Effect of rotating cell culture system on proliferation and stemness maintenance of epidermal stem cells in the elderly

Zhang Min; Dong Xixi; Huang Meng; Wang Chaoxiang; Zhang Luyue; Cao Junkai

doi:10.12307/2025.079

Abstract：BACKGROUND:Due to the aging population and the increase in elderly diseases,research and utilization of epidermal stem cells in the elderly have become increasingly important.However,due to the weak proliferation and drying ability of epidermal stem cells in the elderly,their application in biomedical and clinical research is limited. OBJECTIVE:To investigate the ability of rotating cell culture system to promote the proliferation and maintenance of epidermal stem cells in elderly individuals. METHODS:Epidermal stem cells were obtained from children (7-12 years old) and the elderly (over 60 years old) using traditional planar culture methods.After identification by cell morphology and cell immunofluorescence,the differences in proliferation ability of epidermal stem cells between the children and the elderly were analyzed by flow cytometry,CCK-8 assay,and cell clone formation assay.Elderly epidermal stem cells were cultured using a rotating cell culture system and 3D TableTrix® microcarriers.The cell proliferation and expression of stemness markers were detected by Live/Dead staining,cell doubling efficiency,cell doubling time,and real-time quantitative polymerase chain reaction. RESULTS AND CONCLUSION:(1) Under planar culture conditions,the children group had stronger proliferative ability than the elderly group.(2) Compared with planar culture,the dynamic cultivation of elderly epidermal stem cells using a rotating cell culture system had a higher population doubling (P<0.01) and a shorter population doubling time (P<0.01).(3) The dynamic cultivation of rotating cell culture system promoted the self-assembly of elderly epidermal stem cells to form 3D human epidermal stem cell spheres based on microcarrier scaffolds,which had similar epidermal stem cell proliferation as the children group.(4) After dynamic culture,the expression of stemness marker genes (ITGA6 and ITGB1),basal cell marker genes (K5) and differentiation marker genes (K10 and K1) of elderly epidermal stem cells reached the same level as that of children.The results showed that the dynamic cultivation of rotating cell culture system can not only improve the efficiency of elderly epidermal stem cell culture,promote cell proliferation and self-assembly into 3D cell spheres,but also maintain a certain stemness of elderly epidermal stem cells without complete terminal differentiation.