Establishing Primary Cultures of Adult Mouse Cardiac Fibroblasts: A Comparative Evaluation of Three Experimental Protocols.

Cardiac fibroblasts (cFbs) are important in vitro models for studying myocardial fibrosis, extracellular matrix homeostasis, and tissue remodeling. However, establishing primary cultures from adult mouse hearts remains technically demanding due to the limited proliferative capacity of freshly isolated cells, their susceptibility to enzymatic and mechanical stress during tissue dissociation, and the resulting difficulty in obtaining sufficient numbers of viable fibroblasts for downstream experiments. Even small variations in digestion and early culture conditions can affect cell recovery, purity, and expansion, highlighting the need for optimized and reproducible isolation protocols. Here, we compared three enzymatic strategies for establishing primary adult mouse cardiac fibroblast cultures: sequential digestion with collagenase I and trypsin, retrograde coronary perfusion with collagenase II, and continuous digestion with Liberase DH. The three protocols were evaluated by assessing post-isolation cell viability, the composition of recovered non-myocyte populations by flow cytometry, and the ability of isolated cells to establish and expand during the first five days of culture. Retrograde coronary perfusion generated viable fibroblasts but yielded relatively few cells and is most advantageous when fibroblasts are isolated simultaneously with cardiomyocytes from the same heart. Liberase DH digestion recovered a broader spectrum of non-myocyte populations, making it suitable for immediate phenotypic analyses, although it produced less efficient fibroblast expansion in culture. Sequential collagenase I/trypsin digestion showed the best overall performance, with low mortality, negligible CD31+ endothelial contamination, and robust expansion into confluent spindle-shaped monolayers within five days. Overall, these findings identify sequential collagenase I/trypsin digestion as an effective and reproducible approach for establishing viable adult mouse cardiac fibroblast cultures for downstream functional and molecular studies.