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.
D000818:Animals; D001769:Blood; D001792:Blood Platelets / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000502:physiology; D003470:Culture Media; D005260:Female; D005353:Fibronectins / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013552:Swine
D003470:Culture Media; D005353:Fibronectins; D012710:Serum Albumin, Bovine; D011374:Progesterone; D005640:Follicle Stimulating Hormone; D003094:Collagen