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SUBSTRATE FOR STEM CELL CULTURE, AND CULTURE METHOD USING SAME

Foreign code F130007118
File No. S2011-0831-N0
Posted date Jan 17, 2013
Country WIPO
International application number 2012JP066491
International publication number WO 2013002311
Date of international filing Jun 28, 2012
Date of international publication Jan 3, 2013
Priority data
  • P2011-142403 (Jun 28, 2011) JP
Title SUBSTRATE FOR STEM CELL CULTURE, AND CULTURE METHOD USING SAME
Abstract Provided are a substrate for stem cell cultures having no risk of pathogen infection or undesirable side effects, and a method for using the same. The present invention comprises a substrate for stem cell cultures in which a cell adhesion peptide having a cyclic skeleton structure is bonded to a polymer substrate, and a stem cell culture method using the substrate for stem cell cultures. A substance containing the Arg-Gly-Asp sequence found in fibronectin can be used as the cell adhesion peptide with a cyclic skeleton structure. A polypeptide and/or a polysaccharide can be used for the polymer substrate.
Outline of related art and contending technology BACKGROUND ART
Embryonic stem cells (ES cells) or induced pluripotent stem cells such as stem cells (iPS cells), tissue and organ reconstruction and inside and outside of the living body, instead of the lost tissue and organ transplantation in order to realize the essential for regenerative medicine. The current, while maintaining the pluripotent stem cells for expansion, an animal or human-derived fibroblasts should be used as feeder cells, animal-derived materials such as gelatin and matrigel coated culture vessel is used. These techniques are, or an animal an animal or human fibroblasts from prion-derived material or unknown viruses and other pathogens is at risk of contamination, and prevents achievement of regenerative medicine is one of the factors.
The inventors of the invention, an animal or human-derived material and an alternative to a high level of safety in the creation of a biocompatible material with the aim of earnest studies, which have such properties are provided in a full chemical synthesis of polypeptide synthesis techniques are completed. For example, in patent document 1, pathogen infection or a risk of transmission of the pathogenic agent and which may produce undesirable side effects, such as various physiologically active substances or apatite as a novel polypeptide useful as the carrier of the, - Pro-X-Gly - (X is, represents Pro or Hyp) having the amino acid sequence represented by and cleaved, is -Pro-Hyp(O-Y-Z) -Gly-(Y, carbonyl group, does not have a carbonyl group or a saturated or unsaturated hydrocarbon group, or an aromatic group, a carbonyl group or saturated or unsaturated does not represent a hydrocarbon group, Z represents a carboxyl group) having the amino acid sequence represented by cleaved and a polypeptide comprising the disclosed.
On the other hand, the non-patent document 2, found in fibronectin cell adhesion sequence Gly-Arg-Gly-Asp-Ser and poly Pro-His-Ser-Arg-Asn(Pro-Hyp-Gly) polypeptide materials attached to a mouse fibroblast cell line is the cell adhesion of NIH3T3 and facilitating movement of, and rabbit corneal epithelial cells to promote layered strains have been reported.
Patent Document 2 is, in the culture environment , while retaining differentiation pluripotency in order to maintain a human pluripotent stem cell, a fragment of the coated human pluripotent stem cell culture base material using this culture method has been proposed.
Patent Document 3 is, from the embryonic stem cells to induce the differentiation of hepatocytes efficiently to provide a method and a material thereof for the purpose, as a main component of the crosslinking polysaccharide sponge form of embryonic stem cells culture base material has been proposed.
Is Patent Document 4, feeder cells and feeder cell-derived component in the absence, quantities and safely maintain undifferentiated embryonic stem cells in the culture substrate and cultured for the purpose to provide a method, a porous body made of a nonwoven fabric or the like has been proposed from the culture substrate.
Patent Document 5 is, stem cells, while maintaining its differentiation efficiently to provide a culture substrate for growing the purpose, a substance with a cell adhesion activity characterized in that the stem cell culture base material has been proposed.
Is the non-patent document 3, a cell adhesion sequence Arg-Gly-Asp, the surface of the polymer containing polylactic acid using the same method of combining a method for constructing the bone tissue is reported.
Scope of claims (In Japanese)請求の範囲 [請求項1]
環状骨格構造を有する細胞接着性ペプチドが高分子基材に結合されている幹細胞培養用基材。

[請求項2]
前記環状骨格構造を有する細胞接着性ペプチドがArg-Gly-Asp配列を含む請求項1記載の幹細胞培養用基材。

[請求項3]
前記環状骨格構造を有する細胞接着性ペプチドがcyclo(Arg-Gly-Asp-(D)Phe-Lys)である請求項1記載の幹細胞培養用基材。

[請求項4]
前記高分子基材がポリペプチド及び/又は多糖類である請求項1記載の幹細胞培養用基材。

[請求項5]
前記高分子基材が下記式(1)で表されるアミノ酸配列を有するペプチドユニット(1)と、下記式(2)で表されるアミノ酸配列を有するペプチドユニット(2)とを含むポリペプチドである請求項1記載の幹細胞培養用基材。
       -Pro-X-Gly-             (1)
       -Pro-Hyp(O-Y-Z)-Gly-         (2)
(式中、XはPro又はHypを示し、Yはカルボニル基、又はカルボニル基を有するか若しくは有しない飽和又は不飽和の炭化水素基を示し、Zはカルボキシル基を示す)

[請求項6]
Yが、-(C=O)-(CH 2)n- (式中nは0又は1~18の整数を示す);-(C=O)-(CH 2)n-(CH=CH)m-(CH 2)k- (式中n及びkは独立に0又は1~18の整数を示し、mは1~18の整数を示す);及び-(C=O)-(CH 2)n-(C 6H 4)-(CH 2)k- (式中n及びkは独立に0又は1~18の整数を示し、C 6H 4はフェニレン基を示す)からなる群より選択される1種以上である請求項5記載の幹細胞培養用基材。

[請求項7]
ペプチドユニット(1)とペプチドユニット(2)との割合(モル比)が、(1)/(2)=99.9/0.1~1/99である請求項5記載の幹細胞培養用基材。

[請求項8]
前記高分子基材が、円二色性スペクトルにおいて、波長220~230nmに正のコットン効果を示し、波長195~205nmに負のコットン効果を示す請求項5記載の幹細胞培養用基材。

[請求項9]
前記高分子基材が、ポリペプチドの少なくとも一部が3重らせん構造を形成している請求項5記載の幹細胞培養用基材。

[請求項10]
前記高分子基材が、分子量5×10 3~5×10 6の範囲にピークを示す請求項5記載のポリペプチドである幹細胞培養用基材。

[請求項11]
前記環状骨格構造を有する細胞接着性ペプチドが光分解性リンカーを介して高分子基材に結合されている、請求項1記載の幹細胞培養基材。

[請求項12]
光分解性リンカーが2-ニトロベンゼン骨格、2-ニトロフェノール骨格、ニトロインドール骨格、又はクマリン骨格を有する請求項11記載の幹細胞培養基材。

[請求項13]
請求項1から12のいずれか記載の幹細胞培養用基材上で幹細胞を培養する工程を含む幹細胞の培養方法。

[請求項14]
幹細胞が胚性幹細胞及び/又は誘導多能性幹細胞(iPS細胞)である請求項13記載の培養方法。

[請求項15]
請求項11又は12に記載の幹細胞培養基材上で幹細胞を培養する工程、及び、
前記培養後の培養基材に光を照射することにより、幹細胞を幹細胞培養基材から分離する工程を含む、幹細胞の培養方法。

[請求項16]
請求項5記載の高分子基材のポリペプチドが有するカルボキシル基と、環状骨格構造を有する細胞接着性ペプチドが有するアミノ基とを脱水縮合して得られるアミド結合により、環状骨格構造を有する細胞接着性ペプチドと高分子基材とが結合することを含む幹細胞培養基材の製造方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
  • Inventor
  • TANIHARA, Masao
  • SATO, Narutoku
  • KOTOKU, Tomomi
  • SHIBASAKI, Yoshiaki
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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