成果報告書詳細
管理番号20130000000695
タイトル*平成24年度中間年報 「ヒト幹細胞産業応用促進基盤技術開発 ヒト幹細胞実用化に向けた評価基盤技術の開発 ヒト幹細胞の安定的な培養・保存技術の研究開発」(学校法人福田学園大阪保健医療大学、DSファーマバイオメディカル株式会社、株式会社ツーセル、国立大学法人大阪大学、有限会社スペース・バイオ・ラボラトリーズ、国立大学法人広島大学、株式会社丸菱バイオエンジ)
公開日2014/8/8
報告書年度2012 - 2012
委託先名学校法人福田学園大阪保健医療大学 DSファーマバイオメディカル株式会社 株式会社ツーセル 国立大学法人大阪大学 有限会社スペース・バイオ・ラボラトリーズ 国立大学法人広島大学 株式会社丸菱バイオエンジ
プロジェクト番号P10027
部署名バイオテクノロジー・医療技術部
和文要約
英文要約“Development of the basic systems for cell culture and cell preservation to make the stem cells propagate in a large quantity”Research contents and results by using the supplementary budget for the 2012 fiscal year

1.Development of culture methods to expand synovium-derived mesenchymal stem cells (MSCs) on a large-scale
Analyzing media composition, searching optimum materials of media, and considering procedures of both separating MSCs from human synovial membrane and passaging in detail, we established methods to mass-produce MSCs. Two-three million of MSCs in the cell number can be stably expanded into 1 x 1012 through six passages (P6).
To assess the safety of cellular products made of the expanded MSCs derived from synovial membrane, we carried out the soft agar colony formation assay, chromosome aberration tests, and tumorigenecity testing in severely immunodeficient mice (NOG mice). Results of all these tests suggested the cellular products we had prepared were not transformed into tumor cells.
To acquire MSCs derived from human synovial membrane in a larger quantity, we adopted several devices such as floating culture in spinner flasks, a hollow fiber culture system, microgravity environment in a three-dimensional clinostat, and a special bioreactor manufactured by ATMI, Inc. Although each of these methods could not lead to satisfactory proliferation, X-culture system using specific film succeeded to increase cell proliferation, eighteen times as many cell numbers per volume as conventional dish culture, with one-third or less volume of medium. Therefore, we can say that the X-culture system has the potential to develop a revolutionary automatic cell culture system.
We also tried to search genetic markers which specify and define human synovium- derived MSCs cultured with serum-free medium. Through DNA microarray analysis, we compared the expression patterns of genes in MSCs with those in fibroblast, both cultured with serum-free medium. As a result, sixteen and seven genes were defined with a significant difference as positive and negative markers, respectively.
2.Development of cryopreservation technologies for synovium-derived MSCs
We time-dependently assessed the influence of preservation temperature and components of stock solution on survival rates of MSCs, which had been derived from human synovial membrane and cultured with serum-free medium. We also examined the composition of cell stock solution such as the reduction of the DMSO content percentage.
Concluded.
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