An efficient culture method of trophoblastic cells biopsied from bovine blastocysts
2006
Kan, K.(Yamagata-ken. General Agricultural Research Center (Japan)) | ; Aoyagi, K. | ; Sendai, Y. | ; Hoshi, H.
It is essential for multiple genotype analysis of bovine preimplantation embryos to yield enough amount of their DNA samples without losing embryo viability. This study was aimed to establish optimal culture conditions to support the growth of bovine trophoblastic cells derived from biopsied embryos. The zona pellucida of in vitro produced embryos were partially slit by a glass microblade and the herniated trophoblastic cells were grown after a subsequent culture. The trophoblastic cells were cut by metal microblade and cultured in various types of culture media. In experiment 1, two types of basic media (HPM 199 and ESM-2) were compared for the cell viability of trophoblastic cells in 10% fetal bovine serum (FBS)-containing medium. The percentage of cell viability cultured in ESM-2 (91.2%) was significantly (p0.01) higher than in HPM199 (16.7%) and the estimated mean cell number cultured in ESM-2 (1,682) was greater than in HPM199 (629) after 7 days in culture. In experiment 2, the optimal serum requirement on the cell growth of trophoblastic cells was tested at various concentrations (0, 2.5, 5, 10 %, respectively) in ESM-2 medium after 7 days in culture. The percentage of cell survival (91.2%) in 10% FBScontaining medium was highest among the concentrations of serum tested. The mean cell number (172) in the absence of serum was remarkably low compared to those (1,409, 2,012, 1,682) in the presence of serum (2.5, 5, 10%, respectively). These results have shown that herniated and biopsied trophoblastic cells were fully grown in ESM-2 and 10% FBS. This improved medium also allowed to grow biopsied trophoblastic cells from in vivo derived embryos. This culture system provides high cell viability and growth potential of biopsied trophoblastic cells to determine the multiple genotyping of preimplantation embryos.
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