悬浮搅拌法培养和扩增诱导多能干细胞

A new expansion system for induced pluripotent stem cells in stirred suspension bioreactors

目的建立一种新的搅拌培养法，大量培养、快速扩增诱导多能干细胞（iPSCs）。方法采用搅拌式细胞培养器悬浮培养iPSCs，每4d为1个周期，第4天检测细胞聚集球的直径；经8次传代后，计算细胞倍增时间和细胞数量，经实时定量聚合酶链反应（Real一timePCR）检测多能性标志物0ct4、Nanog和性别决定区Y框蛋白2（SOX2）mRNA表达，免疫荧光法检测0ct4蛋白表达，组织染色法检测碱性磷酸酶（AIJP）活性。结果搅拌式培养iPSCs，细胞倍增时间为（20．82±1．27）h；培养1d后，细胞形成微小聚集球，经1个周期4d的培养，细胞球直径达到最大（184．1±7．2）±m，经传代8次，可以扩增到起始细胞数量的1014倍；Real-timePCR和免疫组织化学结果显示这些iPSCs仍然高表达多能性标记。结论这种新方法能够快速、大量扩增iPSCs，并且这样培养的iPSCs能够保持其多能性。

Objective To establish protocols for expanding human induced pluripotent stem cells （iPSCs） as undifferentiated aggregates in stirred suspension bioreactors and illustrate that such protocols can be adapted for long term culture of human iPSCs. Methods The human iPSCs lines maintained on the Matrigel-coated plates were collected, seeded in stirred suspension bioreactor, and cultured for 8 passages, 4 days each passage. Cell number and viability were calculated and the immunoflurescence analysis and gene expression profiles of pluripotency markers including Oct4, Nanog, sex determining region Y-box 2 （ Sox2 ） and alkaline phosphatase （ALP） were conducted at the start and the end of the experiment. In vitro differentiatiori into hepatocytes was performed at the end of the long term culture. Results The doubling time of iPSC in stirred suspension bioreactors was （ 20. 82 ± 1.27） h, and the cells formed cell aggregates whose diameter got （184. 1 ± 7. 2） μm after suspension culturing for 4 days. Stirred suspension bioreactors yielded 1 x 1014-fold expansion of undifferentiated pluripotent iPSCs over cuhure of 32 days. The iPSCs still expressed the pluripoteney markers over extended culture time in suspension. In vitro differentiation into hepatocytes, further confirmed the functional and undifferentiated pluripotent iPSC aggregates following long-term passaging. Conclusion Stirred suspension bioreactor system represents an efficient process for the large-scale expansion of highly pluripotent human iPSCs populations, which is an important and the first step in their clinical application.