Influence of digestion time on the primary culture of cardimyocytes
- VernacularTitle:不同消化时间对原代心肌细胞的影响
- Author:
Shuangli QU
;
Xiaomin YANG
- Publication Type:Journal Article
- Keywords:
Primary culture;
Cardiomyocytes;
Digestion temperature;
Digestive enzyme;
Digestion time
- From:
Journal of Medical Postgraduates
2015;(6):564-568
- CountryChina
- Language:Chinese
-
Abstract:
Objective Primary myocardial cell culture , as an important technique for in vitro experiment , provides an essen-tial basis for the establishment of models of cardiovascular diseases .This study aimed to investigate the influence of digestion time on primary myocardial cell culture and to find some simple primary culture methods for obtaining myocardial cells with high survival rate , purity, and activity. Methods We used only one collagenase for repeated digestion of myocardial tissue for 8 and 10 minutes, re-spectively , and obtained high-purity cardiomyocytes by differential adhesion and Brdu inhibition .We observed the morphological chan-ges of the cardiomyocytes under the optical microscope , measured their vitality according to the beating frequency in different regions , and counted the living cells after typan blue staining. Finally, we established a model of myocyte hypertrophy for verification of cell activity and compared the cell surface area between the experimental ( angiotensinⅡ) and control ( 10%FBS DMEM [H +] medium ) groups. Results After cultured for 48 hours, the primary cardimyocytes in the 10 min group mostly extended pseudopodia , in-terwoven into a network and gradually forming cell clusters , with obviously powerful beating and contraction .After isolation and purifi-cation, the average survival rate of the cells was >90%, significantly higher in the 10 min group than in the 8 min ([95.4 ±0.8]%vs [93.0 ±0.8]%, P<0.05).The positive expression rate of cardiac α-actin was >95%in both of the groups.After cultured with angiotensinⅡfor 48 hours, the 10 min group showed a significantly increased level of atrial natriuretic peptide (ANP) (20.8 ±0.67 vs 15.8 ±0.48, P<0.05) and cell surface area ([34.77 ±8.43] μm2 vs [14.11 ±4.29] μm2, P<0.05) as compared with the 8 min group. Conclusion Primary culture of cardimyocytes with 8 min digestion is simple and time-saving, and what is most important , can obtain high-quality cardiomyocytes .
