Culture of porcine luteal cells as a substrate for in vitro maturation of porcine cumulus oocyte complexes. Establishment and characterization

Abstract

The aim of this study was to establish and characterize the porcine luteal cells (PLC) culture for the subsequent coculture with porcine COC. The final purpose is to promote the oocyte maturation. The PLC was established using corpora lutea obtained from slaughterhouse ovaries. Corpora lutea were dissected and luteal tissue submitted to a mechanical and enzymatic digestion with collagenase IV. The cell suspension was filtered and centrifuged and the cells obtained were diluted in 15 mL of DMEM-F12 supplemented media. Diluted cells were seeded in 3 culture flasks T25, staying in a controlled environment and changing the medium every 2 days. For the analysis and characterization, the cells were assessed by the Nile red staining to detect intracellular lipids, immunocytochemistry (ICC) for 3β-hydroxy steroid dehidrogenase (3β-HSD) and ELISA for P4 determination. We observed the presence of lipid intracellular droplets. Also, we observed an increase of P4 concentration at 48, 96 y 144 h of primary culture and almost all the cells were positive to the ICC evaluation for 3β-HSD, showing the steroidogenic capacity of the culture cells.

Keywords

• Coculture
• Corpus Luteum
• Porcine
• In Vitro Maturation