Optimal conditions for the study of surface topography of granulosa cell cultures

The purpose of this study was to define optimal substrate conditions for permitting correlation of cell surface morphology with progesterone secretion by luteinized granulosa cells in vitro. Some 3-5 x 10(5) granulosa cells were obtained from preovulatory follicles of PMS-LH treated rats and plated upon carbon, poly-L-lysine or collagen-coated glass coverslips within plastic multiwells or upon Cytodex, Biosilon or poly-L-lysine coated Sephadex beads within siliconized glass roller tubes. Cells were maintained at 36.5 degrees C in 1.0 ml medium with and without 1.0 microgram purified prolactin (r, bPRL). Spent medium was assayed for progesterone by RIA. Cultures were processed for SEM observation of cell surface features. Results indicate that: 1) cells cultured in multiwells upon carbon, poly-L-lysine or glass grow as flattened monolayers but demonstrate considerable tearing distortion following routine processing for SEM, 2) cells cultured upon collagen-coated coverslips or microcarriers within roller tubes maintain a more spherical shape, exhibit the least distortion of surface fine structure and secrete increased amounts of progesterone in the presence of PRL. It is concluded that the use of Cytodex microcarriers for culturing granulosa cells permits optimal conditions for both progesterone secretion and maintenance of surface morphology.