Shen Jian-Liang, Gong Li-Zhong, Cen Jian, Liu Yi, Wang Li-Xing, Yin Wen-Jie, Zhao De-Feng, Ma Wei-Na, Huang You-Zhang
Department of Hematology, Chinese PLA Navy General Hospital, Beijing, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Feb;21(1):181-7. doi: 10.7534/j.issn.1009-2137.2013.01.037.
Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton's jelly derived mesenchymal stem cells (WJ-MSC), and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80°C refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage (PP1). Thus, PP2-PP15 WJ-MSC were obtained by continuous cell subculture. The 1st control passage (CP1) to 15th passage (CP15) represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated cells, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92.6%. There was a statistically significant difference in proliferation index between PP1 and CP1 (P < 0.05), but there were no statistically significant differences between PP2-PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not different. After induced differentiation into adipocyte, osteoblast and neuron, the staining with oil red O, alkaline phosphatase and neuron-specific enolase was performed respectively, and the positive degrees were not clearly different macroscopically between two groups. Relatively high levels of triglyceride, alkaline phosphatase, and neuron-specific enolase in relevant cells could be detected, but had no significant differences between two groups. It is concluded that some WJ-MSC (< 10%) are damaged after cryopreservation, and the biological characteristics of WJ-MSC in cryopreservation group keep constant, as compared with that in non-cryopreservation group.
本研究旨在探讨冷冻保存对脐带来源间充质干细胞（WJ-MSC）生物学特性的影响，为其临床应用及建立WJ-MSC库提供实验依据。原代WJ-MSC通过脐带组织体外培养获得。将连续细胞培养获得的第5代WJ-MSC与冷冻保护剂混合，置于-80°C冰箱冷冻后保存于液氮中。将冻存的WJ-MSC复苏后，经细胞培养获得第1代WJ-MSC作为第1次冻存代（PP1）。以此类推，通过连续细胞传代获得PP2 - PP15代WJ-MSC。第1次对照代（CP1）至第15代（CP15）代表非冻存组传代获得的第6代至第21代WJ-MSC。检测并比较冻存组和对照组WJ-MSC的生物学特性，包括有核细胞回收率、台盼蓝拒染率、CCK-8活性、细胞凋亡、细胞贴壁、增殖指数、细胞表面抗原、细胞周期以及向脂肪细胞、成骨细胞和神经元诱导分化的能力。结果显示，冻存WJ-MSC的有核细胞回收率为98.2%，台盼蓝拒染率为94.3%，CCK-8活性为91.4%，凋亡率为3.9%，贴壁率为92.6%。PP1与CP1的增殖指数差异有统计学意义（P < 0.05），但PP2 - PP15与其相应对照组之间差异无统计学意义。传代细胞高表达CD29、CD44、CD71、CD73、CD90、CD105、CD166和HLA-ABC，低表达CD34、CD45和HLA-DR。两组上述表面抗原的表达差异无统计学意义。两组传代细胞的增殖潜伏期和对数增殖也无差异。诱导分化为脂肪细胞、成骨细胞和神经元后，分别进行油红O、碱性磷酸酶和神经元特异性烯醇化酶染色，两组宏观上阳性程度差异不明显。相关细胞中可检测到较高水平的甘油三酯、碱性磷酸酶和神经元特异性烯醇化酶，但两组间无显著差异。结论是，冷冻保存后部分WJ-MSC（< 10%）受损，与非冷冻保存组相比，冷冻保存组WJ-MSC的生物学特性保持稳定。
