Isolation, Culture, and Osteogenic Induction of Cardiac Fibroblasts from Adult Mouse Hearts.

Cardiac fibroblasts (CFs) are the predominant non-myocyte population in the heart and play essential roles in maintaining myocardial architecture and mediating tissue remodeling and fibrosis. Beyond their well-established fibrogenic functions, CFs exhibit substantial phenotypic plasticity and can acquire osteogenic characteristics, potentially contributing to pathological cardiac calcification following severe myocardial injury. Despite growing interest in the osteogenic potential of CFs, standardized and reproducible methods for their isolation and osteogenic induction remain limited. Here, we present a detailed protocol for the isolation, culture, and in vitro osteogenic differentiation of primary CFs from adult mouse hearts. The procedure combines enzymatic digestion of ventricular tissue, differential plating to enrich CFs, and osteogenic induction using a defined differentiation medium. CF identity and purity are assessed by immunofluorescence staining, while osteogenic differentiation and calcification are evaluated by Alizarin Red S staining and quantitative calcium measurement. This protocol provides a reliable and reproducible in vitro platform for investigating CF osteogenic differentiation and elucidating the cellular and molecular mechanisms underlying pathological cardiac calcification.