Developing a protocol to counteract spontaneous in vitro activation of intestinal fibroblasts using design of experiments.

In vitro studies of intestinal fibrosis are confounded by spontaneous fibroblast activation on tissue culture polystyrene, hindering the investigation of early events that initiate fibrosis. Here, we present a statistically optimized culture protocol that suppresses fibroblast activation while preserving cell viability under standard culture conditions. Using a design of experiments (DOE) framework, we systematically evaluated combinations of extracellular matrix proteins and soluble factors to identify conditions that reduce myofibroblastic marker expression and extracellular matrix production. Fibroblasts cultured under optimized conditions remained spindle-shaped, exhibited low myofibroblastic marker expression, and showed reduced collagen and fibronectin secretion without evidence of cytotoxicity. The protocol was further validated in primary human colonic fibroblasts from both male and female donors, yielding consistent suppression of activation markers with high viability. This accessible and scalable approach provides a reproducible baseline for studying fibroblast activation and supports the use of DOE as a powerful strategy for defining microenvironmental conditions that regulate fibroblast behavior in vitro.