Effect of hypotonic treatment on Sertoli cell purity and function in culture.

Commonly used enzymic methods for the isolation of rat Sertoli cells yield populations containing approximately 15% germ cells. Although the germ cells become eliminated after several media changes, they could interfere with the use of Sertoli cells for critical studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4) was found to eliminate most of the residual contaminating germ cells. The duration of this treatment varied from 1.0 to 10 min, depending on cell density in the culture, the degree of germ cell contamination, and the age of animals used for Sertoli cell isolation. In a study of 95% pure, 7-d Sertoli cell cultures, the hypotonic treatment did not alter the DNA or RNA content per dish or the incorporation of [3H]uridine into total and poly A+ RNA. Also, the hypotonic treatment did not alter specific Sertoli cell functions, i. e., secretion of Sertoli cell factor (inhibin) and stimulation of cAMP levels by follicle stimulating hormone in 2-d cultures. Androgen receptor concentration per dish was also not changed. Changes in several general metabolic parameters observed after hypotonic treatment of 2-d cultures were attributed primarily to loss of contaminating germ cells. Consequently, hypotonic treatment can be used to eliminate contaminating germ cells from the Sertoli cell cultures without apparent detrimental effects on a number of Sertoli cell biochemical parameters. This may be of considerable importance when the purity of Sertoli cells is critical for the interpretation of experimental data.