组织工程与重建外科杂志2025，Vol.21Issue(5)：452-457,6.DOI:10.3969/j.issn.1673-0364.2025.05.007
一种连续高效的人皮肤成纤维细胞培养方法
A continuous and high-efficiency method for culturing human skin fibroblasts
摘要
Abstract
Objective To enhance the isolation efficiency of human skin fibroblasts(hSFs),minimize tissue explant consumption,and shorten the cell culture period.Methods HSFs were isolated using a modified explant culture method(15-min room-temperature trypsin digestion followed by explant culture).After passaging,adherent cells were further expanded using a repeated plating technique(re-seeding tissue explants into new culture dishes).Cells isolated via the modified explant method were designated hSFs-1,while those obtained from the 3rd and 5th replating cycles were termed hSFs-3 and hSFs-5,respectively.Cell proliferation was assessed using CCK-8 assays for hSFs-1,hSFs-3,and hSFs-5.Vimentin expression was evaluated via immunofluorescence,and its protein levels were quantified by Western blot.Cells at passage 1,2,3,and 5 were labeled P1,P2,P3,and P5.Cytokeratin 5(CK5)expression in P1,P2,P3,and P5 cells was analyzed by Western blot.Results hSFs were successfully isolated from tissue explants and subcultured.The repeated adherence method yielded additional hSFs with no significant differences in proliferation capacity among hSFs-1,hSFs-3,and hSFs-5(P>0.05).Vimentin expression was consistent across all groups(P>0.05).Immunofluorescence confirmed the spindle-or star-shaped morphology of hSFs and their universal Vimentin positivity.Notably,CK5 protein levels significantly decreased in P3 and P5 cells(P<0.05).Conclusion After using the modified explant culture method,subsequent application of the repeated plating technique enabled continuous cultivation of hSFs.No significant differences were observed in cell morphology,proliferative capacity,or protein expression between cells isolated by the repeated plating technique and the modified explant culture method.After 3-5 passages,the fibroblasts were significantly purified.关键词
原代培养/人皮肤成纤维细胞/改良组织块培养/重复贴壁法Key words
Primary culture/Human skin fibroblasts/Modified explant culture method/Repeated adherence method分类
生物科学引用本文复制引用
胡金宏,刘鹏超,梁亚坤,翟天慧,樊磊强..一种连续高效的人皮肤成纤维细胞培养方法[J].组织工程与重建外科杂志,2025,21(5):452-457,6.基金项目
2024年河北省在读研究生创新能力培养资助项目(编号:CXZZSS2024121) （编号:CXZZSS2024121）
2024年度承德市应用技术研究与开发暨可持续发展议程创新示范区专项科技计划项目(编号:202404B076). （编号:202404B076）
