WO2014196549A1 - Procédé de culture tridimensionnelle de cellules au moyen de fibre sur fibre et substrat pour ce faire - Google Patents

Procédé de culture tridimensionnelle de cellules au moyen de fibre sur fibre et substrat pour ce faire Download PDF

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WO2014196549A1
WO2014196549A1 PCT/JP2014/064789 JP2014064789W WO2014196549A1 WO 2014196549 A1 WO2014196549 A1 WO 2014196549A1 JP 2014064789 W JP2014064789 W JP 2014064789W WO 2014196549 A1 WO2014196549 A1 WO 2014196549A1
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cells
fiber
cell
culture
pluripotent stem
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謙一郎 亀井
憲夫 中辻
劉　莉
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Kyoto University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • 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.
  • multipotency multipotency
  • feeder cells and various polymers have been used as cell culture substrates.
  • these methods are complicated in preparation and quality is not stable. Therefore, stable culture and supply of human pluripotent stem cells has been difficult.
  • 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.
  • Non-Patent Documents 1 and 2 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.
  • Non-patent Document 3 Non-patent Document 3
  • Non-Patent Documents 4 and 5 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.
  • 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.
  • Non-patent Document 7 it has been reported that human ES cells cannot be maintained and grown in a culture system that does not use feeder cells. On the other hand, there has been no report of using nanofibers composed only of biopolymers as a culture substrate for pluripotent stem cells.
  • 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. .
  • 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.
  • 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.
  • a cell culture substrate comprising a nanofiber made of a biopolymer selected from the group consisting of gelatin, collagen and cellulose on a support.
  • a nanofiber made of a biopolymer selected from the group consisting of gelatin, collagen and cellulose on a support.
  • the biopolymer is gelatin or collagen.
  • the cell is a stem cell.
  • the stem cells are pluripotent stem cells.
  • the pluripotent stem cells are ES cells or iPS cells.
  • the pluripotent stem cells are derived from human.
  • the culture is a maintenance amplification culture of cells.
  • a cell freezing agent comprising the substrate according to any one of [1] to [6] above.
  • a method for culturing cells comprising seeding cells on the substrate according to any one of [1] to [6], and culturing the cells stationary.
  • the culture substrate of the present invention 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.
  • SSEA4 pluripotent stem cell marker
  • SSEA1 differentiation marker
  • FIG. 1 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. 1 Photomicrographs of immunostaining with anti- ⁇ -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.
  • 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.).
  • 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
  • stem cells may be 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.
  • 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.
  • 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.
  • unipotent stem cells include, but are not limited to, muscle stem cells, reproductive stem cells, and dental pulp stem cells.
  • 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.
  • 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.
  • the preparation method of various pluripotent stem cells is demonstrated concretely below, the other well-known method can also be used without a restriction
  • 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.
  • LIF leukemia inhibitory factor
  • bFGF basic fibroblast growth factor
  • 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.
  • ES cells can be generally selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index.
  • gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog
  • OCT-3 / 4, NANOG, and ECAD 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.
  • sperm stem cells are testis-derived pluripotent stem cells that are the origin of sperm formation. Like ES cells, these cells can be induced to differentiate into various types of cells.For example, when transplanted into a mouse blastocyst, a chimeric mouse can be produced (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012).
  • Spermatozoa can replicate in culture medium containing glial cell line-derived neurotrophic factor (GDNF) and repeat passages under the same culture conditions as ES cells. Stem cells can be obtained (Masatake Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 41-46, Yodosha (Tokyo, Japan)).
  • GDNF glial cell line-derived neurotrophic factor
  • Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, and they 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).
  • 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 proliferation ability 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.
  • 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.
  • 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
  • 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, HIV-derived TAT and polyarginine), or microinjection.
  • a cell membrane-permeable peptide for example, HIV-derived TAT and polyarginine
  • Virus vectors include retrovirus vectors, lentivirus vectors (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.
  • artificial chromosome vectors examples include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
  • HAC human artificial chromosomes
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosomes
  • 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), ⁇ -glucuronidase (GUS), FLAG, etc.
  • GFP green fluorescent protein
  • GUS ⁇ -glucuronidase
  • FLAG FLAG
  • 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.
  • RNA it may be introduced into somatic cells by techniques such as lipofection and microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine® (TriLink® Biotechnologies) is used. Yes (Warren L, (2010) Cell Stem Cell. 7: 618-630).
  • a culture solution for inducing iPS cells 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, ⁇ -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) are included.
  • a 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) are included.
  • the somatic cell is brought into contact with the reprogramming factor 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.
  • feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
  • 10% FBS-containing DMEM culture medium including LIF, penicillin / streptomycin, etc.
  • feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
  • 5% CO 2 at 37 ° C. can be suitably included with puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
  • 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.
  • 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).
  • hypoxic conditions oxygen concentration of 0.1% or more and 15% or less
  • 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.
  • 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
  • a culture solution containing the corresponding drug selection The established iPS cells can be selected by culturing with the culture medium.
  • 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 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).
  • an ES cell established from an inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with a nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell.
  • nt ES nuclear transfer ES
  • 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), pp. 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 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.
  • 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.
  • 10 kDa or more preferably 20-70 kDa, more preferably 30 It can be appropriately selected within the range of -40 kDa.
  • 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.
  • the biopolymer is first dissolved in an appropriate solvent.
  • 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.
  • acetic acid is used in the production of gelatin nanofibers.
  • formic acid can be preferably used.
  • 1,1,1,2,2,2-hexafluoro-2-propanol can be used.
  • highly polar ionic liquids can be used in the production of cellulose nanofibers.
  • 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 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.
  • Nanofibers By turning on the power of the syringe pump and applying a voltage, the biopolymer is jetted onto the collector to form nanofibers.
  • 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.
  • 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.
  • the cross-linking treatment simultaneously imparts the functional peptide onto the nanofiber substrate. It is also useful in terms.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the culture substrate 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. .
  • the medium is preferably removed from the culture vessel, replaced with a fresh medium (preferably containing a ROCK inhibitor), and cultured for 1 day.
  • a fresh medium preferably containing a ROCK inhibitor
  • 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.
  • a method for culturing the cell for example, a maintenance amplification method
  • 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.
  • a culture substrate 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.
  • the form of the base material is maintained, so that it becomes easier to separate the base material and the cells.
  • 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.
  • 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.
  • examples of serum-free medium include mTeSR medium containing recombinant animal protein
  • examples of xeno-free medium include TeSR2 medium containing human serum albumin and human bFGF as examples of protein-free medium.
  • E8 medium respectively.
  • the pluripotent stem cells dissociated from the culture substrate of the present invention are subcultured from the adherent culture using the feeder cells and the like according to the present invention.
  • 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.
  • 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.
  • the medium is preferably removed from the culture vessel, replaced with a fresh medium (preferably containing a ROCK inhibitor), and cultured for 1 day.
  • a fresh medium preferably containing a ROCK inhibitor
  • 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.
  • pluripotent stem cells 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.
  • 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 as needed by those skilled in the art.
  • the solution may be any solution that can protect cells under freezing.
  • commercial 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), etc. You can also
  • 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 Catalog56480) ⁇ 99.5% ethanol (Wako) Gauze BEMCOT (registered trademark) S-2 (Asahi Kasei) Culture cover glass 25mm ⁇ and 32mm ⁇ Silicon wafer high-voltage power supply (TECHDEMPAZ Japan) Vacuum pump
  • 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.
  • 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).
  • 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
  • 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
  • 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.
  • BEMCOT registered trademark
  • 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 was 2 ⁇ 10 5 to 3 ⁇ 10 5 cells / sample) was seeded on the fiber-on-fiber.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 16 shows the results of staining with fluorescently labeled antibodies (anti-tubulin III antibody, anti- ⁇ -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)).
  • Example 3 Cryopreservation 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.
  • Example 4 Mass culture of human ES cells using fiber-on-fiber
  • a gas-permeable cell culture bag manufactured by Nipro; FIG. 23A
  • mTeSR-1 culture solution 55 mL of mTeSR-1 culture solution
  • H1 mTeSR-1 culture solution
  • 1.2 ⁇ 10 6 cells were encapsulated and cultured at 5% CO 2 and 37 ° C. for 7 days.
  • the medium was changed twice during the culture period.
  • SSEA4 pluripotent stem cell marker
  • SSEA1 differentiation marker
  • 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.

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Abstract

La présente invention concerne, par exemple : un substrat pour culture de cellules, qui est un support sur lequel sont contenues des nanofibres formées à partir d'un polymère biologique choisi dans l'ensemble consistant en gélatine, collagène et cellulose ; un cryogène pour cellules contenant le substrat ; un procédé de culture de cellules comprenant l'inoculation des cellules sur le substrat et la culture statique des cellules ; le même procédé comprenant la dissociation des cellules et du substrat au moyen d'une solution de dissociation sans enzyme, l'inoculation des cellules sur un autre substrat de culture et ensuite la culture statique des cellules ; en particulier, un procédé comprenant la séparation des cellules issues de la sous-culture en cellules individuelles ; et un procédé de cryoconservation de cellules souches pluripotentes au moyen du cryogène pour cellules.
PCT/JP2014/064789 2013-06-03 2014-06-03 Procédé de culture tridimensionnelle de cellules au moyen de fibre sur fibre et substrat pour ce faire Ceased WO2014196549A1 (fr)

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WO2016068266A1 (fr) * 2014-10-31 2016-05-06 国立大学法人京都大学 Procédé de culture en trois dimensions utilisant un polymère biodégradable et un substrat de culture permettant la transplantation de cellules
CN107149704A (zh) * 2017-04-21 2017-09-12 芜湖扬展新材料科技服务有限公司 一种胎盘干细胞复合再生纤维素组织修复材料的制备方法
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WO2018199194A1 (fr) 2017-04-25 2018-11-01 北海道公立大学法人札幌医科大学 Procédé de production de cellules souches mésenchymateuses, marqueur d'effets thérapeutiques des cellules souches mésenchymateuses, méthode de détermination des effets thérapeutiques des cellules souches mésenchymateuses, et préparation cellulaire contenant des cellules souches mésenchymateuses
WO2018235745A1 (fr) * 2017-06-20 2018-12-27 日本毛織株式会社 Tissu non tissé à fibres longues biocompatible, son procédé de production, échafaudage tridimensionnel pour culture cellulaire, et procédé de culture cellulaire l'utilisant
JP2019172720A (ja) * 2018-03-27 2019-10-10 大日精化工業株式会社 水不溶性成形体の製造方法及び水不溶性成形体
WO2019208688A1 (fr) 2018-04-25 2019-10-31 北海道公立大学法人札幌医科大学 Tapis cellulaire pour transplantation vitale et son procédé de production
WO2019245005A1 (fr) * 2018-06-20 2019-12-26 株式会社 東芝 Dispositif de test, méthode de production dudit dispositif de test, méthode de détection de cellules utilisant ledit dispositif de test, chambre pour ledit dispositif de test, méthode de production de chambre pour ledit dispositif de test, et méthode de test
JPWO2020262458A1 (fr) * 2019-06-28 2020-12-30
WO2021100718A1 (fr) 2019-11-21 2021-05-27 日本毛織株式会社 Agrégat cellulaire, procédé de production d'agrégat cellulaire, kit de production pour agrégat cellulaire et procédé d'évaluation de composé chimique utilisant un agrégat cellulaire
JP2023175783A (ja) * 2017-03-30 2023-12-12 日産化学株式会社 ナノファイバーを用いた細胞培養
WO2024257852A1 (fr) 2023-06-15 2024-12-19 Ｕｂｅ株式会社 Procédé de cryoconservation de cellules, procédé de culture de cellules et substrat de cryoconservation
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JPWO2019245005A1 (ja) * 2018-06-20 2021-05-13 株式会社東芝 検査デバイス、この検査デバイスの製造方法、この検査デバイスを用いた細胞検出方法、この検査デバイス用セル、この検査デバイス用セルの製造方法、及び検査方法
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