Developing a Cell Culture Protocol to Induce Quiescence in Intestinal Fibroblasts Using Design of Experiments...

In vitro studies of intestinal fibrosis are confounded by spontaneous fibroblast activation on rigid substrates, hindering the investigation of the early events that initiate fibrosis. To address this challenge, we applied a design of experiments (DOE) framework to systematically evaluate extracellular matrix proteins and soluble factors that influence fibroblast phenotype. Using CCD-18Co colonic fibroblasts, we identified optimized conditions that reproducibly sustained a quiescent state. Validation experiments confirmed that under these conditions, fibroblasts remained spindle-shaped and viable, with reduced αSMA expression and decreased extracellular matrix secretion. These findings demonstrate that DOE can reveal non-linear interactions between microenvironmental cues while enabling the development of a reproducible culture protocol to maintain fibroblast quiescence. This accessible method will allow researchers to investigate the cellular and molecular signals that trigger intestinal fibrosis in a more physiologically relevant context.