Successful vitrification of human amnion-derived mesenchymal stem cells
Successful vitrification of human amnion-derived mesenchymal stem cells
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DOI：
10.1093/humrep/den202
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发表时间：
2008-08-01
影响因子：
6.1
通讯作者：
Kim, Hae Kwon
中科院分区：
文献类型：
--
作者：
Moon, Jeong Hee;Lee, Jung Ryeol;Kim, Hae Kwon
背景技术背景：需要一种用于人骨髓间充质干细胞（HAM）的冷冻保存方案，因为这些细胞不能在培养中长时间存活。本研究的目的是确定玻璃化冷冻是否是一种有用的冷冻方法，用于储存火腿。方法：采用玻璃化法对HAM进行冻存。台盼蓝染色法观察解冻后HAM的形态学变化及存活率。采用流式细胞术、RT-PCR和免疫细胞化学方法检测胚胎干细胞（ESC）标志物的表达。采用Von Kossa、油红O和阿尔新蓝染色法检测解冻后HAM的分化潜能。结果：HAM的解冻后活力为84.3 +/- 3.2%（平均值+/- SD，n = 10）。解冻的火腿显示出与非玻璃化冷冻的新鲜火腿难以区分的形态特征。表面抗原的表达CD 44、CD 49 d、CD 59、CD 90、CD 105、HLA-ABC强阳性，HLA-G弱阳性; CD 31、CD 34、CD 45、CD 106、CD 117和HLA-DR阴性）和ESC标志物[CK 18、成纤维细胞生长因子-5、加塔-4、神经细胞粘附分子、巢蛋白、Oct-4、干细胞因子、HLA-ABC，波形蛋白、骨形态发生蛋白（BMP）4、肝细胞核因子4 α（HNF-4 α）、Pax-6、甲胎蛋白、Brachyury、BMP-2、TRA-1-60、阶段特异性胚胎抗原（SSEA-3、SSEA-4）]维持在玻璃化-解冻的HAM中。解冻后的HAM在适当的培养条件下仍能分化为成骨细胞、脂肪细胞和软骨细胞。结论：玻璃化法是一种可靠有效的HAM冷冻保存方法。
BACKGROUND: A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. METHODS: HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. RESULTS: The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. CONCLUSIONS: Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.