Primary culturing and biological identification of neonate rat myocardial cells / 解放军医学杂志

ABSTRACT

Objective To find out a way for culturing the myocardial cells of neonatal rats in vitro, and to study their biological characters. Methods Cardiomyocytes from the heart of 1-3 days old Sprague-Dawley rats were prepared by a modified protocol. Hearts were harvested and cut into pieces of about 1mm3 in size, and placed into cell culture bottles with nylon without pre-treatment of digestive enzyme. DMEM supplemented with 10% (v/v) FCS was used for culture, with thymidine (6mg/ml) added to inhibit fibroblast growth. Cells in monolayer were formed on the gauze and the bottom of the culture flask, and then cells were isolated by 0.25% trypsin digestion for 30 seconds. Cell suspension was transferred to 100cm2 cell culture flasks at a density of 104 cells /cm2 for identification, viability test and morphologic observation. The cells obtained were stained with monoclone anti-a-sarcomeric actinin and FITC to evaluate the purity of the myocardial cell preparation by flow cytometry, AO-PI fluorescein stain was used to evaluate the viability and Giemsa staining for examining the morphology of the myocardial cells. Results 24-48 hours after culture, the myocardial cells became adherent to bottle wall to form a sheet, and began to pulsate. The purity of myocardial cells in culture cell population was 94.13%, the ratio of viable myocardial cells was 95.3%, and 95%CI was 91.6%-99%. The output of myocytes from each neonatal heart was 3.3?106 and 95%CI is 2.1? 106- 4.5?106. Conclusion Neonatal myocytes culture in this way can generate large amount of viable single myocardial cells suitable for myocardial research in a short period.