Hormonally active long-term culture of human ovarian cells: initial characterization

At present, to our knowledge, there are no long-term hormonally active cultures of normal human stromal ovarian cells. This report describes a method of developing such a system. Normal human stromal ovarian cells from three patients were cultured in McCoy's 5A tissue culture medium at 37 degrees C, 5% CO2, 95% humidity. The cells rapidly acquired fibroblastic appearance and grew in monolayers. The cells were trypsinized and passaged weekly. Tissue culture medium was collected and assayed for progesterone. The cells were initially producing 33.2 +/- 2.64 ng/mg protein of progesterone. By the eighth to twelfth passage, however, progesterone was no longer detectable in the medium. When the cells of the 12th passage were incubated with cholesterol, progesterone production resumed (8.9 +/- 0.09 ng/mg protein). When pregnenolone was used as a substrate, progesterone production was also present (8.06 +/- 0.24 ng/mg protein). Moreover, in the cells incubated with pregnenolone, progesterone production could be stimulated by adding 50 ng/mL FSH, or 50 ng/mL insulin to the incubation medium. Progesterone content of the medium increased to 11.3 +/- 0.29 ng/mg protein and 10.97 +/- 0.54 ng/mg protein respectively (P less than .05). The cells were also able to convert androstenedione to estrone. When the cells were incubated with 3 mumol/L androstenedione, estrone content of the medium ranged from 318 +/- 13 to 382 +/- 14 pg/mg protein. This finding suggests that aromatase is present in cultured human ovarian cells. These cells have now been maintained for 12 passages. A hormonally active long-term culture of normal human ovarian cells could be a useful tool in studies of ovarian physiology.