A simplified serum-free method for preparation and cultivation of human granulosa-luteal cells

A simplified method for the preparation and long-term cultivation ofgranulosa-luteal cells in serum-free medium is described. The cells wereharvested from women undergoing in-vitro fertilization, enriched bysedimentation and dissociated by enzymatic treatment. We demonstrated, byintroducing a synthetic serum replacement (SSR2), that these primary cellcultures cultivated in monolayers on an extracellular matrix may be used inexperiments exceeding 7 days with low cell loss and cell death. No adverseeffect on progesterone production was found. There was a high diversity inprogesterone production between cells from individual patients. Afterseveral days in culture, the cells were challenged with human chorionicgonadotrophin which revived the rapidly decreasing progesterone production.We were unable to demonstrate an increase in cell number after 7 days ofcultivation when the cells were grown in medium supplemented with eitherserum or SSR2. The mitogens epidermal growth factor and basic fibroblastgrowth factor had no influence on proliferation. We also found that thepresent method prevents leukocyte contamination in the granulosa-lutealcell cultures. Compared with the common method based on the enrichment ofgranulosa-luteal cells on a density gradient (Ficoll/Percoll), this methodsaves time, labour and expense, in addition to augmenting purity.