 1 to 3, the cells are labeled more brightly than in Comparative Examples 1 to 3, and in particular, in Example 1, the cells are labeled more brightly than in Examples 2 and 3.

[0196] (4-2) Flow cytometry

[0197] Prepare an antibody against TRA-1-60, i.e., an antibody labeled with PE fluorescent dye ("TRA-1-60 PE Antibody" manufactured by Thermo Fisher Scientific). In addition, prepare an antibody against SSEA4, i.e., an antibody labeled with PE fluorescent dye ("SSEA4 PE Antibody" manufactured by Thermo Fisher Scientific). Contact P6 cells with these antibodies. Next, use a flow cytometer ("Attune NxTFlow Cytometer" manufactured by Thermo Fisher Scientific) to determine the positivity and MFI of TRA-1-60, as well as the positivity and MFI of SSEA4. It should be noted that MFI refers to the median fluorescence intensity. Specifically, the evaluation was performed according to the following steps.

[0198] Similar to the "Cell Passaging" step, P6 cells 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. Next, 5 × 10 5 The cells of cells were collected into a 1.5 mL tube, and the 1.5 mL tube was centrifuged at 300 × g and 5 minutes. After centrifugation, the supernatant was removed and 1 × DPBS was added to the 1.5 mL tube. Then, centrifugation and supernatant removal were performed again under the conditions described. Then, blocking was performed with DPBS containing 1% by weight BSA. Then, an appropriate amount of antibody solution containing the two antibodies ("TRA-1-60 PEAntibody" manufactured by Thermo Fisher Scientific and "SSEA4 PE Antibody" manufactured by Thermo Fisher Scientific) was added to the 1.5 mL tube and incubated for 1 hour in the dark at room temperature. Then, after the 1.5 mL tube was centrifuged at 300 × g and 5 minutes, the supernatant was removed and washed twice with 1 × DPBS. Then, 0.4 mL of PBS containing 1% by weight BSA was added to the 1.5 mL tube and mixed, and measured by flow cytometry. The proportion of TRA-1-60 positive cells and the proportion of SSEA4 positive cells included in the main cell population developed on the FSC-SSC dot plot were analyzed. The results are shown in the following table.

[0199] (5) Undifferentiated (gene expression marker)

[0200] Using cells from P0, P1, P3, and P7, relative quantification of undifferentiation marker genes expressed in each cell was performed. Relative quantification was performed using a PCR device ("QuatnStudio3 Real-Time PCR System" manufactured by Thermo Fisher Scientific). Specifically, the evaluation was performed according to the following steps. During subculturing, the cell population was peeled off from the culture container and singled, and 2×10 5 The cells of cells were collected in 1.5mL tubes. Then, the "TaqMan Fast Advanced Cells-to-Ct Kit" manufactured by Thermo Fisher Scientific was used to perform cell lysis, cDNA synthesis and quantitative PCR. The target gene expression markers were NANOG and OCT3 / 4, and the "TaqMan Gene Expression Assay" manufactured by ThermoFisher Scientific was used as the respective primers and detection reagents. It should be noted that the relative quantitative value was calculated based on the gene amount of the P0 cells of FL. The results are as follows Figure 5 shown. Figure 5 The result in the order of P0, P1, P3, and P7 from the left to the right is shown in FIG.

[0201] (6) Differentiation ability (formation of embryoid bodies)

[0202] (6-1) Microscope observation

[0203] Differentiation induction of iPS cells:

[0204] A 96-well plate ("Ultra-low Adsorption Round Bottom 96wel Plate" manufactured by Corning) was prepared. In addition, "Essential 6" manufactured by Thermo Fisher Scientific was prepared as a culture medium. The culture medium was added to the wells of the 96-well plate. It should be noted that ROCK inhibitor (Y-27632) was pre-added to the culture medium used for cell inoculation at a final concentration of 10 μmol / L. The P3 cells (iPS cells, single cells) cultured in step (3) were incubated at 1.5×10 4 The cells were inoculated at a number of cells / well, centrifuged at 300×g for 5 minutes, and then cultured in suspension in an incubator at 37°C and a CO2 concentration of 5%. It should be noted that the culture medium was replaced on the second day of inoculation and every other day thereafter.

[0205] Microscope observation:

[0206] From the time of inoculation to the 39th day, the cells in culture were observed under a microscope. The condition of the cells on the 39th day (n=2) was as follows: Figure 6 In Examples 1 to 3, cell aggregation was observed and the formation of embryoid bodies was confirmed. On the other hand, in Comparative Examples 1 to 3, the formation of embryoid bodies was not confirmed.

[0207] (6-2) Gene expression (qPCR)

[0208] As described below, the three germ layer markers in the embryoid bodies obtained in Examples 1 to 3 were evaluated. The single cells at the time of inoculation (cells at the start of culture (day 0)), the cells on the 11th day from the start of culture (embryoid bodies), the cells on the 18th day from the start of culture (embryoid bodies), and the cells on the 39th day from the start of culture (embryoid bodies) were recovered, and these embryoid bodies were processed with an enzyme containing liquid (Accumax) for cell stripping to make single cells. Then, the "TaqMan Fast Advanced Cells-to-Ct Kit" made by Thermo Fisher Scientific was used to perform cell dissolution, cDNA synthesis and quantitative PCR. With regard to the gene expression markers of the target, AFP, FOXA2, GATA4, GATA6, SOX17, and SOX7 were set as endoderm markers, KDR and T were set as mesoderm markers, and NES, PAX6, and SOX1 were set as ectoderm markers. In addition, the "TaqMan Gene Expression Assay" made by Thermo Fisher Scientific was used in each primer and detection reagent. In addition, the relative quantitative value was calculated together with each gene expression using the gene amount of P0 cells as a reference.

[0209] The results of evaluating the gene expression of each marker by qPCR are as follows Figure 7~Figure 9 In addition, the presence or absence of marker rise was used to score, and a radar chart summarizing the differentiation scores for each germ layer was shown in Fig.10 The scores for endoderm, mesoderm, and ectoderm were evaluated as 1, 3, and 2, respectively, relative to the increase of 1 marker.

[0210] The details and results are shown in Table 1 below.

[0211] [Table 1]

[0212]

[0213] In addition, as Experimental Examples 1 to 4, the following mRNA introduction experiments into somatic cells were performed.

[0214] As a culture container, the following container was prepared.

[0215] Culture container (S1) (a plate having a cell scaffold material containing a peptide-containing polyvinyl acetal resin, prepared by the following preparation method (S1))

[0216] Culture container (L1) (a plate coated with "laminin 511E8 fragment (iMatrix-511)" manufactured by NIPPI, "mircroplate with lid, flat bottom, TC treated polystylene" manufactured by IWAKI)

[0217] Culture container (R1) (a plate coated with "RetroNectin (T100A)" manufactured by TAKARABIO, "Falcon Cell Culture 48 well Multiwell Plate (353078)" manufactured by Corning)

[0218] Culture container (V1) (a plate coated with "vitronectin (220-02041)" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., "Falcon Cell Culture 48 well Multiwell Plate (353078)" manufactured by Corning)

[0219] [Method for preparing culture container (S1)]

[0220] 10 μL of the coating solution was applied to a 48-well plate (bottom area of ​​each well: 0.75 cm) by casting. 2 ) and, except for this, a culture container (S1) is obtained in the same manner as in the above-mentioned “method for preparing a culture container (S)”.

[0221] (Experimental Example 1)

[0222] Introduction of mRNA-eGFP into somatic cells:

[0223] As somatic cells, human peripheral blood CD8+ T cells (Lonza "2W-300") were prepared. In addition, 0.2 mL of the culture medium (1) was added per well to the culture container (S1). Next, 1.4×10 5CD8+T cells were cultured at 37°C and 5% CO2 concentration in an incubator for 2 days. Next, 2 μg / well of mRNA-eGFP ("CleanCap EGFP mRNA" manufactured by TriLink BioTechnologies) was added to the culture container (S1) and mRNA-eGFP was introduced into CD8+T cells by plasma gene introduction. It should be noted that the conditions for plasma irradiation are as follows.

[0224] Power frequency: 50kHz

[0225] Discharge voltage: 16kV p-p

[0226] Discharge time: 30msec

[0227] Separation distance between the electrode tip and the upper surface of the culture container: 1 mm

[0228] Next, 0.2 mL of TransAct (manufactured by Milteny) was added per well to the culture container (S1), and culture was carried out for 30 hours.

[0229] Evaluation of introduction efficiency (flow cytometry):

[0230] Cells cultured for 30 hours were detached from the culture container by pipetting to make single cells. The single cells obtained were measured using a flow cytometer (Attune NxT Flow Cytometer manufactured by Thermo Fisher Scientific) to determine the number of live cells (R1) and the number of cells with GPP fluorescence (R2). The efficiency of mRNA-eGFP introduction was calculated by the following formula.

[0231] Import efficiency (%) = (R2 / R1) × 100

[0232] R1: number of viable cells

[0233] R2: The number of cells with GFP fluorescence

[0234] (Experimental Examples 2~4)

[0235] The type of culture container is selected so that Fig.11 The efficiency of mRNA-eGFP introduction was calculated in the same manner as in Experimental Example 1 except that the above-mentioned method was changed.

[0236] (result)

[0237] like Fig.11As shown, when the culture container (S1) is used, the efficiency of introducing mRNA-eGFP into somatic cells is higher than when the culture containers (L1), (V1), and (R1) are used. That is, in the presence of a cell scaffold material containing a peptide-containing polyvinyl acetal resin, the efficiency of introducing mRNA into somatic cells can be further improved compared to the presence of a cell scaffold material containing a protein.

[0238] Furthermore, as Example 4, an experiment for establishing iPS cells using the following mRNA was performed.

[0239] (Example 4)

[0240] Process (1):

[0241] Peripheral blood mononuclear cells (Human PBMC10M Healthy Japanese (Cat#:93210-10M) manufactured by Precision for Medicine) were prepared as somatic cells. In addition, 0.4 mL of the culture medium (1) was added per well to the culture container (S1). Next, 5.0×10 5 Peripheral blood mononuclear cells at 10 cells / well were cultured in an incubator at 37°C and a CO2 concentration of 5% for 5 days. It should be noted that on the third day from the start of the culture, 0.2 mL of the culture medium (1) in the culture container (S1) was replaced with 0.2 mL of fresh culture medium (1).

[0242] Step (2):

[0243] The culture medium (1) was removed from the culture container (S1) after 5 days of culture in step (1). Next, 0.8 μg / well of mRNA ("ReproRNA-OKSGM" manufactured by STEMCELL Technologies, C / N: ST-05931) was added to the culture container (S1), and the mRNA was introduced into the peripheral blood mononuclear cells by the plasma gene introduction method. It should be noted that the conditions for plasma irradiation are as follows.

[0244] Power frequency: 50kHz

[0245] Discharge voltage: 16kV p-p

[0246] Discharge time: 30msec

[0247] Separation distance between the electrode tip and the upper surface of the culture container: 1 mm

[0248] Step (3):

[0249] The following culture media (3A) and (3B) were prepared.

[0250] Culture medium (3A):

[0251] Recombinant B18R Protein (manufactured by STEMCELL Technologies) was added in an amount of 0.175 μg to 1 mL of the culture medium (1), thereby obtaining a culture medium (3A).

[0252] Culture medium (3B):

[0253] Recombinant B18R Protein (manufactured by STEMCELL Technologies) was added in an amount of 0.175 μg to 1 mL of the culture medium (3). Thus, a culture medium (3B) was obtained.

[0254] In the culture container (S1), 0.2 mL of culture medium (3A) was added per well, and the somatic cells introduced with mRNA (reprogramming factor) were cultured for 1 day in an incubator at 37°C and a CO2 concentration of 5%. After 1 day of adherent culture, 0.133 mL of culture medium (3B) was added per well, and cultured in an incubator at 37°C and a CO2 concentration of 5%. From the start of culture in step (3) to the 8th day, 0.135 mL of culture medium (3B) was added per well every 2 to 3 days, and from the start of culture in step (3) to the 9th day and thereafter, the entire amount of culture medium (3B) was replaced with fresh culture medium (3B) every 2 to 3 days.

[0255] (evaluate)

[0256] (1) Microscope observation

[0257] Prepare an antibody against TRA-1-60, i.e., an antibody labeled with the NL557 fluorescent dye ("Human Glo LIVE anti TRA-1-60(R) Northern Lights NL557-conjugated Antibody" manufactured by R&D Systems). In addition, prepare an antibody-containing culture medium (3B) obtained by diluting the antibody culture medium (3B) 50 times. On the 35th day from the start of the culture in step (3), replace the culture medium (3B) in the culture container (S1) with the antibody-containing culture medium (3B). Then, culture for 30 minutes in an incubator at 37°C and a CO2 concentration of 5%. After culturing for 30 minutes, wash the wells of the culture container (S1) with PBS and replace with new culture medium (3B). Use a fluorescence microscope ("BZ-X800" manufactured by KEYENCE) to observe the wells under the fluorescent field or bright field and take pictures. As a result, the colony morphology of iPS cells was observed, and in addition, a large number of cells labeled with antibodies were observed.

[0258] (Example 5)

[0259] As culture containers, the above-mentioned culture container (S1) and culture container (L1), and the following culture containers (S2) and (S3) are prepared.

[0260] Culture container (S2) (a plate having a cell scaffold material containing a peptide-containing polyvinyl acetal resin, prepared by the following preparation method (S2))

[0261] Culture container (S3) (a plate having a cell scaffold material containing a peptide-containing polyvinyl acetal resin, prepared by the following preparation method (S3))

[0262] [Method for preparing culture container (S2)]

[0263] 10 μL of the coating solution was applied to a 96-well plate (bottom area of ​​each well: 0.32 cm) by casting. 2 ) and, except for this, a culture container (S2) is obtained in the same manner as in the above-mentioned “method for preparing a culture container (S)”.

[0264] [Method for preparing culture container (S3)]

[0265] 100 μL of the coating solution was applied to a 12-well plate (bottom area of ​​each well: 3.8 cm) by casting. 2 ) and, except for this, a culture container (S3) is obtained in the same manner as in the above-mentioned “method for preparing a culture container (S)”.

[0266] The following culture media (4A), (4A+), (4B), and (4B+) were prepared.

[0267] Culture medium (4A):

[0268] As a culture medium (4A), "Fibroblast Growth Medium (Ready-to-use)" manufactured by TAKARABIO was used.

[0269] Culture medium (4A+):

[0270] Recombinant B18R Protein (manufactured by STEMCELL Technologies) was added in an amount of 0.175 μg to 1 mL of the culture medium (4A), and a penicillin-streptomycin mixed solution (manufactured by NACALAITESQUE) was added in an amount of 10 μL. Thus, a culture medium (4A+) was obtained.

[0271] Culture medium (4B):

[0272] "StemFit AK02N" manufactured by AJINOMOTO was used as a culture medium (4B).

[0273] Culture medium (4B+):

[0274] Recombinant B18R Protein (manufactured by STEMCELL Technologies) was added in an amount of 0.175 μg to 1 mL of the culture medium (4B), and a penicillin-streptomycin mixed solution (manufactured by NACALAITESQUE) was added in an amount of 10 μL. Thus, a culture medium (4A+) was obtained.

[0275] Process (1):

[0276] As somatic cells, human fibroblasts ("Normal human skin fibroblasts (children)" manufactured by TAKARABIO) were prepared. In addition, 0.2 mL of the culture medium (4A) was added per well to the culture container (S1). Next, 1.0×10 3 Human fibroblasts at 10 cells / well were cultured in an incubator at 37°C and a CO2 concentration of 5% for 6 days. It should be noted that on the third day from the start of the culture, 0.2 mL of the culture medium (4A) in the culture container (S1) was replaced with 0.2 mL of fresh culture medium (4A).

[0277] Step (2):

[0278] The culture medium (4A) was removed from the culture container (S1) after 6 days of culture in step (1). Next, 0.8 μg / well of mRNA ("ReproRNA-OKSGM" manufactured by STEMCELL Technologies, C / N: ST-05931) was added to the culture container (S1), and the mRNA was introduced into human fibroblasts by plasma gene introduction. It should be noted that the conditions for plasma irradiation are as follows.

[0279] Power frequency: 50kHz

[0280] Discharge voltage: 16kV p-p

[0281] Discharge time: 30msec

[0282] Separation distance between the electrode tip and the upper surface of the culture container: 1 mm

[0283] Step (3):

[0284] In the culture container (S1), 0.4 mL of culture medium (4A+) was added per well, and the somatic cells into which the mRNA (reprogramming factor) was introduced were cultured in an incubator at 37°C and a CO2 concentration of 5%. After 1 day of culture, the entire amount of the culture medium was replaced with 0.4 mL of fresh culture medium (4A+). On the 4th day from the start of the culture in step (3), the entire amount of the culture medium was replaced with a fresh culture medium (4A+) containing 0.8 μg of puromycin per 1 mL.

[0285] On the 7th day from the start of the culture in step (3), the entire amount of the culture medium was replaced with a fresh culture medium (4B+). Then, from the start of the culture in step (3) to the 14th day, the entire amount of the culture medium was replaced with a fresh culture medium (4B+) every 3 days.

[0286] The number of iPS cell colonies was confirmed on the 14th day from the start of the culture in step (3). In addition, the colonies were peeled off by pipetting and recovered in the culture container (S2) (P1). 0.1 mL of culture medium (4B) was added to each well of the culture container (S2), and cultured in an incubator at 37°C and a CO2 concentration of 5%. The entire amount of culture medium was replaced with fresh culture medium (4B) every 2 days. On the 6th day from the start of culture in the culture container (S2), a peeling agent ("TrypLE Select" manufactured by Thermo Fisher Scientific) was used to make single cells. From the obtained single cells, 2×10 4The cells of cells were inoculated into a culture container (S3), thereby performing cell passage (P2). It should be noted that the cell passage was performed once a week, and the culture medium used was culture medium (4B) (P3 to P7). During the passage, a ROCK inhibitor (Y-27632) was added to the culture medium (4B) 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 two days.

[0287] (Comparative Example 4)

[0288] Step (1) to step (3) on the 14th day were carried out in the same manner as in Example 5, except that the culture container (L1) was used instead of the culture container (S1).

[0289] (evaluate)

[0290] (1) Undifferentiated (cell surface markers)

[0291] (1-1) Microscope observation

[0292] Using cells on the 14th day after plasma irradiation, the undifferentiation markers on the cell surface were stained according to the following steps to evaluate undifferentiation. Prepare a TRA-1-60 antibody labeled with NL557 fluorescent dye ("Glo LIVE anti TRA-1-60 NL557" manufactured by R&D Systems). In addition, prepare an antibody-containing culture medium obtained by diluting the antibody culture medium (4B) 50 times. Replace the culture medium (4B) in the culture container with the antibody-containing culture medium, and culture it in an incubator at 37°C and a CO2 concentration of 5% for 1 hour. After culturing for 1 hour, wash the wells of the culture container once with 1×DPBS. Use a fluorescence microscope ("BZ-X800" manufactured by KEYENCE) to observe the culture container under the fluorescent field of view and take pictures. The results are as follows. Fig.12 shown. Fig.12 This is the result of photographing the fluorescent field at a magnification of 100 times.

[0293] like Fig.12 As shown, cells labeled with the antibody (TRA-1-60 positive cells) were well obtained in Example 5. On the other hand, TRA-1-60 positive cells were hardly obtained in Comparative Example 4. Therefore, the subsequent evaluation was performed only on the cells obtained in Example 5.

[0294] (1-2) Flow cytometry

[0295] A TRA-1-60 antibody labeled with FITC fluorescent dye ("TRA-1-60 FITCAntibody" manufactured by BD) was prepared. The cells on day 7 of P3 were reacted with the antibody, and the expression of TRA-1-60 was analyzed using a flow cytometer ("Attune NxT Flow Cytometer" manufactured by Thermo Fisher Scientific). Specifically, the evaluation was performed according to the following procedure.

[0296] In the same manner as in the "Cell Passaging" step, the cells on day 7 of P3 were separated into single cells using a stripping agent ("TrypLE Select" manufactured by Thermo Fisher Scientific). 5×10 5 The cells were collected in a 1.5 mL tube and centrifuged at 300 × g for 5 minutes. After centrifugation, the supernatant was removed and Pharmingen Stain Buffer (FBS) manufactured by BD was added to the 1.5 mL tube. Then, centrifugation and supernatant removal were performed again under the conditions described above. Then, Pharmingen Stain Buffer (FBS) manufactured by BD was used for blocking. Then, a solution prepared by diluting 10 μL of the antibody with 100 μL of Pharmingen Stain Buffer (FBS) manufactured by BD was added to the 1.5 mL tube and incubated for 1 hour in the dark at room temperature. Then, after centrifuging the 1.5 mL tube at 300 × g for 5 minutes, the supernatant was removed and washed twice with Pharmingen Stain Buffer (FBS). Then, 0.4 mL of Pharmingen Stain Buffer (FBS) was added to the 1.5 mL tube and mixed, and measured by flow cytometry. The histogram of the fluorescence intensity of FITC fluorescent dye is as follows Fig.13 shown.

[0297] Fig.13 In the figure, the horizontal axis represents the fluorescence intensity of FITC fluorescent dye, and the vertical axis represents the number of cells. Fig.13 The histogram on the left (no coloring) is a histogram obtained when the cells were not reacted with the antibody. Fig.13 The histogram on the right side of the graph (with color) is a histogram when the cells are reacted with the antibody. Fig.13 The maximum value of the fluorescence intensity in the left histogram (without coloring) in is set as the boundary value between the negative area and the positive area in the right histogram (with coloring). Fig.13In the figure, the ratios (%) of the number of cells contained in the negative area and the positive area in the right histogram (colored) are represented as "FITC-A-" and "FITC-A+", respectively.

[0298] (2) Undifferentiated (gene expression markers)

[0299] Using cells from day 7 of P6, relative quantification of undifferentiation marker genes expressed in each cell was performed. Relative quantification was performed using a PCR device ("QuatnStudio3 Real-Time PCR System" manufactured by Thermo Fisher Scientific). Specifically, the evaluation was performed according to the following steps. During subculturing, the cell population was removed from the culture container and singled, and 2×10 5 cells were collected in a 1.5 mL tube. Next, the "TaqMan Fast Advanced Cells-to-Ct Kit" manufactured by Thermo Fisher Scientific was used to perform cell lysis, cDNA synthesis and quantitative PCR. The target gene expression markers were NANOG, OCT3 / 4 and SOX2, and the "TaqMan Gene Expression Assay" manufactured by ThermoFisher Scientific was used as the respective primers and detection reagents. The expression level of each gene expression marker was standardized by the ΔΔCT method using the housekeeping gene 18S. It should be noted that the relative quantitative value was calculated by taking the expression level in the human fibroblast-derived iPS cell line (253G1) established by Kyoto University as 1. The results are as follows Fig.14 shown.

[0300] (3) Differentiation ability

[0301] Differentiation induction of three germ layers of iPS cells:

[0302] As a culture medium, each germ layer differentiation medium (Ectoderm Medium, Mesoderm Medium, Endoderm Medium) of the "Trilineage Differentiation Kit" manufactured by Stemcell Technologies was prepared. The culture medium was added to each well of the culture container (S3). It should be noted that ROCK inhibitor (Y-27632) was pre-added to the culture medium used for cell inoculation in such a way that its final concentration was 10 μmol / L. Similar to the step of "cell passaging", the cells on the 7th day of P7 were made into single cells using a stripping agent ("TrypLE Select" manufactured by Thermo Fisher Scientific). Endoderm differentiation induction and ectoderm differentiation induction were performed at 8×10 5 The number of cells per well was 2×10 cells / well. 5 The cells were inoculated at the number of cells / well, centrifuged at 300×g for 5 minutes, and then cultured in an incubator at 37°C and 5% CO2. It should be noted that the culture medium was replaced once a day. For comparison, the three germ layer differentiation was induced using the iPS cell line (253G1) in the same way.

[0303] (3-1) Immunostaining

[0304] Prepare the following solutions and antibodies.

[0305] [Solution]

[0306] Fixative: 4% paraformaldehyde

[0307] Blocking solution: 0.1% TritonX100, 10% FBS containing PBS

[0308] Wash buffer: PBS containing 0.025% polyoxyethylene (20) sorbitan monolaurate

[0309] [Antibody]

[0310] Primary antibody (ectoderm): Anti-PAX6 antibody (Rabbit IgG)

[0311] Primary antibody (mesoderm): Anti-Brachyury / Bry antibody (Rabbit IgG H&L)

[0312] Primary antibody (endoderm): Anti-SOX17 antibody (Rabbit IgG)

[0313] Secondary antibody (Rabbit): Goat pAb to Rb IgG- Alexa Fluor488

[0314] Immunostaining for endoderm differentiation induction and mesoderm differentiation induction was performed on the 5th day from the start of culture in each differentiation medium, and immunostaining for ectoderm differentiation induction was performed on the 7th day from the start of culture in the differentiation medium. First, after removing the culture medium from the culture container (S3), 0.75mL of PBS was added. Next, PBS was removed, 0.5mL of fixative was added, and the mixture was allowed to stand for 15 minutes. The fixative was removed and washed twice with 0.5mL of PBS. Then, 500μL of blocking solution was added and allowed to stand for 15 minutes. The blocking solution was removed, Wash buffer was added, and the solution was removed after standing for 5 minutes. Wash buffer was added again, and the solution was removed after standing for 5 minutes. Then, the first antibody-containing solution was added at 300μL / well and allowed to stand for 2 hours. Each first antibody-containing solution was prepared by diluting the blocking solution. The first antibody-containing solution was removed, Wash buffer was added at 2mL / well, and the mixture was removed after standing for 5 minutes. Wash buffer was added again, and the solution was removed after standing for 5 minutes. Then, add the solution containing the secondary antibody and incubate in the refrigerator overnight. The next day, dilute 1000 times with Wash buffer and stain with DAPI for 3 minutes. Wash twice with 2mL / well Wash buffer. Next, use a fluorescence microscope ("BZ-X800" manufactured by KEYENCE) to observe the wells under the fluorescent field and take pictures. The iPS cell line (253G1) before differentiation induction was used as a negative control. The results are as follows Fig.15 shown.

[0315] (3-2) Gene expression (qPCR)

[0316] When performing the "(3-1) immunostaining", a stripping agent ("TrypLE Select" manufactured by Thermo Fisher Scientific) is used to recover single cells in the same manner as in the "cell passaging" step. Next, the "TaqMan Fast Advanced Cells-to-Ct Kit" manufactured by Thermo Fisher Scientific is used to dissolve the cells, synthesize cDNA, and perform quantitative PCR. For the gene expression markers of the target, FOXA2 and SOX17 are set as endoderm markers, T is set as a mesoderm marker, and PAX6 is set as an ectoderm marker. In addition, the "TaqMan Gene Expression Assay" manufactured by Thermo Fisher Scientific is used in each primer and detection reagent. The expression level of each gene expression marker is standardized by the ΔΔCT method using the housekeeping gene 18S. The iPS cell line (253G1) before differentiation induction and cells on the 7th day of P7 were used as negative controls. In addition, the relative quantitative value was calculated by taking the expression level in the negative control of the iPS cell line (253G1) as 1 together with the expression of each gene.

[0317] The results of evaluating the gene expression of each marker by qPCR are as follows Fig.16 shown. Fig.16 In the figure, "253G1" and "Example 5" represent the results of the negative control, "253G1(inner)" and "Example 5(inner)" represent the results after induction of endoderm differentiation, "253G1(middle)" and "Example 5(middle)" represent the results after induction of mesoderm differentiation, and "253G1(outer)" and "Example 5(outer)" represent the results after induction of ectoderm differentiation.

Claims

1. A method for producing induced pluripotent stem cells, comprising: A step of introducing reprogramming factors into somatic cells; and A step of culturing the somatic cells into which the reprogramming factors have been introduced in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin.

2. The method for producing induced pluripotent stem cells according to claim 1, comprising, before the step of introducing reprogramming factors into the somatic cells: A step of culturing somatic cells in the presence of a cell scaffold material comprising a peptide-containing polyvinyl acetal resin.

3. The method for producing induced pluripotent stem cells according to claim 1 or 2, wherein: In the step of introducing the reprogramming factors into the somatic cells, the reprogramming factors are introduced into the somatic cells using a Sendai virus vector.

4. The method for producing induced pluripotent stem cells according to claim 1 or 2, wherein: The reprogramming factor is mRNA.

5. The method for producing induced pluripotent stem cells according to claim 4, wherein: The mRNA is introduced into the somatic cells by plasma gene introduction method.

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