1. Human tissue was derived from atrial or ventricular biopsy specimens belonging to patients undergoing heart surgery.
2. Isolated myocardial tissue was cut into 1- to 2-mm3 pieces.
3. Isolated myocardial tissue was washed with Ca2+ -Mg2+–free phosphate-buffered solution (PBS).
4. Isolated myocardial tissue was digested three times for 5 minutes at 37°C with 0.2% trypsin and 0.1% collagenase IV.
5. The
obtained cells were discarded, and the remaining tissue fragments
washed with primary cell culture medium and primary cell culture
supplemented with 10% fetal calf serum, 100 U/mL penicillin G, 100 µg/mL
streptomycin, and 2 mmol/L L-glutamine were cultured at 37°C and 5% CO2.
6. Twenty-four hours later, the medium was again replaced with primary cell medium and supplement.