Serum-free medium enhances growth and differentiation of cultured pig granulosa cells

We have developed new serum-free culture techniques for swine granulosa cells from immature (1-3 mm) follicles. These methods have allowed more detailed examination of factors regulating both replication and cytodifferentiation of these cells. For optimal replication, collagen-coated culture dishes and a highly supplemented nutrient medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-10), containing 5 micrograms/ml transferrin, 300 mU/ml insulin, 40 ng/ml hydrocortisone, 4 mg/ml BSA, and 2.5% (vol/vol) of a platelet extract (PE) was found to be essential. Cultures maintained in this serum-free complete medium (SFCM) grew to confluence and contained as many or more cells than replicate cultures maintained in 10% fetal calf serum (10% FCS) (e.g. SFCM: 1.89 +/- 0.17; 10% FCS: 1.12 +/- 0.02 cells per well X 10(-5) on day 6). In the absence of albumin, PE, or without collagen coating, the cell numbers were, respectively 4.5%, 9.8%, and 5.0% of that observed with complete SFCM. The mitogenic effect of the PE was due to heat-labile as well as heat-stable components and could not be replaced by platelet-derived growth factor. To evaluate cytodifferentiation, cells grown in SFCM were compared with those grown in 10% FCS with regard to progesterone secretion and FSH responsiveness. Basal progesterone levels were higher in SFCM at all stages in culture. FSH stimulated progesterone secretion in both 10% FCS and SFCM. However, FSH responsiveness was diminished after 4 days with 10% FCS, whereas cells in SFCM remained responsive for 10 days. Thus, this system seems to be highly suitable for the study of the regulation of growth and differentiation of granulosa cells.