Culture , Isolation and Passage of Porcine Embryonic Stem Cells
Dong Xiao1,3 Feng Shutang1** Wang Zhanhe1,2 Wang Duanyun1 Zheng Xing3
(1. Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing 100094, China; 2.Beijing Agricultural College, Beijing 102206, China; 3. College of Biological Sciences,China Agricultural University, Beijing 100094, China)
Abstract：Abctract: Embryonic stem (ES) cells are pluripotential cells isolated from culture of preimplantation embryos in vitro. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotential porcine embryo-derived cell lines could be isolated. Porcine embryos from D5,6,7,9,10 after the first day of estrus and insemination (day 0) were recovered by a surgical uterine flush technique with phosphate buffered saline (PBS) supplemented with 1% fetal bovine serum (FBS). A total of 124 porcine embryos were collected. Embryos were cultured at 38 ℃ on the feeder layers prepared from mitotically inactivated permanent mouse embryonic fibroblasts (STO) in the same medium[DMEM(Dulbecco's modified eagle medium) + 10 % NBS (newborn bovine serum)+ 10 %FBS+1 000 IU/mL LIF (leukemia inhibitory factor)+ 30 ng/mL SCF（stem cell growth factor）], the ICM(inner cell masses) growing rate of the embryos collected on day 5, 6, 7, 9, 10 was 0, 12.5%, 59.2%, 100%, 100% respectively; When embryos were cultured in two different mediums [Medium 1:DMEM + 10 % NBS + 10 % FBS + 1 000 IU/mL LIF + 30 ng/mL SCF; Medium 2: DMEM + 10 % NBS + 10 % FBS + 1 000 IU/mL LIF + 30 ng/mL SCF+ 20 ng/mL bFGF (basic fibroblast growth factor)], the result showed that bFGF was not essential in the culture and passage of ES cells; When the diameter of Day10 embryos was considered, we got that the larger embryos were better in ES cell culture, and the highest ES cells passages rate was gotten when the embryos have a diameter over 100 μm. In this experiment, we got ES-like colonies passed 4 times thatexpressed alkaline phosphatase activity.
