Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell

Summary We have carried out systematic studies to optimize and standardize methodology to isolate and culture the adult rat ventricular cardiac muscle cell. Four hearts were perfused simultaneously with a calcium-free medium containing collagenase. The ventricular tissue was then minced and further digested to liberate individual cells. Approximately 16 million rod-shaped muscle cells were obtained. The plating efficiency has been greatly improved by culturing the cells in a conditioned medium prepared from a rabbit corneal cell line. This medium also contained added fetal bovine serum, essential and nonessential amino acids, vitamins, insulin, transferrin, and 25 trace minerals. The culture flasks were precoated with rat-tail collagen. Fibroblast contamination was virtually eliminated by including cytosine arabinoside in the medium during the first 7 d of culture. After this time the cells could be cultured in the absence of serum in a chemically defined medium composed of MEM, vitamins, nonessential amino acids, and trace minerals. They continued to contract spontaneously and do well in this medium for at least 3 d thereafter. This improved methodology resulted in a reproducible culture system with improved plating efficiency. It provided a new and unique system to study the structure and function of the adult mammalian ventricular cardiac muscle cell. This investigation was supported by Grant HL 25873 from the National Institutes of Health, Bethesda, MD.