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Cell Biology International (2009) 33, 796–800 (Printed in Great Britain)

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Three key variables involved in feeder preparation for the maintenance of human embryonic stem cells

Di Zhou, Tiancheng Liu, Xiaoying Zhou and Guangxiu Lu^*

Institute of Reproductive & Stem Cell Engineering, Central South University, National Engineering & Research Center of Human Stem Cells, No. 86 Xiangya Road, Changsha, Hunan 410078, PR China

Abstract

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.

Key words: Embryonic stem cell, Mouse embryonic fibroblast, Mitomycin C.

^*Corresponding author. Fax: +86 731 4497661.

Received 2 November 2008/18 December 2008; accepted 14 April 2009