Development of a novel protocol for isolation and purification of human granulosa cells

Abstract

To develop an optimal method of isolation and purification of human granulosa cells from ovarian follicular fluid. Follicular fluid was collected from patients undergoing oocyte retrieval. A series of isolation and purification techniques was performed, involving density gradient centrifugation and use of different antibody-bead complexes. The highest percent yield of live purified granulosa cells came from density gradient centrifugation using sucrose polymer followed by positive selection of granulosa cells using primary antibody to MISRII and secondary antibody coupled to iron oxide beads. A novel protocol for granulosa cell purification has been developed yielding samples that are largely free of nondesirable cells. This protocol provides a purification solution, especially for patient samples that have significant RBC contamination.