title: [Isolation, purification and primary culture of adult mouse cardiac fibroblasts].
abstract: Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.
authors.affiliation: Department of Vasculocardiology, Second Clinical Medical College, Yangzhou University, Yangzhou 225012, China.
authors.forename: Rujun
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Vasculocardiology, Second Clinical Medical College, Yangzhou University, Yangzhou 225012, China. *Corresponding author, E-mail: yungkzh@163.com.
authors.forename: Kaizheng
authors.initials: K
authors.lastname: Gong
authors.affiliation: Department of Vasculocardiology, Second Clinical Medical College, Yangzhou University, Yangzhou 225012, China.
authors.forename: Zhengang
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000166:cytology*