Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis

• Published in: Bio-protocol Vol. 13; no. 4; p. e4616
• Main Authors: Kumar, Shweta, Nagesh, Dimple, Ramasubbu, Venketsubbu, Prabhashankar, Arathi, Sundaresan, N. Ravi
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 20.02.2023; Bio-protocol LLC
• Subjects: Biology; Methods; Primary culture; Cardiac fibrosis; Cardiac fibroblasts; TGF-β1; Fibrosis
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.4616

Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth.