Isolation of Adult Mouse Cardiac Fibroblasts

Fibroblasts are dynamic cells of mesenchymal origin that regulate tissue homeostasis, extracellular matrix production, and acute wound healing. Fibroblasts respond to tissue injury and inflammation by differentiating into myofibroblasts and secreting extracellular matrix proteins. Fibroblasts are the principal mediators of the fibrotic response in all tissues and organs. Adult primary fibroblasts represent an essential tool for in vitro studies. Although they lack surface markers, fibroblasts are relatively easy to obtain and culture; primary fibroblasts are sensitive heterogeneous cell subpopulations with limited expansion potential and increased differentiation capacity. Adult primary fibroblasts fail to maintain an undifferentiated state ex vivo for long periods and quickly differentiate into myofibroblasts in culture, which necessitates the utilization of these cells either directly after isolation or after a few passages. Herein, we describe a detailed protocol for enzymatic isolation of primary cardiac fibroblasts from adult mouse hearts and their culture and expansion in a serum-containing culture medium. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Preparation of primary adult cardiac fibroblasts for culture and single-cell analysis.