JPWO2014196549A1 - 3D cell culture method using fiber-on-fiber and substrate for the same - Google Patents

Abstract

The present invention includes a substrate for cell culture, comprising a nanofiber made of a biopolymer selected from the group consisting of gelatin, collagen and cellulose on a support, a cell freezing agent containing the substrate, Cell culture method comprising seeding cells on the substrate and statically culturing the cells, dissociating the cells from the substrate using an enzyme-free dissociation solution, and culturing the cells in another culture The method comprising seeding on a substrate and further culturing the cell further, and in particular, the method comprising dispersing the cells into single cells at the time of passage, using the cell freezing agent A method for cryopreserving pluripotent stem cells is provided.

Description

  The present invention relates to a culture medium suitable for culturing cells, for example, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), particularly human pluripotent stem cells. The present invention relates to a material, and a method for culturing stem cells using the material. More specifically, the present invention relates to a cell culture substrate in which nanofibers (bionanofibers) made of biopolymers such as gelatin, collagen, and cellulose are coated on a support such as gauze or sponge, and the culture substrate. The present invention relates to a method for maintaining and amplifying cells, a method for freezing cells, and the like by dispersing cells to a single cell without performing enzyme treatment at the time of subculture using the cell freezing agent containing the material and the culture substrate.

  Human pluripotent stem cells can grow indefinitely under appropriate conditions, and have the property of being able to differentiate into any cell in living tissue (multipotency), so cell transplantation therapy, drug screening, and regenerative medicine Application to various fields is expected. However, in conventional human pluripotent stem cell culture methods, feeder cells and various polymers have been used as cell culture substrates. However, these methods are complicated in preparation and quality is not stable. Therefore, stable culture and supply of human pluripotent stem cells has been difficult. In particular, development of a high-quality, large-scale, fully automatic culture method for human pluripotent stem cells requires a more stable and inexpensive method, but such a method has not yet been established.

  In the conventional two-dimensional culture using culture dishes, the number of culture dishes is required in units of 100, and subculture operations are required from individual culture dishes. It is unsuitable for the development of high-quality, large-volume, fully automated culture methods. Therefore, in order to enable mass culture of pluripotent stem cells in a limited space, three-dimensional culture is essential. So far, culture methods using suspension culture, microbeads, etc. have been developed (Non-Patent Documents 1 and 2), however, shearing stress on the cell surface due to aggregation or agitation of cell mass has become a problem, It has not been put into practical use.

  In recent years, development of novel human pluripotent stem cell culture methods that do not use feeder cells has been actively conducted. Currently, cell culture substrates that are widely used include Matrigel and recombinant proteins (Non-patent Document 3). However, these materials are expensive and have large quality differences between lots. It lacks stability.

Human pluripotent stem cells cultured under these conditions become unstable, resulting in abnormal cell growth rates, transformation to very heterogeneous cell populations, loss of differentiation potential, karyotypic mutations Cause abnormalities.
As an alternative to this, the development of cell culture substrates using polymers such as polymers has also been reported (Non-Patent Documents 4 and 5), and although they have been commercialized, stable products can be obtained. However, it is very expensive and may not be suitable depending on the cell line. Thus, a stable and inexpensive cell culture substrate has not been prepared.

The cell culture substrate is required to supply oxygen and nutrients necessary for the target cell group and to maintain a stable shape. Recently, nanofibers have attracted attention. Nanofibers are ultrafine fibers with a fiber diameter on the order of nanometers, and the structure composed of nanofibers is similar in size to the extracellular matrix, and the cell adhesion is improved by increasing the specific surface area. Since there are advantages such as being possible, a nanofiber made of a synthetic polymer (Non-patent Document 6) or a mixture of a synthetic polymer and a biopolymer such as collagen or gelatin (Non-Patent Documents 6 and 7) is produced. However, it has been reported that human ES cells cannot be maintained and grown in a culture system that does not use feeder cells (Non-patent Document 7).
On the other hand, there has been no report of using nanofibers composed only of biopolymers as a culture substrate for pluripotent stem cells.

  In addition, conventionally, for the passage of human pluripotent stem cells, techniques using enzymes such as collagenase, dispase, trypsin, or mechanical passage methods such as cell strainers and pipetting have been performed. In the method using an enzyme, there is damage to the cell by the enzyme reaction, and the enzyme reaction to the cell is not uniform. Moreover, there is a problem that cells are killed when dispersed to a single cell. On the other hand, the mechanical passage method has many problems due to extremely large cell damage.

Tissue Eng Part C Methods., 16 (4), 573-582 (2010) Curr Protoc Stem Cell Biol Chapter 1, Unit 1C 11 (2010) Nataure Biotechnology, 28 (6): 581-583 (2010) Nataure Biotechnology, 28 (6): 606-610 (2010) Nataure Biotechnology, 28 (6): 611-615 (2010) Advanced Drug Delivery Reviews, 61 (12): 1084-1096 (2009) Journal of Cellular and Molecular Medicine, 13 (9B): 3475-3484 (2009)

  An object of the present invention is to provide an inexpensive novel culture substrate capable of stably supplying a large amount of human pluripotent stem cells, and to provide an inexpensive and simple method for culturing human pluripotent stem cells using the same. Is to provide. Another object of the present invention is to provide a substrate for culturing human pluripotent stem cells that does not require enzyme treatment at the time of passage and does not die even if dispersed in a single cell, Thus, it is to provide a more uniform culture of human pluripotent stem cells. Furthermore, another object of the present invention is to provide a cell freezing agent containing the culture substrate and a cell freezing method.

  The present inventors have focused on using a biomaterial that is highly biocompatible and inexpensive as a substrate for culturing human pluripotent stem cells, and using electrospinning to convert the biomaterial into nanofibers. I was devising. Therefore, in order to achieve the above object, an attempt was made to apply the nanofiber to a support such as gauze or sponge in order to further increase the physical strength of the nanofiber. The obtained culture substrate was named “fiber on fiber”.

Fiber-on-fiber can be folded and used because its shape can be changed flexibly. Therefore, the present inventors placed a suspension of human ES cells or human iPS cells dispersed into single cells on four fiber-on-fibers, folded them and cultured them in a medium for ES cells. . As a result, human pluripotent stem cells cultured on this fiber-on-fiber substrate showed approximately twice the number of cells compared to culture on matrigel-coated dishes. The cell density was higher than that. In addition, since gauze, sponge, etc. are more porous than glass / plastic substrates, etc., when the fiber-on-fiber is immersed in the culture solution, the culture solution will naturally permeate so that the culture solution to the cells Supply has also improved.
This fiber-on-fiber is flexible in shape, so there is no need to select a container, and it can be cultured in any container as long as the nutrients reach the cells. That is, stem cells such as pluripotent stem cells It has become clear that desired cells such as can be easily cultured in large quantities. Furthermore, the present inventors have confirmed that pluripotent stem cells maintain pluripotent and normal karyotype even after long-term subculture using this fiber-on-fiber substrate. Moreover, even when freezing and thawing operations were performed using a fiber-on-fiber substrate, it was found that pluripotent stem cells formed colonies and the cell viability was high, and the present invention was completed.

That is, the present invention is as follows.
[1] A cell culture substrate comprising a nanofiber made of a biopolymer selected from the group consisting of gelatin, collagen and cellulose on a support.
[2] The substrate according to the above [1], wherein the nanofiber is crosslinked.
[3] The substrate according to [1] or [2] above, wherein the biopolymer is gelatin or collagen.
[4] The substrate according to [1] or [2] above, wherein the biopolymer is gelatin.
[5] The base material according to any one of the above [1] to [4], wherein the nanofiber is obtained by an electrospinning method.
[6] The substrate according to any one of [1] to [5], wherein the support is selected from the group consisting of gauze and sponge.
[7] The base material according to any one of the above [1] to [6], wherein the cell is a stem cell.
[8] The substrate according to [7] above, wherein the stem cells are pluripotent stem cells.
[9] The substrate according to [8] above, wherein the pluripotent stem cells are ES cells or iPS cells.
[10] The substrate according to [8] or [9] above, wherein the pluripotent stem cells are derived from human.
[11] The substrate according to any one of [1] to [10] above, wherein the culture is a maintenance amplification culture of cells.
[12] A cell freezing agent comprising the substrate according to any one of [1] to [6] above.
[13] A method for culturing a cell, comprising seeding a cell on the substrate according to any one of [1] to [6], and culturing the cell stationary.
[14] Dissociate cells from the substrate using a dissociation solution that does not contain an enzyme, seed the cells on the substrate according to any one of [1] to [6] above, and further leave the cells still The method according to [13] above, which is cultured.
[15] The method described in [14] above, wherein the cells are dispersed into single cells at the time of passage.
[16] The method according to any one of [13] to [15] above, wherein the cells are cultured in a serum-free medium.
[17] The method described in [16] above, wherein the serum-free medium is a xeno-free medium.
[18] The method described in [16] above, wherein the serum-free medium is a protein-free medium.
[19] The method according to any one of [13] to [18] above, wherein the cell is a stem cell.
[20] The method described in [19] above, wherein the stem cell is a pluripotent stem cell.
[21] The method described in [20] above, wherein the pluripotent stem cells are ES cells or iPS cells.
[22] The method described in [20] or [21] above, wherein the pluripotent stem cell is derived from a human.
[23] The method according to any one of [13] to [22] above, wherein the culture is a maintenance amplification culture of cells.
[24] A method for cryopreserving cells using the freezing agent according to [12].

Since the culture substrate of the present invention has high physical strength and is flexible in shape, three-dimensional culture is possible, and a large amount of cells can be supplied while realizing space saving. In addition, since the culture substrate of the present invention is highly biocompatible and inexpensive, stable supply is facilitated. Furthermore, since the shape of the culture substrate of the present invention can be easily changed, it can be stored frozen regardless of the container.
In addition, when the culture substrate of the present invention is used, since the culture solution penetrates when the culture solution is immersed, the culture solution can be easily supplied to the cells.

They are the electron micrograph (left) of the fiber on fiber obtained by apply | coating nanofiber on cotton gauze, and the electron micrograph (right) of cotton gauze. It is a photograph when a fiber-on-fiber is immersed in a culture solution and human ES cells (H9) are seeded thereon. It is an electron micrograph of human ES cells (H9) cultured for 5 days on fiber-on-fiber. It is the photograph which dye | stained the alkaline phosphatase which is a pluripotent stem cell marker for the human ES cell (H9) cultured on the fiber on fiber. It is a microscope picture of human ES cells (H9) stained with alkaline phosphatase. It is a photograph of a control test in which human ES cells (H9) were cultured on cotton gauze and then stained with alkaline phosphatase. It is a figure which shows the result of the flow cytometry which shows the expression of the pluripotent stem cell marker (SSEA4) in the human ES cell (H9) cultured on the fiber on fiber. It is an immune cell staining photograph showing expression of pluripotent stem cell markers in human ES cells (H9) cultured on fiber-on-fiber. This is a photograph of three-dimensional culture by folding one or four fibers on fiber in a 35-mm dish. It is a figure showing the number of cells after seeding human ES cells (H9) on one or four fiber-on-fibers, folding them, and performing three-dimensional culture. This is a picture of folding 32 fiber-on-fibers and mass-culturing (100 mL) human pluripotent stem cells. It is an alkaline phosphatase staining photograph of human ES cells (H9) subcultured 10 times on fiber-on-fiber. It is a figure which shows the result of the flow cytometry which shows the expression of the pluripotent stem cell marker of the human ES cell (H9) subcultured 10 times on a fiber on fiber. Flow site showing the expression of pluripotent stem cell marker (SSEA4) and differentiation marker (SSEA1) of human ES cells (H1, H9) and human iPS cells (253G1) subcultured 20 times or more on fiber-on-fiber It is a figure which shows the result of a measurement. It is a figure which shows the result of the karyotype analysis of the human ES cell (H9) and the human iPS cell (253G1) which were cultured on the fiber on fiber for a long period (passage 10 times or more). A. It is a figure which shows that the human ES cell (H9) cultured on the fiber on fiber can be differentiated into all germ layers of ectoderm (left), mesoderm (center), and endoderm (right). The upper row shows micrographs stained with fluorescent labeled antibodies (anti-tubulin III antibody, anti-α-SMA antibody, anti-SOX17 antibody in order from the left) for each germ layer detection marker, and the lower row is a micrograph of DAPI-stained cells. Indicates. B. Shows that human ES cells (H1) subcultured 10 times or more on fiber-on-fiber can differentiate into all germ layers of mesoderm (left), ectoderm (center), and endoderm (right) FIG. In order from the left, photomicrographs of immunostaining with anti-α-SMA antibody, anti-tubulin III antibody, and anti-SOX17 antibody (nuclear double staining with DAPI) are shown. It is a figure which shows the teratoma formation ability in the human ES cell (H1, H9) and the human iPS cell (253G1) which were subcultured 10 times or more on a fiber on fiber. It was confirmed to differentiate into all three germ layers of neuroepithelium (ectodermal), cartilage (mesoderm) and endopulmonary lobe (gut-like epithelium). It is a photograph when human ES cells (H9) cultured on fiber-on-fiber are changed in shape with tweezers and inserted into a tube. It is an electron micrograph of human ES cells (H9) cultured on fiber-on-fiber before freezing (left) and 4 days after thawing (right). It is a figure which shows the result of the flow cytometry which shows the expression of the pluripotent stem cell marker in the cell after freezing and thawing | decompressing the human ES cell (H1) cultured on the fiber on fiber. It is a photograph in which cells after freezing and thawing of human ES cells (H1) cultured on fiber-on-fiber were stained with alkaline phosphatase. It is a figure which shows the cell viability after putting the human ES cell (H1) culture | cultivated on the fiber on fiber in a cryopreservation liquid, and freezing and thawing | decompressing. A. It is a photograph of a mass culturing apparatus for human pluripotent stem cells using fiber-on-fiber as a culture substrate. B. It is a figure which shows the result of the flow cytometry which shows expression of the pluripotent stem cell marker (SSEA4) and differentiation marker (SSEA1) of the human ES cell (H1) mass-cultured with the apparatus of A.

  The present invention relates to a cell culture substrate comprising nanofibers composed of a biopolymer selected from the group consisting of gelatin, collagen and cellulose (hereinafter sometimes abbreviated as the culture substrate of the present invention). )I will provide a.

  Cells to which the culture substrate of the present invention can be applied are not particularly limited, and any cell that can be cultivated stationary (for example, lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), Hair cells, hepatocytes, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells, etc.), differentiated cells such as brain cells, lung cells, kidney cells, adipocytes, undifferentiated tissue precursor cells and stem cells Etc.).

  In a preferred embodiment, stem cells are mentioned. Stem cells are not particularly limited as long as they have the ability to differentiate into self-replicating cells and other types of cells (other than stem cells), and are pluripotent stem cells that can differentiate into all three germ layers, generally beyond germ layers It can be applied to both pluripotent stem cells that cannot be differentiated but can be differentiated into various cell tumors, and unipotent stem cells that are limited to one type of differentiateable cell tumor.

  The pluripotent stem cell is not particularly limited as long as it is an undifferentiated cell having `` self-renewal ability '' that can proliferate while maintaining an undifferentiated state and `` differentiated pluripotency '' that can differentiate into all three germ layers. ES cells, iPS cells, embryonic germ cells (EG) cells derived from primordial germ cells, multipotent germline stem (mGS) cells isolated during the establishment of GS cells from testicular tissue, multipotents isolated from bone marrow Examples include adult progenitor cells (MAPC), cultured fibroblasts, bone marrow stem cell-derived pluripotent cells (Muse cells), and the like. The ES cell may be a nuclear transplanted ES (ntES) cell produced by nuclear reprogramming from a somatic cell. ES cells or iPS cells are preferred.

  Examples of stem cells having multipotency include, but are not limited to, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, and the like. Examples of the unipotent stem cells include, but are not limited to, muscle stem cells, reproductive stem cells, and dental pulp stem cells.

When the cells cultured by the method of the present invention are differentiated cells, tissue progenitor cells, pluripotent stem cells, or unipotent stem cells, these cells can be obtained by any known method according to any method in which they exist. It can be isolated from mammalian tissue. The isolated cells can be applied as they are as primary cultured cells, or can be applied after maintenance culture by a method known per se. Moreover, various cell lines obtained by immortalizing these cultured cells can also be used.
On the other hand, when the cell is a pluripotent stem cell, the method of the present invention can be applied in any mammal in which any pluripotent stem cell is established or can be established, for example, human, Examples include mouse, monkey, pig, rat, dog and the like, preferably human or mouse, more preferably human. Although the preparation method of various pluripotent stem cells is demonstrated concretely below, the other well-known method can also be used without a restriction | limiting.

I. Preparation of pluripotent stem cells
ES cells can be established by removing an inner cell mass from a blastocyst of a fertilized egg of a subject animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining human and monkey ES cells, see, for example, US 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485.

As a culture solution for ES cell production, for example, DMEM / F-12 culture solution supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / mL bFGF ( Alternatively, human ES cells can be maintained in a humid atmosphere of 37 ° C, 2% CO ₂ /98% air using a synthetic medium (mTeSR, Stem Pro, etc.) (O. Fumitaka et al. (2008) Nat. Biotechnol., 26: 215-224). ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / mL collagenase in PBS containing 1 mM CaCl ₂ and 20% KSR. Can be performed using IV.

  In general, ES cells can be selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index. In particular, in the selection of human ES cells, expression of gene markers such as OCT-3 / 4, NANOG, ECAD and the like can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443 -452).

  Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute of Regenerative Medicine, Kyoto University (Kyoto, Japan) Is possible.

  A sperm stem cell is a testis-derived pluripotent stem cell, which is a cell that is the origin for spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is capable of self-replication in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and by repeating subculture under the same culture conditions as ES cells, Stem cells can be obtained (Massunori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), pages 41 to 46, Yodosha (Tokyo, Japan)).

  Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, and are primitive in the presence of substances such as LIF, bFGF, and stem cell factor. It can be established by culturing germ cells (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550-551).

  Artificial pluripotent stem (iPS) cells can be created by introducing specific reprogramming factors into somatic cells in the form of DNA or protein, such as almost the same characteristics as ES cells, such as differentiation pluripotency And an artificial stem cell derived from a somatic cell having the ability to proliferate by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131 : 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); WO 2007/069666). The reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or non-coding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be constituted by a low molecular compound. Examples of genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination. As combinations of reprogramming factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Ju dson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. 2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-174 , Han J, et al. (2010), Nature. 463: 1096-1100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-229 is exemplified.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (registered trademark) (Millipore), HuSH 29 mer shRNA Constructs against HDAC1 (OriGene), etc.], MEK inhibitors (eg, PD184352, PD98059, U0126, SL327 and PD0325901) , Glycogen synthase kinase-3 inhibitors (for example, Bio and CHIR99021), DNA methyltransferase inhibitors (for example, 5-azacytidine), histone methyltransferase inhibitors (for example, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2 , Nucleic acid expression inhibitors such as siRNA and shRNA against SetDBl and G9a), L-channel calcium agonist (eg Bayk8644), butyric acid, TGFβ inhibitor or ALK5 inhibitor (eg LY364947, SB43) 1542, 616453 and A-83-01), p53 inhibitors (eg siRNA and shRNA against p53), ARID3A inhibitors (eg siRNA and shRNA against ARID3A), miR-291-3p, miR-294, miR-295 and miRNA such as mir-302, Wnt Signaling (eg soluble Wnt3a), neuropeptide Y, prostaglandins (eg prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, Factors used for the purpose of improving establishment efficiency such as DMRTBl are also included, and in this specification, factors used for improving establishment efficiency are not distinguished from initialization factors. Shall.

  In the form of a protein, the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, TAT and polyarginine derived from HIV), or microinjection.

  On the other hand, in the case of DNA, it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, a polyadenylation site, etc., so that a nuclear reprogramming substance can be expressed. Selectable marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can be included. In addition, the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.

  In the case of RNA form, it may be introduced into somatic cells by techniques such as lipofection and microinjection, and RNA containing 5-methylcytidine and pseudoridine (TriLink Biotechnologies) is used to suppress degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630).

  As a culture solution for iPS cell induction, for example, DMEM, DMEM / F12 or DME culture solution containing 10-15% FBS (in addition to these culture solutions, LIF, penicillin / streptomycin, puromycin, L-glutamine) , Non-essential amino acids, β-mercaptoethanol, etc.) or a commercially available culture medium [eg, culture medium for mouse ES cell culture (TX-WES culture medium, Thrombo X), primate ES cells Culture medium for culture (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology) and the like are included.

As an example of the culture method, for example, in the presence of 5% CO ₂ at 37 ° C., the somatic cell and the reprogramming factor are contacted on DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact of the somatic cells with the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.

Alternatively, 10% FBS-containing DMEM culture medium (including LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) in the presence of 5% CO ₂ at 37 ° C. And can be appropriately cultured with puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) to produce ES-like colonies after about 25 to about 30 days or more . Desirably, instead of feeder cells, somatic cells to be initialized themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or an extracellular matrix (for example, Laminin ( WO2009 / 123349) and Matrigel (BD)) are exemplified.

In addition, a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106: 15720-15725). Furthermore, in order to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
During the culture, the culture medium is exchanged with a fresh culture medium once a day from the second day onward. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

  iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture solution containing the corresponding drug (selection The established iPS cells can be selected by culturing with the culture medium. In addition, if the marker gene is a fluorescent protein gene, iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.

  ES cells derived from cloned embryos obtained by nuclear transfer (nt ES cells) have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740). -743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell. For the production of nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52). Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.

  Multilineage-differentiating Stress Enduring cells (Muse cells) are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time. Preferably, it is a pluripotent cell obtained by trypsin treatment for 8 hours or 16 hours and then suspension culture, and is positive for SSEA-3 and CD105.

II. Biopolymer The biopolymer used in the culture substrate of the present invention is selected from the group consisting of gelatin, collagen and cellulose.
Gelatin is mainly produced from cow bone, cow skin, and pig skin, but it may be made from fish skin and scales such as salmon, and its origin is not particularly limited. Methods for extracting and purifying gelatin from these raw materials are well known. Commercially available gelatin can also be used.
Collagen can be purified from collagen raw material before modification with acid or alkali in the course of gelatin production. There is no particular limitation on the origin of collagen. Commercially available collagen such as those commercially available as a coating substrate for cell culture can also be used.
Cellulose can be extracted and purified from plants by a known method, and any cellulose may be used.
The molecular weight of the biopolymer is not particularly limited, but if the molecular weight is small, nanofibers may not be formed by electrospinning. For example, in the case of gelatin, 10 kDa or more, preferably 20-70 kDa, more preferably 30 It can be appropriately selected within the range of -40 kDa.

III. Production of nanofibers The method of producing nanofibers from these biopolymers is not particularly limited, and examples include electrospinning, conjugate melt spinning, and meltblowing. A spinning method is preferably used.
In the case of the electrospinning method, the biopolymer is first dissolved in an appropriate solvent. As the solvent used here, any solvent can be used regardless of whether it is an inorganic solvent or an organic solvent as long as it can dissolve gelatin, collagen, and cellulose. For example, in the production of gelatin nanofibers, acetic acid is used. And formic acid can be preferably used. In the production of collagen nanofibers, for example, 1,1,1,2,2,2-hexafluoro-2-propanol can be used. Alternatively, highly polar ionic liquids can be used in the production of cellulose nanofibers.
The concentration of the biopolymer solution is not particularly limited, but in order to obtain a preferable fiber diameter and uniformity, for example, when using an acetic acid solution of gelatin, 5-15 w / v%, preferably 8-12 w It is desirable to use in the concentration range of / v%.

  The electrospinning method can be carried out according to a method known per se. The principle of the electrospinning method is to spray the material with electric force to form nano-sized fibers. A biopolymer solution is filled in a syringe, and a syringe pump is connected to a tip provided with a nozzle such as an injection needle to give a flow rate. A collector that collects nanofibers at an appropriate distance from the nozzle (a flat plate or a take-up type can be used. A support described later is placed on a flat collector and the nanofiber is directly placed on the support. A fiber can also be formed to form the culture substrate of the present invention), and the positive electrode of the power source is connected to the nozzle side and the negative electrode is connected to the collector side. By turning on the power of the syringe pump and applying a voltage, the biopolymer is jetted onto the collector to form nanofibers. Here, the fiber form and the fiber diameter vary depending on the voltage, the distance from the nozzle to the collector, the inner diameter of the nozzle, etc., but those skilled in the art can appropriately select these to have a desired fiber diameter and be uniform. Nanofibers can be produced. For example, various conditions used in examples described later can be employed, and the conditions described in Non-Patent Documents 4 and 5 described above can be appropriately used.

  The nanofibers produced as described above may be those having a fiber diameter of 1-1000 nm, preferably 10-800 nm, more preferably 50-500 nm.

In order to impart suitable three-dimensional properties to the nanofiber and facilitate cell dissociation during passage, the produced nanofiber is preferably crosslinked using an appropriate crosslinking agent. The type of the crosslinking agent is not particularly limited, but preferred crosslinking agents include water-soluble carbodiimide (WSC), N-hydroxysuccinimide (NHS) and the like. Two or more kinds of crosslinking agents may be mixed and used. The crosslinking treatment can be performed, for example, by dissolving a crosslinking agent in an appropriate solvent and immersing the nanofibers obtained in the crosslinking agent solution. A person skilled in the art can appropriately set the solution concentration and the crosslinking treatment time according to the type of the crosslinking agent.
In addition, if a known peptide that imparts functionality to the cross-linking agent and the culture substrate is conjugated, the cross-linking treatment simultaneously imparts the functional peptide onto the nanofiber substrate. It is also useful in terms.

IV. Fabrication of fiber-on-fiber A fiber-on-fiber can be fabricated by applying the nanofibers produced as described above onto a support.
The method of coating is not limited as long as the nanofibers are uniformly coated on the support, but a method of generating nanofibers on the support by an electrospinning method that is simple and has wide applicability is preferably used.

  The support is preferably flexible and strong. The type of the support is not particularly limited, and preferred examples of the support include gauze and sponge. These materials are not particularly limited, but are preferably biocompatible polymers such as cotton, linen, collagen sponge, biological cellulose derivatives, silicone polymers, segmented polyurethanes, etc. It is not limited to.

V. Culture of cells using fiber-on-fiber substrate The culture substrate of the present invention (fiber-on-fiber) containing the nanofibers made of biopolymer on the support thus obtained. The fiber base material) is used for culturing various cells including stem cells such as pluripotent stem cells (for example, maintenance amplification culture, differentiation induction culture, dedifferentiation induction culture, etc.). Therefore, the present invention also provides a method for culturing the cells by seeding the cells, preferably stem cells, more preferably pluripotent stem cells on the culture substrate of the present invention, and culturing the cells stationary, preferably A maintenance amplification culture method is provided.
In the following, the present invention will be described more specifically by taking a method for maintaining and culturing pluripotent stem cells as an example, but when differentiation induction from pluripotent stem cells or other stem cells to various differentiated cells, tissue precursor cells or In the case where tissue stem cells or differentiated cells are dedifferentiated to a more undifferentiated state, or other stem cells, tissue precursor cells or differentiated cells are maintained and amplified, conventional methods are used respectively. By applying the culture substrate of the present invention in place of the existing culture substrate, it can be carried out easily.

First, pluripotent stem cells that have been established and adhered and cultured on a matrix such as feeder cells, Matrigel, collagen, etc. are dissociated by enzyme treatment, and preferably a ROCK inhibitor (for example, Y- 27632 and the like can be used in the same manner as described above as a culture medium for pluripotent stem cells in I. Preferably a serum-free medium, more preferably a pluripotent culture A stem cell is a medium that does not contain a protein derived from a different animal (Xeno-free), more preferably a medium that does not contain a protein such as serum albumin or bFGF is used, and is suspended in a culture vessel (eg, a dish). , Petri dishes, tissue culture dishes, multi dishes, micro plates, micro well plates, multi plates, multi wells Rate, chamber slides were placed dish, tube, tray, in culture back, etc.), on culture substrate of the present invention, about 0.5 × 10 ⁴ - about 10 × 10 ⁴ cells / cm ^2, preferably about Seed to a cell density of 2 × 10 ⁴ -about 6 × 10 ⁴ cells / cm ² . Prior to seeding of pluripotent stem cells, the culture substrate is preferably impregnated with a medium having the same composition as the above medium (no ROCK inhibitor is required) and pre-incubated under the same conditions as in the main culture. .

After seeding pluripotent stem cells, the medium is preferably removed from the culture vessel, replaced with a fresh medium (preferably containing a ROCK inhibitor), and cultured for 1 day. The culture is performed, for example, in a CO ₂ incubator under an atmosphere having a CO ₂ concentration of about 1 to about 10%, preferably about 2 to about 5% at about 30 to about 40 ° C., preferably about 37 ° C. It is desirable to replace the medium with no ROCK inhibitor the next day, and thereafter replace with a fresh medium every 1-2 days. The culture is performed for 1-7 days, preferably 3-6 days, more preferably 4-5 days.

The present invention also dissociates cells (eg, stem cells such as pluripotent stem cells) from a substrate using a dissociation solution that does not contain an enzyme, and reseeds the cells on the culture substrate of the present invention. Provided is a method for culturing the cell (for example, a maintenance amplification method) by further culturing the cell. Human pluripotent stem cells may be subcultured as a cell mass of a certain size because there is a problem that cell death tends to occur when they are made into single cells by the conventional subculture method. When a culture substrate is used, cells can be easily dissociated from the substrate using a dissociation solution that does not contain an enzyme, and can be dispersed to a single cell by a slight pipetting operation. If the above-mentioned cross-linked base material is used, the form of the base material is maintained, so that it becomes easier to separate the base material and the cells.
As the dissociation solution not containing an enzyme, a dissociation solution conventionally used in mechanically dissociating cells can be used in the same manner, and examples thereof include Hank's solution and a solution in which citric acid and EDTA are combined. It is done.

  A notable point of the present invention is that when human pluripotent stem cells are dispersed into single cells, the rate of cell death is significantly suppressed in single-cell pluripotent stem cells. It is done. This is because a more uniform cell population of human pluripotent stem cells can be prepared. Therefore, the present invention also suppresses cell death by dispersing pluripotent stem cells into single cells without performing enzyme treatment at the time of subculture using the culture substrate of the present invention. A method for maintaining and amplifying pluripotent stem cells is provided. In order to disperse the cells dissociated from the substrate into single cells, it is only necessary to gently pipette the cells about 10 times in a medium containing a ROCK inhibitor. According to this method, since the death of single cells is remarkably suppressed, it is sufficient to add a ROCK inhibitor to the medium for about 1 day. Since it is desirable to avoid contact with the ROCK inhibitor for a long period of time from the viewpoint of safety, the effect of the present invention is extremely significant.

Stem cells, especially human stem cells, are expected to be applied to transplantation medicine and the like. Therefore, in order to enable safe transplantation, it is necessary to avoid contamination of viruses and other contaminants harmful to the human body as much as possible. Therefore, particularly in the maintenance amplification culture of human stem cells, it is desired to use a serum-free medium, more preferably a xeno-free medium containing no xenogeneic component, and more preferably a protein-free medium. If subculture is continued using the culture substrate of the present invention, a growth efficiency comparable to that of a serum-containing medium or the like can be obtained in any of these media.
Here, examples of serum-free medium include mTeSR medium containing recombinant animal protein, and examples of xeno-free medium include TeSR2 medium containing human serum albumin and human bFGF as examples of protein-free medium. And E8 medium, respectively.

The pluripotent stem cells dissociated from the culture substrate of the present invention (preferably dispersed into single cells) are subcultured from the adherent culture using the feeder cells and the like according to the present invention. As with the transfer onto the culture substrate, the cell density is about 0.5 × 10 ^{4 to} about 10 × 10 ⁴ cells / cm ² , preferably about 2 × 10 ^{4 to} about 6 × 10 ⁴ cells / cm ^2. Sow on a new culture substrate. Similarly to the above, this culture substrate is also impregnated with a medium having the same composition as the main culture (ROCK inhibitor is not required) prior to seeding with pluripotent stem cells, and preincubated under the same conditions as in the main culture. It is desirable to keep it.

After re-seeding the pluripotent stem cells, the medium is preferably removed from the culture vessel, replaced with a fresh medium (preferably containing a ROCK inhibitor), and cultured for 1 day. The culture is performed, for example, in a CO ₂ incubator under an atmosphere having a CO ₂ concentration of about 1 to about 10%, preferably about 2 to about 5%, at about 30 to about 40 ° C., preferably about 37 ° C. It is desirable to replace the medium with no ROCK inhibitor the next day, and thereafter replace with a fresh medium every 1-2 days. The culture is performed for 1-7 days, preferably 3-6 days, more preferably 4-5 days.

  By repeatedly performing the above operation, pluripotent stem cells can be maintained and amplified with extremely good proliferation efficiency in a state where pluripotency and normal traits are maintained over a long period of time. In this way, it is possible to stably amplify high-quality pluripotent stem cells in large quantities, and supply a sufficient amount of pluripotent stem cells as a source of differentiated cells for cell transplantation therapy and drug screening Can do.

VI. Cryopreservation of cells using fiber-on-fiber substrate The cells cultured on the fiber-on-fiber substrate can be cryopreserved by inserting the substrate together with the substrate. The container only needs to be suitable for freezing, and is not limited in capacity, shape (tube, bag, ampoule, vial, etc.). A person skilled in the art can appropriately select a suitable container. Moreover, those skilled in the art can change the shape of the substrate after culturing with tweezers and insert it into the container.

  A cell freezing solution can be added to the cells for freezing as required by those skilled in the art. The solution may be any solution that can protect cells under freezing. For example, commercially available products such as mFreSR (Veritas), cryopreservation solution for primate ES cells (Reprocell), CRYO-GOLD Human ESC / iPSC Cryopreservation Medium (System Bioscience), Cell Banker 3 (Juji Field) You can also

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to these Examples.

Example 1 Preparation of gelatin nanofiber (1) Material gelatin solution / gelatin (SIGMA G2625 MW: 30 kDa; Nippi Nippi High Grade Gelatin AP MW: 8 kDa)
・ Glacial acetic acid (AA; SIGMA P-338826)
・ Anhydrous ethyl acetate (EA; SIGMA P270989)

Cross-linking buffer / water-soluble carbodiimide (WSC; DOJINDO Catalog 344-03633)
・ N-hydroxysuccinimide (NHS; SIGMA Catalog 56480)
・ 99.5% ethanol (Wako)

Gauze BEMCOT (registered trademark) S-2 (Asahi Kasei)
Culture cover glass 25mmφ and 32mmφ
Silicon wafer high-voltage power supply (TECHDEMPAZ Japan)
Vacuum pump

(2) Operation procedure
Preparation of 1 mL of 10% w / v gelatin solution (AA: EA = 3: 2)
A 2 mL tube was charged with 0.1 g of 30 kDa gelatin (final concentration 10% w / v) and 0.2 mL of sterile distilled water. Next, 0.42 mL of glacial acetic acid (final concentration 42% w / v) and 0.31 mL of anhydrous ethyl acetate (final concentration 28% w / v) were added in a fume hood, and the tube was vortexed and stirred well. When the gelatin was sufficiently dissolved, the tube was set in a rotor and mixed by inversion overnight (room temperature: 20 ° C or higher).

Application of gelatin nanofibers to the support by electrospinning method The gelatin solutions of various concentrations prepared as described above are put into a syringe equipped with a 23G blunt needle (Nipro), air bubbles are removed, and then the microsyringe pump is used. The flow rate was set at 0.2 mL / h. Two culture cover glasses were placed side by side in the center of the silicon wafer, and part of both ends of the glass was fixed with cello tape. The silicon wafer was fixed vertically in a vise and placed at a distance of about 10 cm from the needle of the syringe set in the micro syringe pump. Attach the + electrode (red line) to the brandt needle, the-electrode (green line) to the silicon wafer, turn on the microsyringe pump, apply a voltage of 11 kV, and spout the fiber onto the gauze or glass on the silicon wafer. It was. The voltage was stopped, the silicon wafer was rotated 180 degrees, and the fiber was ejected again for the same time. After fiber ejection, the gauze (fiber on fiber) or glass (control nanofiber) on the wafer was gently removed and placed in a petri dish. This petri dish was placed in a desiccator and dried all day and night while applying a vacuum pump.

Preparation of 0.2 M WSC / NHS cross-linking buffer (40 mL)
A 50 mL Falcon tube was charged with 1.52 g of WSC and 0.92 g of NHS. 30 mL of 99.5% ethanol was added to the tube and vortexed to dissolve the reagent, and then quantified with 99.5% ethanol to 40 mL and vortexed again.

Gelatin nanofibers (fiber-on-fiber or control nanofiber) dried with a cross-linking desiccator were immersed in a cross-linking buffer in an amount sufficient to immerse the surface for 4 hours. The nanofibers were taken out and washed by immersing them in 99.5% ethanol for 5 to 10 minutes (this operation was repeated twice). Next, the nanofibers were air-dried on a petri dish laid with Kimwipe, and then placed in a desiccator and allowed to dry overnight.

Example 2 Method for Passing Human Pluripotent Stem Cells onto Fiber-on-Fiber (1) Material
mTeSR 1 STEM CELL Veritas ST-05850
Y-27632 Wako 257-00511 (1 mg) 255-200513 (5 mg)
Cell Dissociation Buffer enzyme-free, Hanks'-based GIBCO 13150-016
TrypLE Express GIBCO 12605-010
Human embryonic stem cells: H9, H1
Human induced pluripotent stem cells: 253G1

(2) Operation procedure
Pre-treatment of nanofibers Fiber-on-fiber prepared by spraying gelatin nanofibers with a diameter of 300nm ± 100nm onto cotton gauze (BEMCOT (registered trademark) S-2) was set in a 35 mm dish (6-well plate). Then, it was washed 3 times with 1 mL of 99.5% ethanol and sterilized. The third time, it was carefully aspirated and dried in a clean bench. Fiber on fiber was immersed in the medium and incubated at 37 ° C. 2 mL of mTeSR 1 was placed in a 35-mm dish.

Migration of human pluripotent stem cells from MEF feeder onto nanofiber
Add 2 mL of enzyme dissociation solution TrypLE Express to a human pluripotent stem cell colony (60 mm dish) on the MEF feeder, incubate as it is, shake the dish about 2 minutes later, and the MEF should have peeled off under the microscope After confirming that the colonies were rounded, the enzyme dissociation solution was removed by aspiration (rinsed with 1-2 mL of mTeSR as necessary). Cells were collected with 4 mL of mTeSR 1 (mTeSR 1 (+ Y27632)) containing 10 μM Y-27632 and pipetted about 10 times to make a single cell. After counting the number of cells, the mixture was centrifuged at 1000 rpm for 3 minutes, the supernatant was removed by aspiration, and resuspended to the required cell concentration with mTeSR 1 (+ Y27632). The medium on the fiber-on-fiber that had been pretreated was removed by suction, and 1 to 1.5 mL (cell density: 2 × 10 ^{5 to} 3 × 10 ⁵ cells / sample) was seeded on the fiber-on-fiber. On the next day, the medium was replaced with 2 mL of mTeSR 1 (+ Y27632). From the second day, the medium was cultured with mTeSR 1 not containing Y-27632, and the medium was changed every day.

Passage from nanofiber to nanofiber
After rinsing the cells twice with PBS, add 1 mL of Cell Dissociation Buffer, enzyme-free cell dissociation buffer, and incubate for 5 minutes at 37 ° C. Then, remove the dissociated solution by suction (add 1 mL when using TrypLE Express) Then, after removing it immediately, it was incubated for about 2 minutes). Cells were collected with 2 mL of mTeSR1 (+ Y27632) (1 mL x 2) and pipetted about 10 times to make a single cell. Subsequent operations were performed in the same manner as the transition from the MEF feeder.

Use with multiple fiber-on-fiber folded
Three-dimensional culture was performed by folding four fibers on fiber in a 35-mm dish. It became possible to cultivate a larger number of cells with the amount of the culture solution used in planar culture (3 mL medium in a 35-mm dish).

(3) Results
Structure of Fiber on Fiber FIG. 1 shows a scanning electron micrograph of the fiber on fiber obtained by the above-described method. It was found that gelatin nanofibers were reticulated between cotton gauze fibers. In the subsequent experiments, the fiber-on-fiber was used.

Culture of human pluripotent stem cells on fiber-on-fiber Fiber-on-fiber was immersed in a culture solution, and human pluripotent stem cells (H9 human ES cells) were cultured thereon (FIG. 2). The results are shown in FIG. It was confirmed that human pluripotent stem cells form colonies. The results of staining the cells with alkaline phosphatase, a pluripotent stem cell marker, are shown in FIG. A colony stained in red was observed, and it was confirmed that human pluripotent stem cells strongly expressed alkaline phosphatase even after culture. Moreover, the stained cells were uniformly dispersed on the fibers (FIG. 5). In addition, the shape of the fiber-on-fiber can be freely changed by picking up the fiber-on-fiber with tweezers etc. at any timing during culture, after culture, and after staining. It was possible. That is, it was shown that fiber-on-fiber can culture human pluripotent stem cells while maintaining a flexible shape.

  In contrast, in a control test in which human ES cells (H9) were cultured on cotton gauze and then stained with alkaline phosphatase, several colonies were confirmed, but the number of colonies as shown in FIG. 4 was confirmed. Could not be done (FIG. 6). From this result, it was shown that cell adhesion and cell proliferation were promoted by the presence of gelatin nanofibers.

Quantitative expression analysis of pluripotent stem cell marker by flow cytometry Expression of pluripotent stem cell marker (SSEA4) on human ES cells (H9) cultured on fiber-on-fiber Analysis was performed using a meter (FIG. 7). It was confirmed that 98.7% of the cells strongly expressed SSEA4. It was also confirmed that the cell group was uniform.

Confirmation of pluripotent stem cell marker expression by immune cell staining method After culturing human ES cells (H9) on fiber-on-fiber, immuno-staining of undifferentiated marker (TRA-1-60) Expression was examined. For comparison, expression of the marker in human ES cells (H9) passaged once on Matrigel was also examined. The results are shown in FIG. It was confirmed that the undifferentiation marker was strongly expressed in human ES cells (H9).

Using multiple fiber-on-fiber folded
Three-dimensional culture was performed by folding one or four fiber-on-fibers in a 35-mm dish (FIG. 9), and the number of cells was measured. The results are shown in FIG. As a control, nanofibers coated on a flat substrate were used. It was possible to cultivate a larger number of cells with the amount of culture solution used in planar culture (3 mL medium in a 35-mm dish).
Furthermore, as shown in Fig. 11, 32 fiber-on-fibers are folded, and human pluripotent stem cells are cultured in large quantities (100 mL). It became possible to do.

Long-term culture of human pluripotent stem cells on fiber-on-fiber Human ES cells (H9) were subcultured 10 times on fiber-on-fiber, and the result of alkaline phosphatase staining is shown in FIG. It was confirmed that the undifferentiated marker was strongly expressed even after long-term culture.

Quantitative expression analysis of pluripotent stem cell markers by flow cytometry Expression of pluripotent stem cell marker (SSEA4) in cells after 10 passages of human ES cells (H9) on fiber-on-fiber Analysis was performed using flow cytometry. For comparison, expression of the marker in human ES cells (H9) passaged once on Matrigel was also examined. The results are shown in FIG. It was confirmed that SSEA4 was strongly expressed in the majority of human ES cells (H9) cultured on fiber-on-fiber. It was also confirmed that the cell population was more uniform than that cultured on Matrigel.
Furthermore, pluripotent stem cell marker (SSEA4) and differentiation marker (SSEA1) in cells after subculturing human ES cells (H1, H9) and human iPS cells (253G1) more than 20 times on fiber-on-fiber The expression of was analyzed using flow cytometry. The results are shown in FIG. It was confirmed that 98.4% (H1), 98.6% (H9) and 96.3% (253G1) cells strongly expressed SSEA4. On the other hand, in any pluripotent stem cell, almost no cells expressing SSEA1 were confirmed.

Karyotype analysis of human pluripotent stem cells cultured for a long time on fiber-on-fiber About human ES cells (H9) and human iPS cells (253G1) subcultured 10 times or more on fiber-on-fiber The karyotype was analyzed by multicolor FISH. The results are shown in FIG. It was confirmed that both cells maintained normal karyotype even after passage and culture 10 times or more on fiber-on-fiber (FIG. 15).

Confirmation of differentiation potential of human pluripotent stem cells cultured on fiber-on-fiber for a long time (passage 10 times or more) Human pluripotency cultured on fiber-on-fiber by embryoid body formation The differentiation potential of sex stem cells was confirmed. FIG. 16 shows the results of staining with fluorescently labeled antibodies (anti-tubulin III antibody, anti-α-SMA antibody, and anti-SOX17 antibody, respectively) for detection markers of ectoderm, mesoderm, and endoderm. It has been confirmed that human pluripotent stem cells can be differentiated into ectoderm, mesoderm and endoderm even after being passaged and cultured more than 10 times on fiber-on-fiber (human ES cells). (H9, FIG. 16A), human ES cells (H1, FIG. 16B)).

Confirmation of teratoma-forming ability of human pluripotent stem cells cultured on fiber-on-fiber for a long time (passage 10 times or more) Human ES cells passaged and cultured 10 times or more on fiber-on-fiber (H1, H9) or human iPS cells (253G1) 1 × 10 ⁶ cells suspended in Materigel / DMEM-F12 medium, immunodeficient mice (CB-17 / Icr-scid / scid Jcl, female, 6-8 weeks old, 3 mice) Was injected between the subcutaneous and peritoneum. After 4 to 8 weeks, the tumor was excised, fixed and embedded in paraffin, and the section specimen was stained with hematoxylin-eosin. The results are shown in FIG. It was confirmed that any human pluripotent stem cell can be differentiated into all germ layers.

Example 3 Cryopreservation of human pluripotent stem cells cultured on fiber-on-fiber Human pluripotency cultured on frozen-thawed fiber-on-fiber of human pluripotent stem cells cultured on fiber-on-fiber Sexual stem cells were inserted into a tube in a different shape using tweezers and frozen (FIG. 18). The morphology of the cells before freezing and 4 days after thawing is shown in FIG. It was confirmed that the cells formed colonies even after freezing and thawing.

Quantitative expression analysis of pluripotent stem cell markers after freezing and thawing by flow cytometry Human ES cells (H1) cultured on fiber-on-fiber in a commercially available cell cryopreservation solution (Cell Banker 3, mFreSR) Then, expression of pluripotent stem cell markers (SSEA4, TRA-1-60) and differentiation marker (SSEA1) in cells thawed after freezing for 2 days was analyzed using flow cytometry. The results are shown in FIG. SSEA4 and TRA-1-60 were strongly expressed in the majority of human ES cells (H1) cultured on fiber-on-fiber, whereas cells expressing SSEA1 were rarely confirmed. .

Confirmation of pluripotent stem cell marker expression after freezing and thawing by immune cell staining method Human ES cells (H1) cultured on fiber-on-fiber were frozen for 3 days, and the thawed cells were stained with alkaline phosphatase. The results are shown in FIG. It was confirmed that the undifferentiated marker was strongly expressed even after freezing and thawing.

Number of cells after freezing and thawing of human pluripotent stem cells cultured on fiber on fiber Human ES cells (H1) cultured on fiber on fiber in cell cryopreservation solution (Cell Banker 3, mFreSR) The cells were frozen for 2 days or more and thawed, and the number of cells was measured. The results are shown in FIG. Using any cell cryopreservation solution, high survival rates of about 75% (Cell Banker 3) and about 85% (mFreSR) were shown.

Example 4 Mass culture of human ES cells using fiber-on-fiber
Human ES cells (H1) 1.2 × 10 ⁶ cells cultured on a fiber-on-fiber were enclosed in a gas-permeable cell culture bag (Nipro; FIG. 23A) containing 55 mL of mTeSR-1 culture solution. The cells were cultured for 7 days at 37 ° C. in% CO ₂ . The medium was changed twice during the culture period. When the number of cells was measured after completion of the culture, it was 9.55 × 10 ⁷ cells / 55 mL (= 3.47 × 10 ⁹ cells / L), and the number of cells increased 79.6 times in 7 days. The expression of pluripotent stem cell marker (SSEA4) and differentiation marker (SSEA1) in cells after mass culture was analyzed using flow cytometry. The results are shown in FIG. 23B. It was confirmed that 99.7% of the cells strongly expressed SSEA4. On the other hand, almost no cells expressing SSEA1 were confirmed.

  As the efficiency rate at which cells can be continuously cultured and proliferated, the production rate of the present invention reaches 10 times every 5 days. This efficiency rate is much superior to that of about 5 times that in the previously reported dispersed culture of human pluripotent stem cells. Compared with conventional manual adhesion culture methods (about 4 times every 4 days, or about 3 times every 3 days) at the laboratory level, the growth rate is also excellent. The fiber-on-fiber method developed this time is a method that can increase the number of cells per unit volume in the medium by culturing in 3D cells. Practical mass culture of human pluripotent stem cells The road to Furthermore, since a polymer having high biocompatibility is used as the support, application to cell transplantation therapy can be pioneered.

  While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. For example, three-dimensional culture is possible, and it is possible to supply a large amount of cells while realizing space saving, and the culture solution penetrates when the culture solution is immersed, so that the supply of the culture solution to the cells becomes easy The features of the culture substrate of the present invention, such as that described above, bring about advantageous effects not only in the maintenance amplification culture of stem cells including pluripotent stem cells but also in any culture of arbitrary cells. Thus, it is intended that the present invention be implemented in ways other than those described in detail herein. Accordingly, the present invention includes all modifications encompassed within the spirit and scope of the appended claims.

  The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

  This application is based on Japanese Patent Application No. 2013-117242 filed in Japan on June 3, 2013, the contents of which are hereby incorporated by reference.

Claims (24)

  A cell culture substrate comprising a nanofiber made of a biopolymer selected from the group consisting of gelatin, collagen and cellulose on a support.   The substrate according to claim 1, wherein the nanofibers are crosslinked.   The substrate according to claim 1 or 2, wherein the biopolymer is gelatin or collagen.   The substrate according to claim 1 or 2, wherein the biopolymer is gelatin.   The substrate according to any one of claims 1 to 4, wherein the nanofiber is obtained by an electrospinning method.   The substrate according to any one of claims 1 to 5, wherein the support is selected from the group consisting of gauze and sponge.   The substrate according to any one of claims 1 to 6, wherein the cell is a stem cell.   The substrate according to claim 7, wherein the stem cells are pluripotent stem cells.   The base material according to claim 8, wherein the pluripotent stem cell is an ES cell or an iPS cell.   The substrate according to claim 8 or 9, wherein the pluripotent stem cell is derived from a human.   The substrate according to any one of claims 1 to 10, wherein the culture is a maintenance amplification culture of cells.   The cell freezing agent containing the base material of any one of Claims 1-6.   A cell culturing method, comprising seeding cells on the substrate according to any one of claims 1 to 6, and culturing the cells stationary.   Dissociating the cells from the substrate using a dissociation solution that does not contain the enzyme, seeding the cells on the substrate according to any one of claims 1 to 6, and further culturing the cells statically 14. A method according to claim 13, characterized by.   15. A method according to claim 14, characterized in that the cells are dispersed into single cells at the time of passage.   The method according to any one of claims 13 to 15, wherein the cells are cultured in a serum-free medium.   The method according to claim 16, wherein the serum-free medium is a xeno-free medium.   The method according to claim 16, wherein the serum-free medium is a protein-free medium.   The method according to any one of claims 13 to 18, wherein the cell is a stem cell.   The method according to claim 19, wherein the stem cell is a pluripotent stem cell.   The method according to claim 20, wherein the pluripotent stem cell is an ES cell or an iPS cell.   The method according to claim 20 or 21, wherein the pluripotent stem cell is derived from a human.   The method according to any one of claims 13 to 22, wherein the culture is a maintenance amplification culture of cells.   A method for cryopreserving cells using the freezing agent according to claim 12.