Zhou Di, Liu Tiancheng, Zhou Xiaoying, Lu Guangxiu
Institute of Reproductive & Stem Cell Engineering, Central South University, National Engineering & Research Center of Human Stem Cells, Hunan, PR China.
Cell Biol Int. 2009 Jul;33(7):796-800. doi: 10.1016/j.cellbi.2009.04.008. Epub 2009 Apr 23.
Although the development of a feeder-free culture system for future applications of human embryonic stem cells (hESCs), at present the regular culture system uses mitotically inactivated mouse embryonic fibroblasts (mEFs) as feeder cells for maintaining undifferentiated hESCs. Mitomycin C (MMC) is used to inactivate mEFs, but this causes DNA damage, and it is unclear whether MMC remains in the culture system after several washes. Three variables have been evaluated with respect to feeder preparation and MMC involvement, including mEF exposure to MMC, density of feeder cells, and different wash steps during the preparation of feeder cells. These variables are critical to the subsequent planting of hESCs because remnants of MMC would be unsafe with respect to long-term culture of hESCs The novel data here evaluates the remnant amounts of MMC in a hESCs culture system using HPLC/MS/MS. The ultimate objective of this study is the control of MMC within a safe range.
尽管正在开发用于人类胚胎干细胞（hESCs）未来应用的无饲养层培养系统，但目前常规培养系统使用经丝裂霉素C（MMC）处理使其有丝分裂失活的小鼠胚胎成纤维细胞（mEFs）作为饲养细胞来维持hESCs的未分化状态。MMC用于使mEFs失活，但这会导致DNA损伤，并且在多次洗涤后MMC是否仍残留在培养系统中尚不清楚。已针对饲养细胞制备和MMC的使用评估了三个变量，包括mEFs对MMC的暴露、饲养细胞的密度以及饲养细胞制备过程中的不同洗涤步骤。这些变量对于随后接种hESCs至关重要，因为MMC残留对于hESCs的长期培养而言是不安全的。本文的新数据使用高效液相色谱/串联质谱法（HPLC/MS/MS）评估了hESCs培养系统中MMC的残留量。本研究的最终目标是将MMC控制在安全范围内。
