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

目的：1)检测人骨髓间充质干细胞（MSCs）在两次传代培养过程中的表面标记谱；在原代培养和传代培养（第二代）和低温保存和传代培养（第三代）期间。2)检验MSC冷冻保存的安全性。方法：1)在每个传代结束时分析干细胞标志物CD105、CD133、CD166和CD271的表达情况。2)比较CD271在各时间点的表达水平。体外检测MSC在成骨分化过程中的生存能力。3)比较骨髓洗涤过程存在或不存在情况下MSCs的体外成骨潜能。4)用g带法对冷冻保存的MSCs进行染色体分析。结果：1)MSCs第2、3代CD105、CD133、CD166抗原100%阳性。2)两代CD271（+）和CD271（-）细胞的表达相似，CD271 (+)/CD271（-）细胞的比例没有差异。3) CD271（+）细胞和CD271（-）细胞在原代培养和低温保存后都保持了其多系分化潜能。4)第2代CD271（+）细胞和第3代CD271（-）细胞的成骨细胞活力显著提高。5)骨髓洗涤处理显著提高骨细胞体外活力。6) g带分析显示，原代培养和低温保存（5年、7年和10年）各组传代均无染色体异常。然而，冷冻保存5年、7年和10年的MSCs细胞活力下降。结论：CD271（+）间充质干细胞冷冻保存后成骨潜能降低。g带染色体分析未发现与低温保存相关的异常。在原代培养前对骨髓进行清洗可能是制备间充质干细胞的适当步骤。

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.