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

Abstract

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.

Keywords: Cardiac fibroblasts; Cardiac fibrosis; Fibrosis; Primary culture; TGF-β1.

Figure 1.. Diagrammatic representation of the major steps involved in the isolation and culture of primary cardiac fibroblasts from murine pup hearts.

The figure was designed using images adapted from Servier Medical Art by Servier. Original images are licensed under a Creative Commons Attribution 3.0 Unported License.

Figure 2.. Cultured primary cardiac fibroblasts treated with TGF-β1.

(A) Bright-field image of attached neonatal rat cardiac fibroblasts on poly-L-lysine-coated dishes. Scale bar: 500 μm. (B) Bright-field image of primary cardiac fibroblasts with and without TGF-β1 treatment for 24 h. Scale bar: 100 μm. (C) Primary cardiac fibroblasts were treated with TGF-β1 for 24 h and stained with α-smooth muscle actin (α-SMA) as a marker for myofibroblasts, and fibronectin 1 (FN1), collagen 1a (Col1a), and Collagen 3a (Col3a) as fibrotic markers. Hoechst 33342 was used to stain the nuclei blue. Scale bar: 50 μm.

Figure 3.. Molecular analysis of cardiac fibroblasts treated with TGF-β1.

(A) Western blot analysis of neonatal rat cardiac fibroblasts upon 24 h of TGF-β1 treatment. Α-SMA was used as a myofibroblast marker, and FN1, Col1a, and Col3a were used as fibrotic markers. Actin was used as the loading control. (B) Relative mRNA levels of α-SMA, FN1, Col1a, and Col3a in neonatal rat primary cardiac fibroblasts treated with TGF-β1 for 24 h. Actin has been used for normalization (as an internal control). Student’s t-test used for statistical analysis (*p ≤ 0.05). Data presented as mean ± S.D.