Optimize Concentration Of Porcine Follicular Fluid Proteins (pFF) Of Medium Size Follicle On Primary Granulosa Cell (pGC) Growth
The experimental design to optimize concentration of porcine follicular fluid (pFF) of medium size follicle (4-6 mm in diameter) was required for in vitro cell culture growth. Therefore, porcine granulosa cells (pGC) also obtained from medium size follicle were cultured in M199 medium with Earle's salts (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% heat-treated fetal bovine serum (HTFCS), 2.2 mg/mL NaHCO3, 0.25 mM pyruvate, 1 µg/mL LH, 1 µg/mL estradiol with ethanol, and 50 µg/mL gentamycin sulfate at a high humidified atmosphere with 5% CO2 in 95% air atmosphere at 37°C for 48 h. We found that, all pGC were exhibited from round shape to fusiform shapes with fibroblast-like morphology. They were then treated with medium follicle at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins in culture medium without FCS for 24 h. The pGCs viability was evaluated by MTT assay and analyzed by one-way ANOVA. Percent viability of pCG treated pFF in all concentration of medium follicle showed cell viability higher than control (p<0.05). At treated groups of pFF at 200, 400, 500, and 600 µg proteins showed cell viability higher than control groups (p<0.05) and positive control groups (medium with 10% FCS+ hormone) but not significant. Interestingly, treated group of pFF at 200 µg protein was reached the highest cell viability. Porcine GCs were developed to fibroblast shape longer than control group. This finding implies that the pFF at 200 µg protein could optimize concentration use for cell viability instead of FCS and could apply in cell biotechnology research.
Acknowledgements: The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.
Keywords: Porcinefollicular fluid protein (pFF), porcine granulosa cells (pGC), MTT assay