Abstract：During culturing embryonic stem cells (ESCs), mouse embryonic fibroblasts (MEFs) are used as feeder layer, generally. However, MEFs in limited passages are available for feeder cells. Therefore, it takes much time for preparing MEFs, and expends lots of animals. It is important to find stable cell lines as substitution of MEFs for saving expenditure and reducing workload during ESCs culture. L-Wnt3a cells are immortal cell line that stably secrets activated Wnt-3A protein into culture medium. Recently, it is unclear if the cells can be used as feeder cells for culturing ESCs. In present study, L-Wnt3a cells were used to co-culture with mouse (Mus musculus) ESCs for feeder layer, and its conditional medium was examined to culture ESCs without feeder layer. The results showed that treatment L-Wnt3a cells with mitomycin C for 4 h (10 μg/mL) was effectively for inhibiting proliferation and keeping their viability. Transfer single C57BL/6 mouse blastocyst to wells of plate containing a feeder layer of mitotically inactivated L-Wnt3a cells, primary colonies were formed and an embryonic stem cell line was established. A conditional medium was prepared from L-Wnt3a. ESCs in the conditional medium could proliferate and keep pluripotency when removing feeder layer. Feeder-free cultured ESCs in L-Wnt3a conditioned medium were ALP positive, and immunostaining showed that the cells expressed pluripotent markers, Oct4, Sox2, Nanog, E-cadherin and SSEA-1. It formed embryoid body in vitro and teratoma in vivo, and differentiated into three germinal layers. When injecting feeder-free ESCs into blastocoels, the chimeric offsprings were achieved. In conclusion, the mouse ESCs were successfully isolated and cultured in L-Wnt3a feeder layer. The cells were available cell line for replacing MEFs as feeder cells. It refrained from limited passages and heavy works in MEFs culture. Feeder-free ESCs could be cultured in L-Wnt3a conditional medium, it properly avoides the negative effect of feeder layer during culturing ESCs.
