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RH Wheelock, P Russell; Assessment of Primary Cell Cultures Isolated from Human Trabecular Meshwork as a Model for Studying Glucocorticoid-mediated Gene Expression Changes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1026.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the suitability of primary cell cultures derived from human trabecular meshwork (TM) for gene expression analysis and identify molecular pathways that might play a role in glucocorticoid-triggered pathological events leading to open angle glaucoma. Methods: TM cells were isolated from whole human eyes obtained from the National Disease Research Interchange within 48 hours of enucleation. Cells were maintained in 10% fetal bovine serum, and initial outgrowth cultures from dissected TM were allowed to grow for 4-8 weeks prior to initial passage by trypsinization. Confluent cultures within five passages of initial isolation were treated with 100 nM dexamethasone or vehicle for 9 days. Total RNA was extracted and cDNA microarray analysis was performed on samples from 6 different primary cell isolates using the Micromax© system (Perkin Elmer Life Sciences). Possible glucocorticoid-regulated genes were identified and selected for confirmation by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Results: TM cultures isolated by our methods shared common characteristics with those described in the scientific literature, as well as with cultures obtained from other investigators. However, close examination revealed a variety of morphologically distinct cell subtypes that could be discerned in different culture isolates and which persisted in progressive passages. Microarray analysis indicated that our isolates showed gene expression characteristics of TM and endothelial cells. TM-associated genes previously reported to be up-regulated by glucocorticoid exposure, including myocilin, anti-alphachymotrypsin-1, and αB-crystallin were among the most dramatically and consistently modulated genes on the 2400 gene array. However, the detection and magnitude of changes in expression of these genes varied considerably from isolate to isolate. Conclusion: A variety of potentially different cell types can be isolated by commonly used methods for human TM cell culture. Whether these constituents all derive from the TM or include cell contaminants from neighboring tissues, their re-occurrence in primary cell isolates indicates that they may commonly contribute to the variability that we have observed in our microarray studies on chronic glucocorticoid effects on human TM cells and perhaps underscore the need for detailed molecular characterization of TM-derived primary cells used in gene expression studies.
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