Wang Yiru, Bai Jing, Chen Jie, Liu Lifeng, Wang Yu
Institute of Geriatric Cardiology, General Hospital of Chinese PLA, Beijing, 100853, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Feb;26(2):141-5.
To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs), to investigate a better cryopreservation protocol of HAFMSCs and to observe the bio-characteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and clinical applications.
HAFMSCs were isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method. HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO at cryoprotectant) in liquid nitrogen for 12 weeks. The bio-characteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adipogenic and osteogenic differentiation abilities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservation.
At 12 weeks after cryopreservation, different protocols had different effects on the cell viability; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed "S" shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adipogenic differentiation, the lipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralization were verified by von Kossa staining. There was no significant difference (P > 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservation.
HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabilities and no changes of the bio-characteristics and differentiation potentialities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.
分离培养人羊水间充质干细胞（HAFMSCs），探索更佳的HAFMSCs冷冻保存方案，观察冷冻保存后HAFMSCs的生物学特性及多能性，为进一步的基础研究和临床应用提供依据。
采用改良两步法从孕中期孕妇羊水中分离HAFMSCs。将HAFMSCs用不同冷冻保存方案（冷冻保护剂中含不同比例的胎牛血清和二甲基亚砜）在液氮中冷冻保存12周。分析冷冻保存后HAFMSCs的生物学特性。采用MTT法观察生长特性并绘制生长曲线。利用流式细胞术检测HAFMSCs的表面抗原，包括CD29、CD34、CD44、CD45、CD73和CD90。观察HAFMSCs的成脂和成骨分化能力。比较冷冻保存前后HAFMSCs中Oct-4和Nanog的mRNA水平。
冷冻保存12周后，不同方案对细胞活力影响不同；较好的冷冻保护剂配方为50% DMEM、40%胎牛血清和10%二甲基亚砜。冷冻保存后，细胞增殖迅速，生长曲线呈“S”形，与冷冻保存前的细胞相同。表型显示，HAFMSCs的表面标志物CD29、CD44、CD73和CD90呈阳性，CD34和CD45呈阴性。成脂分化21天后，油红O染色观察到脂滴。成骨分化21天后，冯库萨染色证实有钙矿化。冷冻保存前后Oct-4和Nanog的mRNA水平无显著差异（P>0.05）。
HAFMSCs在体外具有快速增殖和多能性。冷冻保存12周后，细胞活力高，生物学特性和分化潜能无变化。含50% DMEM、40%胎牛血清和10%二甲基亚砜的冷冻保护剂是较好的冷冻保存方案。
