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Human ovarian surface epithelial cells in culture : characterization and matrix interrelationships

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Title: Human ovarian surface epithelial cells in culture : characterization and matrix interrelationships
Author: Kruk, Patricia Ann
Degree Doctor of Philosophy - PhD
Program Cell and Development Biology
Copyright Date: 1992
Abstract: The human ovarian surface epithelium (HOSE) is thought to give rise to over 85% of human ovarian carcinomas. In spite of its clinical importance, experimental systems for investigations of HOSE are lacking and available culture methods have yielded limited success. In this study, HOSE from normal ovarian biopsy specimens was used: 1) to improve and simplify the methodology for HOSE culture; 2) to further characterize HOSE cells; 3) to characterize and isolate an ovarian-derived extracellular matrix (ECM); and 4) to study the interactions between cultured HOSE cells and ECM as a means to examine the dynamic, pleomorphic, and morphogenetic nature of HOSE. Improved tissue culture techniques, developed during the course of this study, allowed for a better method for the culture of HOSE cells and the decontamination of mold-infected cultures. An improved explantation method was developed which takes advantage of the tenuous attachment of HOSE to underlying tissues: the surface epithelium is scraped off the ovarian surface, generating epithelial fragments which produce monolayers in culture, with little contamination by other cell types. The scrape method is superior to the explant method previously described in terms of speed, simplicity, higher purity of cultures, and increased cell yield. This improved culture system provided conditions for the in depth studies of HOSE described herein. A technique for the elimination of mold contamination by a simple one-step Percoll gradient centrifugation was developed, using the fungus Ciadosporium as a prototype and intentionally infected cell lines. Centrifugation in Percoll did not affect cell morphology, growth, or the cells’ ability to produce ECM. Percoll decontamination may be a generally applicable technique for decontamination of mold infected cultures. Characterization of HOSE cells in culture showed that they are variably positive for mucin and contain lipid, vimentin, and keratin subtypes #7, 8, 1 8 and 19 like their in vivo counterparts. Additionally, HOSE cells produce basement membrane and stromal ECM components as demonstrated by the presence of laminin and collagen types I, Ill, and IV in HOSE cultures. ECM derived from rat ovarian surface epithelial cell cultures (ROSE 1 99-ECM) also consisted of basement membrane and stromal matrix components as shown by the presence of laminin, fibronectin, and collagen types I and Ill. This predominantly fibrillar ECM supports the attachment, spreading, and even growth of several cell types. Although HOSE cells remain epithelial, but dispersed throughout the ROSE 1 99-ECM, HOSE cells plated on a combination of ROSE 1 99-ECM and collagen gel contract the matrices into organoids providing evidence that HOSE cells are capable of physically remodelling ECM. Examination of HOSE-ECM interactions revealed that substrata influence HOSE morphology, growth, and proteolytic activities. On plastic and fibrin clots, HOSE cells formed epithelial monolayers while they are spindle-shaped on rat tail tendonderived collagen gels. On Matrigel, the cells form aggregates that penetrate and lyse the gel. HOSE cells grow rapidly on plastic, remain stationary on collagen gels and fibrin clots and eventually decrease in number when maintained on Matrigel. The different morphologic phenotypes did not translate into the expression of different integrins at the cell surfaces except for collagen gels. With the exception of cells on collagen gels, HOSE surveyed for various integrins express receptors for vitronectin (VNR), collagen, laminin, and fibronectin (VLA-2,-3,-5, and 131), but lack 134 and VLA-6, a laminin receptor. There appears to be a downregulation of integrins when cells are maintained on collagen gels. Conditioned medium of HOSE cultures on these substrata contains neutral proteases: 1.) chymotrypsin-like and elastase-like activities that are inversely related to growth; and 2.) a 30 KD and a 42 KD gelatinase. These results suggest that normal HOSE cells play an active role in ECM remodelling, that is, its deposition, degradation, and physical reorganization. These capabilities may be important for normal ovarian development and normal adult functioning such as ovulation. Furthermore, while proteolytic activities and invasiveness are characteristics of the malignant phenotype, they also appear to be part of the normal HOSE phenotype. With regards to why HOSE is a preferred site of cancer development, it is perhaps aberrations of these functions that contribute to pathological states.
URI: http://hdl.handle.net/2429/3287
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]

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