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THREE-DIMENSIONAL CULTURE METHOD USING BIODEGRADABLE POLYMER AND CULTURE SUBSTRATE ENABLING CELL TRANSPLANTATION

Foreign code F180009640
File No. 4720
Posted date Nov 21, 2018
Country WIPO
International application number 2015JP080641
International publication number WO 2016068266
Date of international filing Oct 30, 2015
Date of international publication May 6, 2016
Priority data
  • P2014-223702 (Oct 31, 2014) JP
Title THREE-DIMENSIONAL CULTURE METHOD USING BIODEGRADABLE POLYMER AND CULTURE SUBSTRATE ENABLING CELL TRANSPLANTATION
Abstract The present invention provides a cell culture substrate which includes a nanofiber, made of a biodegradable polymer, on a support made of a biodegradable polymer. Further, provided is a cell culture method which is characterized by performing inoculation of a cell on the substrate and stationary culture of the cell. Moreover, provided is a cell transplantation therapeutic agent which includes the substrate and the cell cultured on the substrate.
Outline of related art and contending technology BACKGROUND ART
Human pluripotent stem cell under appropriate conditions are capable of differentiation in Northern, which cells in the living organism tissue (pluripotency) have properties that can be from, cell transplantation therapy, drug discovery, in various fields including regenerative medicine is expected to be applied to. However, conventional human pluripotent stem cell culture method, feeder cells or various types of polymer and the like have been used as a cell culture, these methods on the preparation operation is complicated because it is not necessary and is stable in quality, stable human pluripotent stem cell culture, it is difficult to supply. In particular, human pluripotent stem cell of high quality, a large amount, the development of a fully automated culture method, more stable, and inexpensive method for necessary, such a method still has not been established.
Culture dishes used hitherto been used in 2 dimensional culture, the culture dish is required in a unit of one sheet 100, the individual cells from the culture dish is required for passage in view of the operation, the quality of the human pluripotent stem cell, a large amount, the development of a fully automated culture method is not suited for. Therefore, pluripotent stem cell in a limited space in order to allow a large amount of the culture, and the 3 dimensional culture is essential. Heretofore, cultured in suspension culture or micro beads have been method has been developed (non-patent document 1, 2), the aggregation or cell mass by stirring at the cell surface and a problem such as the shear stress, has not yet been put to a practical use.
In recent years, feeder cells is not used in the development of new human pluripotent stem cell culture has been actively performed. The current, the cells have been widely used as a culture substrate, and the recombinant protein Matrigel (non-patent document 3) and the like, these materials may have a higher cost, in addition, a large difference between lots in the quality due to the lack of stability such as.
Under such conditions in cultured human pluripotent stem cell becomes an unstable state, as a result, the proliferation rate of abnormal, highly non-uniform cell population to the alteration, loss of differentiation, the karyotype of a causes a failure such as a mutation. As an alternative to this, a polymer is used such as a polymer is also reported the development of cell culturing substrate (non-patent document 4, 5), such as a product has been, a stable product can be obtained, very expensive and, in some cases also is not suitable for some cell lines such as, stable, in order to fabricate an inexpensive cell culturing substrate have not yet been accomplished.
Cell culturing substrate is, in a cell of interest to supply oxygen and nutrients necessary for the group, in addition stable for retaining is a voltage, the nanofiber has been attracting attention in recent years. The nanofibers, the fiber diameter is on the order of nanometers and of ultrafine fibers, the structural body from the nanofibers which approximate the size of the extracellular matrix, cell adhesion by an increase in specific surface area is improved, 3 dimensional culture because of their advantages such as enabling the from, or synthetic polymers (non-patent document 6), synthetic polymers such as gelatin and collagen mixture biopolymers (non-patent document 6, 7) to produce the nanofibers including but, not used-culture system with feeder cells, human ES cells were able to grow not been reported (non-patent document 7).
However, conventional, human pluripotent stem cell passages is, collagenase, dispase, or the method using enzymes such as trypsin, or passaged cell strainer production with pipetting method has been performed, a technique using the enzyme, and damage to the cells by the enzyme reaction, enzyme reaction and the non-uniform with respect to the cells. In addition, a single cell to distribute the cells die and there is a problem. On the other hand, mechanical passaging method, cell damage is very large, often a problem.
The inventors of the present invention, human pluripotent stem cell culture base material serving as the, inexpensive biocompatible and focused on the biological material, using electrospinning, nanofibers can be a biological material has been developed (patent document 1). The nanofibers on a substrate and the cultured human pluripotent stem cells, cultured on matrigel the same excellent proliferation. In addition, the nanofibers using a substrate and passages are done in, without performing the enzyme treatment, only slight pipetting operation can be dispersed to single cells, as seen in the conventional method markedly inhibited cell death can be revealed.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 生分解性ポリマーからなる支持体上に、生分解性ポリマーからなるナノファイバーを含有してなる、細胞の培養用基材。

[請求項2]
 支持体を構成する生分解性ポリマーが合成ポリマーである、請求項1記載の基材。

[請求項3]
 合成ポリマーがポリグリコール酸(PGA)である、請求項2記載の基材。

[請求項4]
 支持体が不織布である、請求項1~3のいずれか1項に記載の基材。

[請求項5]
 ナノファイバーを構成する生分解性ポリマーがゼラチン又は合成ポリマーである、請求項1~4のいずれか1項に記載の基材。

[請求項6]
 合成ポリマーがPGAである、請求項5記載の基材。

[請求項7]
 細胞が多能性幹細胞である、請求項1~6のいずれか1項に記載の基材。

[請求項8]
 請求項1~6のいずれか1項に記載の基材上に細胞を播種し、該細胞を静置培養することを特徴とする、細胞の培養方法。

[請求項9]
 細胞が多能性幹細胞である、請求項8記載の方法。

[請求項10]
 請求項1~6のいずれか1項に記載の基材と、該基材上で培養した細胞とを含んでなる、細胞移植療法剤。

[請求項11]
 細胞が多能性幹細胞から分化誘導されたものである、請求項10記載の剤。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KYOTO UNIVERSITY
  • GUNZE LIMITED
  • Inventor
  • KAMEI, Kenichiro
  • LIU, Li
  • NAKATSUJI, Norio
  • CHEN, Yong
  • SATO, Hideki
  • SUZUKI, Masakazu
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN 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 IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA 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 KM ML MR NE SN ST TD TG
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