Simultaneous separation of primary cardiomyocytes and cardiac fibroblasts from neonatal rats with density gradient centrifugation.
- Author:
Xi CHEN
1
;
Rui XU
1
;
Yi-Nan JIANG
2
;
Wei-Na ZHU
1
;
Yao-Hui WANG
3
Author Information
1. Henan Key Laboratory of Engineering Antibody Medicine, Henan International United Laboratory of Antibody Medicine, Key Laboratory of Cellular and Molecular Immunology, Medical College of Henan University, Kaifeng 475004, China.
2. Basic Medical College of Zhengzhou University, Zhengzhou 450001, China.
3. Henan Key Laboratory of Engineering Antibody Medicine, Henan International United Laboratory of Antibody Medicine, Key Laboratory of Cellular and Molecular Immunology, Medical College of Henan University, Kaifeng 475004, China. wangyaohui2001@sina.com.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Separation;
methods;
Cell Survival;
Centrifugation, Density Gradient;
Fibroblasts;
Heart Ventricles;
cytology;
Myocytes, Cardiac;
Povidone;
Rats;
Rats, Wistar;
Silicon Dioxide
- From:
Acta Physiologica Sinica
2015;67(4):423-430
- CountryChina
- Language:Chinese
-
Abstract:
To improve a fast and high-quality isolation method for culturing the primary cardiomyocyte and fibroblast in vitro, the neonatal Wistar rats were decapitated accordingly and left ventricles were isolated under the sterile condition. The ventricles were chopped and digested in the enzyme solution containing 0.5 mg/mL type II collagenase. During this process, the digesting time, frequency and stirring speed, centrifuging frequency and speed were strictly controlled. The cardiomyocytes were separated from the cardiac fibroblast by using the Percoll density gradient centrifugation. The cell viability was tested by staining with 0.2% trypan blue. The purity of cardiomyocytes and fibroblasts were determined by immunoflourescent staining with anti-cTnI, anti-Vimentin and anti-α-SMA antibodies. The results indicated that with this protocol, the viability and purity of cardiomyocytes were 92% and 95%. The automobile pulse of the adhered cardiomyocyte was visible. For fibroblasts, the cell viability and purity were 96% and 94%. Our results demonstrate that this advanced isolation method is reproducible, and can simultaneously produce high-quality primary cardiomyocytes and fibroblasts for the future study.
