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UBC Theses and Dissertations

Application of macromolecular crowding in gingival fibroblast cultures Ramalingam, Rajesvaran


Macromolecular crowding (MMC) supplies synthetic polymeric crowders into cell culture media to better mimic macromolecular concentrations found in tissues. Their use may improve cell culture models for research and provide tools for regenerative therapy. We hypothesized that compared to traditional culture methods (nMMC), MMC can produce improved cultures and cell-derived matrices (CDMs) from cultured human gingival fibroblasts, a cell type associated with fast and scarless wound healing. Our specific aims were to compare fibroblast cultures generated with or without MMC, develop an improved decellularization method to generate CDMs from the cultures, and test if MMC can generate fibroblast cultures under reduced serum concentrations. To this end, fibroblasts were cultured under MMC (medium supplemented with Ficoll 70/400) and nMMC conditions supplemented with 10% or 2% FBS up to 14 days. Accumulation of proteins and specific ECM molecules in cultures and CDMs were studied by Bradford assay and by immunostaining and image analysis. Cell proliferation, morphology, and orientation were studied by image analysis and gene expression was analyzed by RT-qPCR. Results showed that MMC significantly reduced cell growth, but increased accumulation of collagen I and IV, cellular fibronectin, laminin 1, and tenascin C, while suppressing level of SPARC. Out of 62 genes studied, MMC significantly modulated expression of 20 genes compared to nMMC. Interestingly, MMC suppressed myofibroblast markers and promoted deposition of basement membrane molecules by fibroblasts. Decellularization with a novel method using latrunculin B followed by deoxycholate and DNase treatment produced CDMs that showed efficient removal of cellular elements. Molecular composition of cell cultures under MMC was replicated in CDMs, although decellularization caused some loss of ECM molecules. As expected, culture in low serum reduced cell growth and total protein accumulation. However, abundance of the above ECM molecules relative to cell numbers was significantly higher in MMC cultures and CDMs with 2% compared to 10% FBS. Culture in 2% FBS caused gene expression changes resembling a more profibrotic cell phenotype. Overall, the use of MMC and the novel decellularization technique rendered human gingival fibroblast cultures with distinct gene expression and CDMs with enhanced ECM molecule accumulation.

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