Study On The Optimization Of Dermal Fibroblast Culture In Vitro And Its Promotion Of Wound Healing

Objective The purpose of this study was to explore the optimal method for digestion and culture of skin tissue in vitro,analyze the effects of different schemes on the number and activity of primary cells obtained,as well as inquiring into the effect of cell activity on wound healing in animal models.Methods Human skin tissues were taken and digested in vitro by three methods:collagenase + trypsin(Group A),single trypsin(Group B)and tissue adherence(Group C).The effects of different tissue digestion methods on cell acquisition quantity and cell activity were evaluated by the following experiments.Evaluation methods: 1)The time of cell dissociation from the tissue and the number of cells after the first passage were observed,and the growth curve was plotted;2)The main components of skin cells were identified by immunofluorescence detection of Vimentin expression;3)CCK8 method was used to detect cell activity and analyze the effects of different tissue digestion methods on cell activity;4)The expression of cell surface markers CD34,CD45 and CD44 was detected by flow cytometry to evaluate the effect of different tissue digestion methods on the proportion of stem cells in skin cells and fibroblast aging;5)The local acute wound surface model of nu/nu nude mice was established,and skin cells,aged skin cells and normal saline were applied to the wound surface every day.The effect of skin cell activity on the wound surface was verified by comparing the wound healing time and the amount of collagen in the healing tissues。Results 1)Skin tissues were digested by three methods: collagenase + trypsin(group A),trypsin(group B),and tissue adherent(group C),and fibroblasts were all successfully cultured.After three times of subculture,fibroblasts showed dominant growth,and the cells extended into spindle shape and polygon shape.Compared with the trypsin and tissue adherence methods,trypsin + collagenase digestion method was adopted to achieve the highest efficiency of cell dissociation from the tissue block,and adult fibroblasts were observed radially dissociating from the tissue block after 36 hours.After 48 h of cell culture,the degree of fusion reached more than 90 percent,and the cell yield obtained was the highest,with a statistically significant difference(P<0.01).2)Immunofluorescence was used to detect the expression of Vimentin in skin cells,and it was found that almost all cells expressed Vimentin.After HE staining,the cells were observed to be long spindle or polygon,with dark blue nuclei and light blue cytoplasm,indicating that the skin cells weremainly fibroblasts.3)CCK-8 method was used to analyze the activity of skin cells in different digestive groups.It was found that the proliferation activity of cells in group A was significantly higher than that in group B and C,and the difference was statistically significant(P<0.05).4)Flow cytometry was used to detect CD44,A marker on the cell surface,to evaluate the activity of skin cells.It was found that CD44 positive expression rate was the highest(100%)in vitro digestion of trypsin + collagenase(group A),followed by 98.6% in vitro digestion of trypsin(group B),and the lowest CD44 expression rate was91.5% in adherents(group C).The CD34 and CD45 expressions of the cells obtained by the three methods were negative.These results indicated that the cell activity in vitro obtained by different methods was different.The cell activity of trypsin + collagenase digestion method was the strongest,followed by trypsin,and the cell activity of adherent method was the lowest.5)The acute wound surface model of nude mice was successfully established.And the normal cultured primary skin cells,aged skin cells and NS control were applied to the wound surface every day,and the wound healing time of different treatment groups was recorded.It was found that the normal cultured primary skin cells could achieve wound healing in 4 days,while the NS control group needed 7 days to complete wound healing,and the aging fibroblast group needed 10 days to complete wound healing,with statistically significant differences(P<0.05 or P<0.01).HE staining was carried out on wound scar tissue after 14 days of wound healing.The results showed that in the treatment group of normal skin cells,the collagen content of wound healing tissue was high,the collagen fibers grew and thickened,and the alignment was more close to the normal skin tissue of non-wound skin,while in the treatment group of aging skin cells,the wound tissue was thinner,the dermis had the least collagen,and the collagen was sparse and disordered.The collagen content of scar skin in normal cell treatment group was higher than that in normal saline group and aging cell treatment group,and the difference was statistically significant(P <0.05 or P <0.01)Conclusion 1)This experiment successfully verified that the complex enzyme(trypsin +collagenase)in vitro digestion method can efficiently obtain skin primary cells;2)After immunofluorescence identification,the skin cells were mainly fibroblasts and contained a small number of stem cell-like mesenchymal cells;3)The number and activity of primary skin cells obtained by the complex enzyme digestion method was better than the traditional trypsin digestion method and the cell adherence method,but the activity of the primary cells obtained by the three tissue digestion methods in this experiment reached the peak atthe 4th and 5th generation,which was the best period for cell therapy,and then the cell activity gradually decreased with the increase of passage in vitro,The 4th and 5th generations cultured in vitro have stronger activity,which is a better window period for cell therapy.;4)The mouse wound healing model proved that the activity of skin cells is an important factor in cell therapy,and the aging skin cell population may not be beneficial to wound healing.