Isolation, long-term culture, and characterization of rat pancreatic fibroblastoid/stellate cells

Investigation of pancreatic interstitial fibroblasts has proven difficult i n situ. We have established a method for the isolation of pancreatic fibrob lastoid/stellate cells by outgrowth from pancreatic tissue explanted into c ulture dishes. This technique gives a high yield of viable cells from small tissue samples. Outgrown fibroblastoid cells were established as a primary cell line and characterized during long-term culture. We investigated the development of stellate cell markers, i.e. fat storage, expression of desmi n, and ct-smooth muscle actin (alpha SMA), over weeks in culture. alpha SMA , investigated by indirect immunofluorescence staining and Western blot ana lysis, revealed a constant rise in expression during routine culture. After 13 passages, approximately 100% of cells were positive for alpha SMA expre ssion, indicating a myofibroblast type of differentiation in vitro.