Establishment and Long-Term Maintenance of Bovine Embryonic Stem Cell Lines Using Mouse and Bovine Mixed Feeder Cells and Their Survival after Cryopreservation

Abstract

To investigate the efficiency of establishment and maintenance of bovine embryonic stem (ES) cell lines, the inner cell mass (ICM) of embryos derived from in vitro production was isolated for ES cell preparation by removal of trophoblasts with protease. Feeder cells from bovine, mouse and mixed fibroblasts were produced from mouse and bovine embryonic fibroblasts. The feeder cells were selected and purified through 30 passages. Stabilized feeder cells with their good contact and forming monolayer were used for long-term culture of bovine ES cell lines. The efficiency of bovine ES cell attachment on mouse or mixed feeder cell monolayer increased when compared to those using bovine feeder cells. Bovine ES cell lines were cultured and maintained in undifferentiated state longer on mixed fibroblasts (258±19 days) than bovine (225±6 days) or mouse (196±49 days) fibroblasts. The ES cell lines were identified as pluripotent by alkaline phosphatase (AP) staining and forming the embryoid body. Vitrification procedure for cryopreservation of ES cells showed a higher survival rate than the conventional freezing method. These data suggest that mixed fibroblast feeder cells were more efficient for long-term culture of bovine ES cell lines than using only mouse or bovine fibroblasts.