Improvement of Method for Isolation of Primary Myocardial Cells in Neonatal Rats

Li Yang; Wang Li; Wang Xiaohui; Bai Feng; Liu Huirong

doi:10.16289/j.cnki.1002-0837.2016.03.002

Abstract：Objective To improve the traditional isolation method of primary cardiomyocytes in neonatal rats to get neonatal rat cardiomyocytes with good activity,high purity and good reactivity.Methods Neonatal Wistar rats myocardial tissue within 24 h were obtained with broken head-open chest method,then digested using 0.25％ trypsin without EDTA mixing collagenase Ⅱ,as well as using temperature magnetic stirrer and a rotor in tissue suspension during digestion process.Cell shape observed under inverted phase contrast microscope every day.Using cardiac cell-specific protein α-Sarcomeric actin (αt-actin) monoclonal antibody,the cultured myocardial cells were identified with immunofluorescence.Primary neonatal rat cardiomyocytes were treated with clinical common drugs,isoprenaline and noradrenalin,then cell responses were observed.Results Myocardial cells were confirmed for some cells beating after 24 h culturation and gradually expanded after 48 h,through 20 d continuous observation,no significant changes of cardiomyocytes beat frequencies and shapes were found from 3 rd to 14 th day.The number of red blood cells in the primary cardiac cells obtained with the method of the broken head-open chest was significantly reduced.Immunofluorescence identification showed the purity of primary neonatal rat cardiomyocytes achieved with new method was up to 95％.In addition,isoprenaline and noradrenalin,could make cell response sensitively.Conclusion Primary neonatal rat myocardial cells isolation with the broken head-open chest method are more sensitive and active,and of higher purity,it can meet the requirements of most experiments.