Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells.

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.