The Isolatin, Culturae And Identification Of KB Mouse' ES Cells

KB mouse is a mostly used trail animal in our country. Most of the established mouse ES cells lines were from129, C57BL/6J and BALB/C mouse but very few reports have showed the establishment of KB mouse ES cells lines. So it is very important to establish its ES cells lines for stem cell research in our country.The major object of this paper is to find a more suitable culturing system for KB mouse embryonic stem cells. ES cells were gained from the KB mouse embryo and were cultured in vitro as well as determinated. Some factors influencing the efficiency of the isolation of mouse ES cells have been discussed. Our work may be useful to further work on establishing of Kunbai mouse ES cells lines. The results showed:1. 446 embryos were obtained from Kunbai mouse and 323 ICMs grew out. ICMs were plated on mitomycin-inactivated feeders and cultured in a humidified environment of 5% CO2 in the air with temperature at 37℃. ES-like colonies were gained from 72 ICM and had maintained undifferentiation for 5 passages.2. Embryos were cultured on MEF, STO, STO1 (STO cells were inactivated by mitomycin and frozen and thawed when needed) and PEF feeders. There were no distinct differences on the ratio of hatched embryos and growth of ICM. ICM then were separated onto these four kinds of feeders. Some differences existed on forming of ES colonies and passaging (P< 0.05). MEF is the best feeders, STO and STO1 were better than PEF.3. Buffalo rat liver cells conditioned medium (BRL-CM) were used to isolate and culture the ES cells. Embryos cultured with this CM could attach on the feeders and ICM could grow out normally. ICMs then were separated onto STO feeders or without feeders and cultured with BRL-CM, ES colonies only gained when BRL-CM combined together with feeders. It shows that the BRL-CM can not replace the feeder to support KM ES cells'self-renewing completely.4. Different methods were used to disperse the ES cells. The Trypsin-EDTA with final concentration at 0.25% is not suitable for separating ES cells. The Trypsin-EDTA at concentration of 0.10% combined with mechanical ways is suitable for separating ES cells. Also a neutral protease Dispase at concentration of 2.4 U/mL with mechanical ways or not are suitable for separating ES cells.