title: [Primary culture and biological characteristics of cardiac fibroblasts of adult mice].
abstract: OBJECTIVE
  To establish a set of reliable methods of isolation,  culture and characterization of cardiac fibroblasts from adult mice.
  
  
  METHODS
  Fibroblasts were isolated from adult mice and cultured using combined trypsin-type II collagenase digestion method or tissue block  culture method. The cell growth of cardiac fibroblasts was observed under an  inverted  phase contrast  microscope. Cell viability of the different cell passages was evaluated by Trypan blue staining. The proliferation of cardiac fibroblasts was analyzed by   growth  curve,  MTT assay and DNA cell cycle analysis,  respectively,  to observe the effects of different culture media on the growth of cardiac fibroblasts. The expressions of vimentin,  fibronectin and discoidin domain receptor 2  (DDR2) on the cardiac fibroblasts were observed by immunofluorescence staining.
  
  
  RESULTS
  After 1 d culture with the enzyme digestion method,  the adherent cells were ellipse  observed under an inverted phase contrast     microscope and after 3 d,  cells  were  spindle  and    grew quickly. But only after 4 d isolation and culture by the block    culture method,  it was observed that cells grew from the border of tissues  and after 7 d,  the cells started to amplify gradually. The cell viability rate of both culture methods were more than 97%.  Growth    curve   of the third passage presented a "S" shape by both methods. MTT assay showed the optimal cell proliferation value on day 3 to day 5. The ratio of G0-G1 phase and S+G2+M phase were respectively 62.61% and 30.87% by enzyme digestion method,  and 69.24% and 28.05% by block  culture method. Log phase was found in cardiac fibroblasts cultured in HG/DMED supplemented with 10% fetal bovine serum. The cell colonies like a swirl were observed at the third passage by HE staining. Under a fluorescent microscope,  the cells were highly positive for the expressions of vimentin,  fibronectin and DDR2 which were the classical phenotype of the fibroblasts.
  
  
  CONCLUSIONS
  The cardiac fibroblasts could be effectively obtained by both enzyme digestion and block  culture methods. Compare to the block culture method,  the cells isolated by the combined enzyme digestion method expressed the higher levels of fibroblasts markers such as vimentin,  fibronectin and DDR2.
authors.affiliation: Department of Molecular Biology, Capital Medical University, Beijing, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xin
authors.forename: Sa
authors.initials: S
authors.lastname: Liu
authors.forename: Xiu-fang
authors.initials: XF
authors.lastname: Xu
authors.forename: Wen-bin
authors.initials: WB
authors.lastname: Li
authors.forename: Yi-min
authors.initials: YM
authors.lastname: Huang
authors.forename: Yi
authors.initials: Y
authors.lastname: Luo
authors.forename: Yu-jie
authors.initials: YJ
authors.lastname: Zhou
mesh_terms: D000375:Aging; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002469:Cell Separation; D003470:Culture Media; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000166:cytology*
chemical_list: D003470:Culture Media