Fascia explants enable real-time dynamic studies of wound fibroblast ancestry in living tissues

Aberrant wound healing is at the root of many fibrotic diseases and post-operative complications and understanding the mechanism will increase therapeutic options. Mammalian wound healing is the responsibility of specialized wound fibroblasts that originate from fascia connective tissues and drive inflammation, scar formation and tissue contraction. Monitoring the actions of these wound fibroblast precursors, in deep fascia, in real-time, has been challenging in laboratory animals. Here we isolate the fascia layer and culture it free-floating in cell culture dishes to analyze the causative events of inflammation, scarring and contraction. In this fascia explant (FE) system, wound fibroblast precursors can be systematically interrogated, in their natural living microenvironment. FEs are floating ~4 mm fascia tissue explants that can be cultured for up to seven days, to enable the study of and the process whereby fibroblast precursors differentiate to cause pro-inflammation, connective tissue contraction and scar-like tissue formation. The system is highly versatile and scalable and can be co-cultured with other cell types, e.g. immune cells, to perform chemical screening to interrogate the various facets of the wound repair process. This protocol offers researchers a valuable tool for exploring critical mechanisms of wound repair, enabling direct assessment of drugs on healing outcomes and advancing our understanding of fibroblast dynamics and interactions with their complex tissue environments.