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.