Comparison of two methods for isolation and culture of human foreskin fibroblasts

The efficient acquisition and purification of fibroblasts as ideal seed cells are very important. For optimization of the isolation and culture of human foreskin fibroblasts (HFF), we compared the improved tissue culture method (ITCM) and the enzyme digestion method (EDM).

In ITCM, the skin tissue was digested with 0.1% Type II collagenase overnight at 4 ℃, the epidermis was separated from the dermis and digested again with 0.25% trypsin at room temperature for 15 min, and then the tissue block was attached to the culture dish. In EDM, the skin tissue was digested with 0.25% trypsin overnight at 4 ℃, the epidermis was separated from the dermis and digested with 0.1% Type II collagenase overnight at 4 ℃, the tissue block was filtered and squeezed together with the enzyme mixture, the filter was rinsed with medium containing fetal bovine serum, and the cell suspension was cultured. Both ITCM and EDM used 2 digestion enzymes, but the order, digestion time, and temperature of the 2 enzymes were different. The final inoculations of ITCM and EDM in the dishes for subsequent culture were tissue blocks and cell suspensions, respectively. In this study, HFF cells were isolated and cultured with ITCM and EDM, and the cell morphology was observed from Passage 0 to Passage 3 in the ITCM and EDM groups. The cell purity was identified by staining for vimentin, CD68, and Pan-keratin. The growth curves of Passage 3 were plotted to compare the proliferation ability of the 2 groups. Passage 3 HFF cells in the ITCM and EDM groups were irradiated with medium-wave ultraviolet (UVB) at an energy value of 120 mJ/cm² to establish a light damage model. The experiments were grouped into an UVB group and a control group (Control) according to the presence or absence of UVB irradiation. Platelet-poor plasma (PPP) was extracted by secondary centrifugation, and the HFF cells of ITCM and EDM groups were cultured in groups using complete medium containing different concentrations (0, 2.5%, 5.0%, and 10.0%) of PPP, and the proliferation of damaged cells was detected by cell counting kit-8 after 24 h of PPP incubation.

A large number of HFF could be observed in the ITCM group up to day 3, which was less affected by impurities; the observation of HFF morphology in the EDM group was affected by more impurities. By day 9, cells in both ITCM and EDM groups could be passaged; HFF isolated and cultured in vitro by the 2 methods showed long spindle-shaped, swirling growth. The positive rates of vimentin in the ITCM and EDM groups when HFF cells were cultured up to Passage 2 were significantly different [(97.36±0.76)% vs (99.4±0.56)%, P<0.01)]. The positive rates of CD68 were also significantly different [(70.8±0.46)% vs (78.37±0.75)%, P<0.01]. The expressions of pan-keratin in the ITCM group and the EDM group were positive and negative, respectively. There was no difference in vimentin and pan-keratin staining results between the ITCM group and the EDM group when HFF were cultured to Passage 3. The positive rates of CD68 between the ITCM group and the EDM group were significantly different [(74.73±1.37)% vs (85.27±2.63)%, P<0.001]. The proliferative capacity of HFF cells in Passage 3 was significantly higher in the EDM group than that in the ITCM group (P<0.05). After UVB (120 mJ/cm²) irradiation, HFFs procured by the 2 isolation methods showed damage. The damage repair test demonstrated that the 2.5% PPP+UVB irradiation group showed significantly higher repair competence than the other groups (all P<0.05).

In contrast with HFFs isolated via ITCM, HFF cells isolated by EDM have a faster purification rate and a stronger proliferative capacity. Therapy with PPP can moderately repair UVB-induced damage to HFFs. The results provide a theoretical basis for clinical treatment studies in the future.