OVARIAN MESOTHELIAL AND EXTRAMESOTHELIAL CELLS IN INTERACTIVE CULTURE

The ovarian mesothelium (OM) represents the tissue of origin of ovaria n epithelial cancer. To gain insight into the regulation of this tissu e, OM organoids and submesothelial ovarian stromal cells (SC) were iso lated from New Zealand White rabbits by a stepwise tissue dispersal te chnique, while granulosa cells (GC) were aspirated from mature follicl es (14 +/- 4 groups/animal), OM and SC dispersal were sequentially acc omplished by: a) I-h incubation in collagenase type I (300 U/ml), gent le scraping of the ovarian surface, and 1 g sedimentation of OM organo ids (equivalent to 0.93 +/- 0.40 X 10(6) cells/animal) on 5% bovine se rum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%-300 U/ml) under periodical resuspension and gentle scraping of SC (1.40 +/ - 0.25 X 10(6)/animal) from OM-denuded ovaries. After a week-long in v itro expansion, OM cells (OMC) were cultured alone and with SC or GC w ithin monocameral vessels or bicameral transfilter vessels in serumles s, fibronectinrich (4 mu g/ml) HL-1 medium. After 7 d of contact cell- cell interaction, cytokeratin-positive OMC became surrounded by fibrob lastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly po sitive GC. Filter-bound OMC humorally interacting with underlying SC o r GC displayed a biphasic, epithelioid and spindle, morphology with un iversal cytokeratin expression. Bromo-2'-deoxyuridine (BrdU) immunoper oxidase revealed mean cell proliferation indices of 14.88% for OMC cul tured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monoc ameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vess els over GC or SC, respectively. This model provides an experimental t ool for investigating the unexplored role of stromal-mesothelial inter action in OM pathobiology.