Development of buffalo ear skin fibroblast cell line for somatic cell nuclear transfer

Abstract

Fibroblast is the principal active cell of connective tissue. It can be used as a useful tool for reproductive and therapeutic studies for its ability to easily grow in culture, create a preferable environment to growth and can be used as both for primary and permanent cell lines. Somatic cell nuclear transfer (SCNT) is commonly used for complete genetic reprogramming of a fully differentiated cell (e.g., fibroblast). SCNT is influential means for studying genomic imprinting, nuclear-cytoplasmic interaction, totipotency, and the contribution of paternal and maternal genomes to developing embryos. For reproductive cloning, SCNT is a fundamental step. So, the objective of this study was to adapt buffalo fibroblast cell culture protocol in order to develop fibroblast cell line for SCNT. River type buffalo ear skin tissues were used for developing primary cell culture to establish fibroblast cell line. Cells were grown in vitro. The general morphology and growth of cell population and presence of any microbial contaminants were checked regularly under an inverted microscope in phase contrast. Cells were counted by using hemocytometers. Sliced tissue was sown in 15 culture dishes to create primary and subcultures, and cells were cultivated in 6 of those dishes. Cell confluence varied from 70 to 80%. There were performed subcultures. Cell confluence in passages 1 through 10 varied from 73 to 90%. Cell concentration was 2.72 x 105, 2.68 x 105, and 2.65 x 105 per ml in passages 1, 5, and 10, respectively. Viable cells ranged from 84-93% of the total cells in passages 1 through 10. Different passages of cultivated fibroblast cells showed significantly varying viability (p< 0.05). Cultured fibroblast cell lines could now be preserved using cryotechnology.