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 immor talization of primary cultures of porcine granulosa cells. Three hundred st able cell lines were obtained from three independent immortalization trials . Two of these cell lines retained the steroidogenic capabilities character istic of granulosa cells, such as de novo synthesis of progesterone and con version of androstenedione into estradiol-17 beta. All the stable cell line s expressed the P450arom and 3 beta HSD genes, confirming their granulosa o rigin. Moreover, the steroidogenic stable granulosa cells also expressed St AR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells ove rcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures gr own in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal c alf serum (FCS). Medium 199 is a formulation richer in nutrients compared t o MEM or DM EM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spont aneous immortalization of granulosa cells may be facilitated by using a ric h culture formulation supplemented with low concentrations of serum deficie nt in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the e xpression of the steroidogenic genes. Therefore, we believe that they are u seful models to study the molecular mechanism involved in granulosa cell di fferentiation and steroidogenesis. (C) 2000 Wiley-Liss, Inc.