Isolation，culture and identification of human skin epidermal stem cellexosomes
10.19405/j.cnki.issn1000-1492.2023.02.009
- Author:
Biyou Li
1
;
Jie Ma
2
;
Qiyu Zhang
3
;
Huabing Zhang
4
;
Yunping Zhu
1
Author Information
1. Dept of Biochemistry and Molecular Biology，Basic Medical College，Anhui Medical University，Hefei 230032;National Center for Protein Sciences ( Beijing) ，the State Key Lab of Proteomics，Beijing Institute of Lifeomics，Beijing 102206
2. National Center for Protein Sciences ( Beijing) ，the State Key Lab of Proteomics，Beijing Institute of Lifeomics，Beijing 102206
3. Dept of Dermatology，Peking Union Medical College Hospital，Beijing 100730
4. Dept of Biochemistry and Molecular Biology，Basic Medical College，Anhui Medical University，Hefei 230032;
- Publication Type:Journal Article
- Keywords:
exosomes;
human epidermal stem cells;
ultracentrifugation;
regenerative medicine
- From:
Acta Universitatis Medicinalis Anhui
2023;58(2):224-229
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore method for isolating and culturing human epidermal stem cells ( EPSCs) in vitro and isolating and purifying epidermal stem cell exsomes ( EPSCs-Exo) by optimizing the technical process．
Method:Firstly，the improved separating enzyme was used to isolate the EPSCs derived from human skin tissue．Then，an improved serum-free culture medium and 10 specific factors were combined to construct optimized 2D culture medium which could stimulate the growth of EPSCs，promote the secretion of EPSCs-Exo，maintain the stemness and proliferation of EPSCs，and delay the differentiation and maturation of EPSCs． Further，the conditions of differential centrifugation was optimized，and then the human EPSCs-Exo was successfully extracted with high efficiency and high purity．
Results:The human skin tissue was confirmed with the expressions of markers for epidermal cells． EPSCs were verified with high expression levels of integrin-α6，integrin-β1，P63 and CK19 by immunofluorescence staining and Western blot． The nanoparticle tracking analysis results showed the particles separated for
EPSCs supernatant was saucepan with the detected diameter between 30 － 150 nm． The Western blot results showed the positive expression of membrane markers Tsg101，CD9 and CD63 and the negative expression of intracellular markers Calnexin and GAPDH．
Conclusion:The results show that the human-derived EPSCs have been successfully isolated and cultured in vitro，and the EPSCs-Exo have been successfully isolated and identified．
- Full text:2024071414445285695人皮肤表皮干细胞外泌体的分离培养与鉴定_李碧优.pdf
