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

doi:10.12307/2025.079

Abstract

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.

Key words: human epidermal stem cell, the elderly, rotating cell culture system, microcarriers, 3D dynamics culture, stemness, proliferation

CLC Number:

Zhang Min, Dong Xixi, Huang Meng, Wang Chaoxiang, Zhang Luyue, Cao Junkai. Effect of rotating cell culture system on proliferation and stemness maintenance of epidermal stem cells in the elderly[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(13): 2698-2705.

Figures/Tables 4

2.1 儿童和老年人表皮干细胞的形态学观察和细胞免疫荧光鉴定结果如图1A所示，儿童组和老年组表皮干细胞形态无明显差异，均呈边缘透亮的多边形细胞形态，细胞核较大，细胞排列紧密呈铺路石样。在老年组中小而紧密的细胞之间，夹杂着一些大而扁平的细胞。原代细胞长至融合时，对儿童组和老年组表皮干细胞的特异性标记物CK19、Integrin-β1和p63进行细胞免疫荧光鉴定，如图1B所示，3个标记物均强表达，且儿童组几乎全部表达。结合细胞学形态观察，实验成功分离培养了老年人和儿童表皮干细胞。 2.2 平面培养的儿童和老年人表皮干细胞的增殖情况如图2A所示，儿童组第1代细胞中有65.3%表达CD49fbri/CD71dim，29.8%表达CD49fbri/CD71bri，老年组则有31.7%表达CD49fbri/CD71dim，63.7%表达CD49fbri/CD71bri。经统计分析，两组细胞CD49fbri/CD71dim表达率差异有显著性意义(P < 0.01)，见图2B。以上结果表明，传代后细胞的干性表达有所降低，但儿童组仍较老年组具有更强的增殖能力，老年组增殖能力较弱。如图2C所示，CCK-8检测发现儿童组第1代表皮干细胞在第1，3，5，7天时的增殖能力均高于老年组，差异有显著性意义(P < 0.05)。两组第1代细胞在接种相同细胞数的情况下，儿童组可在7 d达到约90%的细胞融合，而老年组则需12 d，细胞增殖速度明显较慢。 2.3 平面培养的儿童和老年人表皮干细胞克隆形成能力为了进一步检验两组细胞增殖能力的差异，对儿童组第3代表皮干细胞和老年组第1代表皮干细胞进行了细胞克隆形成实验。如图3所示，老年组表皮干细胞形成的细胞克隆小而稀疏，而儿童组在第3代仍可形成远大于老年人的细胞克隆。经统计分析，儿童组第3代表皮干细胞克隆形成效率大于老年组第1代表皮干细胞，具有更强的细胞克隆能力。"

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