Chen Lin, Xie Xiaoyan, Xi Jiafei, Lyu Yang, Tian Yu, Liu Daqing, Yue Wen, Li Yanhua, Nan Xue, Li Siting, Fan Zeng, Pei Xuetao
South China Research Center for Stem Cell & Regenerative Medicine; The Lab of Stem Cell and Regenerative Medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
Zhonghua Xue Ye Xue Za Zhi. 2016 Jan;37(1):45-50. doi: 10.3760/cma.j.issn.0253-2727.2016.01.009.
To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).
UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.
With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.
This non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.
探索脐血单个核细胞（MNCs）来源的红系祖细胞（EPCs）体外生成及冻存技术。
选取脐血作为EPCs来源。用羟乙基淀粉（HES）沉淀红细胞。通过Ficoll密度梯度离心分离MNCs。在添加干细胞生长因子、胰岛素生长因子、促红细胞生成素、Fms样酪氨酸激酶配体、转铁蛋白和地塞米松的悬浮培养体系中，体外从MNCs生成红系祖细胞。通过形态学分析和CD71/CD235a表达谱评估细胞成熟情况。使用不同的冻存培养基对体外诱导的细胞进行冻存。细胞复苏后检测细胞存活率、表型及增殖曲线。
随着培养时间延长，细胞总数显著增加，同时CD71和CD235阳性细胞群比例升高。诱导14天后，细胞数量达到起始数量的约110倍，细胞活力为（88.92±0.95）%。细胞表面标志物CD71和CD71/CD235的比例分别为（86.77±9.11）%和（64.47±16.67）%。随着诱导时间延长，瑞氏-吉姆萨染色显示，早幼红细胞大多出现在第10天，晚幼红细胞出现在第14天。第14天出现红色细胞团，表明血红蛋白生成增多。集落形成试验显示，诱导第7天的红系集落高于未诱导细胞（每2000个细胞中分别为326.00±97.96和61.60±20.03）。随着培养时间延长，红系集落数量减少。用不同的冻存液保存诱导的EPCs，其中10%二甲基亚砜（DMSO）优于5% DMSO。此外，10% DMSO + 2%人血清白蛋白（HSA）与10% DMSO + 5% HSA效果无差异。50%血浆与2% HSA联合使用效果更佳。
这种无血清培养基能有效诱导和扩增EPCs，10% DMSO + 2% HSA + 50%血浆似乎是脐血来源EPCs理想的冻存液。
