Fetal bovine serum (FBS) enhances proliferation and colonization of caprine spermatogonial stem cells

Abstract

Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. This study was undertaken to investigate the effect of serum concentration on colony formation and development of different types of SSC colonies with respect to passage number. Cells were isolated from pre-pubertal buck testes by two step enzymatic digestion method. The filtered cells were enriched by differential adherence selection method. Cells were then randomly divided into 8 groups, depending on concentration of FBS in culture medium ranging from 0% to 35%. In experiment 1, effect of different concentrations of FBS on total number pSSCs with reference to differential plating was observed while in experiment 2, effect of different concentrations of FBS on types of pSSC colonies with respect to passage number was observed. No colony formation was observed in control group (0% FBS) while significantly higher number of single, paired, cluster and rosette colonies observed were with 20% FBS group in differential 2 (D2) as compared to other groups. Alkaline phosphatase staining and immunocytochemistry staining (PGP9.5 and OCT4) were positive in SSCs colonies. The growth rate of the culture was significantly and consistently higher with 20% FBS.