Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps.

Objective: To determine the degree of blood cell contamination in GC preparations; to assess techniques of GC purification; and to determine possible effects of contaminating cells and purification techniques on cultured GC in terms of steroid hormone production.

Design: Contamination of GC by white blood cells was assessed by Wright's stain and immunohistochemistry. Purification was attempted by: (1) Ficoll density centrifugation (to remove polymorphonuclear leukocytes [PML]); (2) incubation in tissue culture plastic dishes (to remove adherent monocyte/macrophages); and (3) incubation in the presence of high salt (to remove lymphocytes).

Results: Ficoll density centrifugation reduced PML to 2% of total cells, incubation in plastic dishes reduced monocyte/macrophage contamination from 6% to 7% down to less than 1%, and high-salt incubation reduced lymphocyte contamination from 10% to 12% down to 4%. Granulosa cells plated after preparation with addition of high salt showed increased progesterone production, which could not be entirely explained by the removal of contaminating lymphocytes.

Conclusion: These results indicate that the human GC culture system is more complex than is often assumed, and methods that remove a majority of these white blood cells are presented.