Application and improvement of calcific heart valve interstitial cell culture method

Objective To compare the quality of cells obtained by two different methods:the modified two-stage collagenase digestion method versus the traditional enzyme digestion method.Methods Cells obtained by the modified two-stage collagenase digestion method were studied as the experiment group,and those obtained bv the traditional enzyme digestion method served as the control group.The time when the cells began to adhere and the quantity of adherent cells after 24 h were observed.Immunocytochemical staining and flow cytometry were performed to analyze the phenotype of calcific valvular interstitial cells.Results The quantity of adherent cells after 24 h in the experiment group was four times of that in the control group.The difference was statistically significant (P ＜ 0.05).Flow cytometry confirmed alive primary valve interstitial cells accounted for 94.39％ in the experimental group.Cell phenotype detection revealed 99.2％ were positive for vimentin,and 53.3％ were positive for α-smooth muscle actin (α-SMA).Conclusion In terms of the quantity of cells,cell viability and cell phenotype,the cells obtained by the modified two-stage collagenase digestion method are superior to those obtained by traditional enzyme digestion and can be used in vitro experiments. Key words: Calcified;  Valve interstitial cells;  Cell culture