A reliable and easy method to isolate a pure population of bovine granulosa cells from slaughterhouse ovaries for in vitro studies

In a growing follicle, granulosa cells (GCs) are the major cellular component and perform several important functions. GCs are an excellent choice for in vitro experimentation to understand their functions in vivo. However, the isolation of a pure fraction of GCs from slaughterhouse ovaries remains a challenge. The current study describes a simpler yet reliable method for isolating GCs from slaughterhouse ovaries of domestic animals. For this, bovine ovaries were collected from a nearby slaughterhouse and GCs were collected by aspiration method, one fraction of fresh GCs was snap frozen and stored at -80°C for further analysis and another fraction was used in culture purpose. The purity of GCs was determined by quantifying follicle stimulating hormone receptor (FSHR) and CYP17A1 transcripts and analyzing the cultured cells under a microscope. The viability of GCs ranged from 66 – 35 % depending on the time and media used in transportation-processing. The results showed that FSHR gene was highly expressed as evidenced by the presence of a strong band while CYP17A1 is completely absent in the GCs isolated using the current method. In addition, the characteristics of the cultured cells further confirm the purity of isolated GCs. The current method isolates a pure population of GCs from slaughterhouse ovary without any contamination of thecal cells.