Ovarian Mesothelial and Extramesothelial Cells in Interactive Culture

• Published in: In vitro cellular & developmental biology. Animal Vol. 31; no. 4; pp. 300 - 309
• Main Authors: Gloria Giacomini, Nicosia, Santo V., Beatriz O. Saunders, Caroline Fultz, Sun, X., Valerio M. Jasonni
• Format: Journal Article
• Language: English
• Published: Germany Society for In Vitro Biology 01-04-1995
• Subjects: Animals; Cell culture techniques; Cell growth; Cell Separation; Cells, Cultured; Cellular Models; Cultured cells; Epithelial Cells; Epithelium; Female; Granulosa cells; Granulosa Cells - cytology; Interstitial cells; Keratins; Ovaries; Ovary - cytology; Rabbits; Stromal cells; Stromal Cells - cytology; Vimentin
• ISSN: 1071-2690; 1543-706X
• DOI: 10.1007/BF02634005

The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles ($14 \pm 4 groups/animal$). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to$0.93 \pm 0.40 \times 10^6 cells/animal$) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%-300 U/ml) under periodical resuspension and gentle scraping of SC ($1.40 \pm 0.25 \times 10^6/animal$) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich ($4\mug/ml$) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentinweakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2'-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.