Isolation，culture and identification of human skin epidermal stem cellexosomes
10.19405/j.cnki.issn1000-1492.2023.02.009
- Author:
Biyou Li
1
,
2
;
Jie Ma
2
;
Qiyu Zhang
3
;
Huabing Zhang
1
;
Yunping Zhu
1
,
2
Author Information
1. Dept of Biochemistry and Molecular Biology，Basic Medical College，Anhui Medical University，Hefei 230032
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
- 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
