Biological Research on Morphological Features of in vitro Porcine Granulosa Cells Culture

Abstract

The purpose of this research was to study the morphology and culturing porcine granulosa cells (GC) collected from large white pig ovaries with sterile technique. The primary cell culture methods were utilized based on M199 formula (supplemented with 10% HTFBS, 15 µg/mL FSH, 1 µg/mL LH, 1 µg/mL estradiol, 2.2 mg/mL NaHCO₃, 0.25 mM pyruvate and 50 µg/mL gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C) at 0, 24, 44-48, 72, 96 and 120 h (short-term culture). The result of culturing GC, using M199 based on cell morphology study, exhibited that, in the beginning, cells were round-shaped and non-ciliated cells. Porcine granulosa cells showed healthy characteristic and changed their morphology from round shape to fibroblast-like morphology, then extending and adhering to the surface of the petri dish. After 24 h, they multiplied and spread 100% all over the culture dish and then turned sharp at both head and bottom and expanded. As cultured for 48, 72, 96, 120 h. they were expanded more, fibroblast-like shape, epithelial-like isolated and cluster group cells under microscopy. The viability of GC culture for 0, 24, 48, 72, 96 and 120 h were 100, 98, 95, 92, 90, and 86%, respectively. The advantages of this research were: that it enabled us to culture GC cells collected from the slaughterhouse efficiently by using short-term culture methods in culture medium; that we were capable of studying the morphological features, as well as the variance of the cell shape in the laboratory; sub-culture cells and long-term culture can possibly be further developed as granulosa cell lines; and that the cells could be utilized practically in innovative biological research field, thus helping economize costs and eliminate the animal experiments correctly in accordance with moral norms.