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Quality and safety of cryopreserved human mesenchymal stem cell(MSC) during serial subculture

连续传代培养中冻存人间充质干细胞（MSC）的质量和安全性

基本信息

批准号：
18592198
负责人：
YAMAZAKI Yasuharu
金额：
$ 2.49万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18592198/
关键词：
tissue engineering human bone marrow cell autologous serum culture

项目摘要

Objectives:1) To examine surface marker profile of human bone marrow-derived mesenchymal stem cells (MSCs) during the two subculture processes; during the primary culture and subculture (the 2nd passage) and during cryopreservation and subculture (the 3rd passage).2) To examine the safety of cryopreserved MSC.Methods:1) The expression of the stem cell markers, CD105, CD133, CD166 and CD271, was analyzed at the end of each individual passage.2) The expression level of CD271 at each time point was compared. The viability of MSC during osteogenic differentiation was examined in vitro.3) In vitro osteogenic potential of MSCs was compared in the presence or absence of bone marrow washing process.4) Chromosome analysis was performed using the cryopreserved MSCs by G-banding.Results :1) MSCs were 100% positive for CD105, CD133 and CD166 antigens at the 2nd and 3rd passages.2) The expression of CD271 (+) and CD271 (-) cells was similar at either passage, without difference in the ratio of CD271 (+)/CD271 (-) cells.3) Both CD271 (+) and CD271 (-) cells preserved their multilineage differentiation potential, even after the primary culture or even after cryopreservation.4) Viability of osteocytes was significantly higher for CD271(+)cells at the 2nd passage and for CD271 (-) at the 3rd passage.5) Washing process of the bone marrow increased significantly in vitro viability of osteocytes.6) G-banding analysis showed no chromosomal abnormality at each passage after primary culture and after cryopreservation (5, 7 and 10 years). However, there was a decline in cell viability for the MSCs that had been cryopreserved for 5, 7 and 10 years.Conclusion :Osteogenic potential of CD271 (+) MSCs decreased after cryopreservation. G-band chromosome analysis revealed no cryopreservation-related abnormality. Washing of the bone marrow before primary culture may be an appropriate step for preparation of MSC.

目的：1）检测人骨髓间充质干细胞（MSCs）在原代培养和传代（第2代）以及冻存和传代（第3代）两个传代过程中的表面标志物变化; 2）检测冻存MSCs的安全性。结果：（1）骨髓间充质干细胞体外成骨分化能力的检测：（2）骨髓间充质干细胞体外成骨分化能力的检测：（3）骨髓间充质干细胞体外成骨分化能力的检测：（4）骨髓间充质干细胞体外成骨分化能力的检测：（5）骨髓间充质干细胞体外成骨分化能力的检测：（6）骨髓间充质干细胞体外成骨分化能力的检测：（7）骨髓间充质干细胞体外成骨分化能力的检测：（8）骨髓间充质干细胞体外成骨分化能力的检测：（10）1）第2代和第3代骨髓间充质干细胞表达CD 105、CD 133和CD 166抗原的阳性率均为100%; 2）第2代和第3代骨髓间充质干细胞表达CD 271（+）和CD 271（-）的阳性率相似，CD 271（+）/CD 271（-）细胞比例无差异。3）CD 271（+）和CD 271（-）细胞均保持其多向分化潜能，即使在原代培养后或甚至在冷冻保存后。4）骨细胞的活力对于第2代的CD 271（+）细胞和第3代的CD 271（-）细胞显著更高。5）6）骨髓原代培养及冻存5、7、10年后G显带分析均未见染色体异常。结论：CD 271（+）MSCs冻存后成骨能力下降。染色体G带分析未发现冻存相关异常。在原代培养前对骨髓进行洗涤可能是制备MSC的适当步骤。