195 DEVELOPMENT OF INNER CELL MASS AND FORMATION OF EMBRYOID BODIES ON A GELATIN-COATED DISH AND ON THE FEEDER LAYER IN BUFFALO (BUBALUS BUBALIS)

Abstract

Isolation and culture of embryo-derived cell lines have been reported in many mammals, however, there is not even a single report toward initiation of such work in buffalo (Bubalus bubalis). Therefore the present study was carried out to isolate the inner cell mass from in vitro-produced buffalo blastocysts and to grow this inner cell mass for formation of embryoid bodies in a gelatin-coated dish and on an homologous fetal fibroblast feeder layer. Immature buffalo oocytes were isolated from the slaughterhouse ovaries. In vitro production of blastocysts was carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). A total of 26 buffalo blastocysts were produced in vitro. These blastocysts were transferred into a 100-mL drop of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) for further culturing. Thirteen blastocysts hatched on the next day of culture. The hatched mass was separated, suspended in Dulbecco's phosphate buffer saline (DPBS, without Ca⁺⁺ and Mg⁺⁺) containing 5% FBS + 0.25% trypsin and examined under the zoom stereomicroscope until disappearance of the trophectoderm cells. The remaining cells of seven blastocysts were cultured in DMEM supplemented with 20% FBS on 0.2% gelatin coated culture dish (group 1), and cells of six blastocysts in DMEM medium supplemented with 20% FBS were cultured on a mitomycin-c-treated (10 µL/mL) feeder layer (group 2), for 14 days. The isolated cells attached to the bottom of the dish in both the groups, spreading was noticed on Day 5 of the culture in group 1 and on Day 3 of culture in group 2. The attached cells were trypsinized using DPBS with 0.25% trypsin, isolated, and subcultured further. Attachment and spreading was noticed only in group 2 subcultured cells. The cellular integrity was homogeneous and the plasma membrane was clearly visible in group 2, but not in group 1. Less than 10% of the attached cells formed embryoid bodies in group 1, where as more than 30% attached cells in group 2 formed embryoid bodies; the latter expressed alkaline phosphatase activity and were blue after staining. These results indicate that the culturing of the inner cell mass on an homologous fetal fibroblast feeder layer is a better choice for production of embryonic stem cells in buffaloes.