A SIMPLIFIED SERUM-FREE METHOD FOR PREPARATION AND CULTIVATION OF HUMAN GRANULOSA-LUTEAL CELLS

A simplified method for the preparation and long-term cultivation of g ranulosa-luteal cells in serum-free medium is described, The cells wer e harvested from women undergoing in-vitro fertilization, enriched by sedimentation and dissociated by enzymatic treatment. We demonstrated, by introducing a synthetic serum replacement (SSR2(TM)), that these p rimary cell cultures cultivated in monolayers on an extracellular matr ix may be used in experiments exceeding 7 days with low cell loss and cell death, No adverse effect on progesterone production was found, Th ere was a high diversity in progesterone production between cells from individual patients. After several days in culture, the cells were ch allenged with human chorionic gonadotrophin which revived the rapidly decreasing progesterone production, We were unable to demonstrate an i ncrease in cell number after 7 days of cultivation when the cells were grown in medium supplemented with either serum or SSR2(TM). The mitog ens epidermal growth factor and basic fibroblast growth factor had no influence on proliferation, We also found that the present method prev ents leukocyte contamination in the granulosa-luteal cell cultures, Co mpared with the common method based on the enrichment of granulosa-lut eal cells on a density gradient (Ficoll(TM)/Percoll(TM)), this method saves time, labour and expense, in addition to augmenting purity.