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Activated pancreatic stellate cells (PSCs) are responsible for the fibrotic matrix of chronic pancreatitis and pancreatic cancer. In vitro protocols examining PSC biology have usually involved PSCs cultured on plastic, a non-physiological surface. However, PSCs cultured on physiological matrices e.g. MatrigelTM (normal basement membrane) and collagen (fibrotic pancreas), may have distinctly different behaviours compared to cells cultured on plastic. Therefore, we aimed to i) compare PSC gene expression after culture on plastic, MatrigelTM and collagen I; ii) validate the gene array data for transgelin, the most highly dysregulated gene in PSCs grown on activating versus non-activating matrices, at mRNA and protein levels; iii) examine the role of transgelin in PSC function; and iv) assess transgelin expression in human chronic pancreatitis sections. Culture of PSCs on different matrices significantly affected their gene expression pattern. 146, 619 and 432 genes respectively were differentially expressed (p < 0.001) in PSCs cultured on collagen I vs MatrigelTM, MatrigelTM vs plastic and collagen I vs plastic. The highest fold change (12.5 fold upregulation) in gene expression in cells on collagen I vs MatrigelTM, was observed for transgelin (an actin stress fibre associated protein). Transgelin was significantly increased in activated PSCs versus quiescent PSCs. Silencing transgelin expression decreased PSC proliferation and also reduced platelet derived growth factor (PDGF)-induced PSC migration. Notably, transgelin was highly expressed in chronic pancreatitis in stromal areas and peri-acinar spaces but was absent in acinar cells. These findings suggest that transgelin is a potentially useful target protein to modulate PSC function so as to ameliorate pancreatic fibrosis.