Technology Of Bovine Stem Cells

Bovine embryonic stem (ES) cell lines has failed to establish. During the culture process, bovine ES cells growth could be influenced by the selection of culture medium, selection of feeder layer, obtaintion of ICM and so on. It is important to determine a good culture method and passage way for culturing bovine ES cells. In the present study, the growth of bovine ES-like cells derived from ICM of bovine IVF embryos was observed by several ways that include preparation of feeder layer, confirmation of the passage way, supplemention of differentiation factors in the culture medium. The bovine ES-like cells were evaluated by a series of detections including morphological evaulation, alkaline phosphatase staining, formation of embryoid bodies, immunofluorescence staining.1. The mouse embryonic fibroblast cells were isolated by trypsin digestion way from mouse fetuses; The bovine fetal fibroblast cells were successfully isolated by attachment of tissue pieces from bovine fetuses. Three different passage ways and four different unfreezed methods were used to culture fibroblast cells. The results indicated that the first passage way (not centrifugated) can promote the fibroblast cells growth and have the higher stickup average rates of cells; the fourth unfreezed method (not centrifugated, changed the culture medium after the cell adhibited 5-6hs) can promote the fibroblast cells growth and have the higher stickup average rates of cells. The two methods to culture cells were beneficial to the fibroblast cells growth.2. DMEM F12 culture medium supplemented with 10%FBS,0.1mMβ-mercaptoethanol,0.1 mM nonessential amino acids, 100IU/ml penicillin, 0.05mg/ml streptomycin,20ng/ml LIF and lOng/ml bFGF was used to culture bovine ES-like cells with the two different feed layers. The bovine embryonic stem (ES)-like cells were evaluated by two detections including morphological evaulation, alkaline phosphatase staining. The results indicated that in the same culture conditions the stickup average rates of the bovine embryos cultured with the feed layers of mouse embryonic fibroblast cells were significant higher than that cultured with the feed layers of the bovine embryonic fibroblast cells (P<0.05) and in better developed state. In conclusion the mouse embryonic fibroblast cells were beneficial to culture bovine embryonic stem (ES)-like cells.3. DMEM F12 culture medium supplemented with 10%FBS, 0.1mMβ-mercaptoethanol,0.1mM nonessential amino acids,100IU/ml penicillin,0.05mg/ml streptomycin,20ng/ml LIF and 10ng/ml bFGF was used to culture bovine ES-like cells with the mouse embryonic fibroblast cell feeder layers in the two different passage ways including passage of machine way and trypsin digestion way. The results indicated that the effects of passage of machine way was better than that of trypsin digestion way in the same culture conditions.4. The mouse embryonic fibroblast cells feeder layer and passage of machine were used to culture bovine embryonic stem (ES)-like cells. The bovine ES-like cells were cultured in the medium supplemented with 20ng/ml LIF, 10ng/ml SCF+20ng/ml LIF and lOng/ml SCF respectively. The results indicated that in the same culture conditions the stickup average rates of the bovine embryos cultured with the lOng/ml SCF+20ng/ml LIF culture medium were significantly higher than that with the lOng/ml SCF culture medium (P<0.05), and there was no significant different effects between culture with 20ng/ml LIF and culture with 10ng/ml SCF+20ng/ml LIF or 10ng/ml SCF. In the bovine ES-like cell subculture, the 20ng/ml LIF culture medium and the 10ng/ml SCF+20ng/ml LIF culture medium did not show obviously difference, but the bovine ES-like cell cultured with lOng/ml SCF+20ng/ml LIF culture medium grew faster than that with 20ng/ml LIF culture medium, and the bovine ES-like cell cultured with the two culture media have the differentiation capacity. The bovine ES-like cells cultured in culture medium with the lOng/ml SCF differentiate disappear after the fifth passage. The results indicated that a certain concentration of LIF and SCF are advantageous for culturing bovine ES-like cell.