中国组织工程研究2017，Vol.21Issue(17)：2631-2637,7.DOI:10.3969/j.issn.2095-4344.2017.17.002
超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件
Optimization of repeated freeze-thaw and ultrasonication for collection of lysate of adipose-derived stem cells
摘要
Abstract
BACKGROUND: It has been believed mesenchymal stem cells (MSCs) play a role in treatment through paracrine mechanism. Various side effects such as embolism, tumorigenesis and immunological reaction caused by direct injection of MSCs can be avoided by extracting MSC lysate. However, there is a larger difference in current collection methods and standards of MSC lysate. OBJECTIVE: To compare repeated freeze-thaw and ultrasonication for the collection of lysate of MSCs. METHODS: Adipose-derived mesenchymal stem cells (ADMSCs) were isolated from the abdominal subcutaneous fat of healthy individuals, and purified with adherence screening method, followed by in vitro amplification using fetal bovine serum medium. The common surface makers of these cells were tested by flow cytometry (1×109, 2×109, 4×109/L). Repeated freeze-thaw and ultrasonication were employed for cell cytoclasis at three different densities respectively in saline and double distilled water, and a comprehensive comparison was performed on cytoclasis rate and the content of protein in cell lysate between the two methods. RESULTS AND CONCLUSION: (1) ADMSCs obtained from in vitro isolated human adipose tissue grew in a swirl or radial pattern with a homogenous size and neat arrangement. CD44, CD90, CD105 and other commonly used surface markers were highly expressed. (2) The study for optimization of lysate collection revealed that the higher cell density implicated a longer time for cell wall disruption and cytoclasis, as well as significantly increased cytoclasis rate. (3) BCA protein assay showed that the highest content of protein was obtained in saline solvent using ultrasonication method. Comprehensive analysis on the results leads to a conclusion that ultrasonication method with saline as the solvent is the optimized method for extraction of ADMSCs lysate, and the cell concentration of less than 4×109/L is recommended.关键词
干细胞/脂肪干细胞/间充质干细胞/脂肪间充质干细胞/超声/反复冻融/细胞裂解液/细胞治疗/细胞外膜泡分类
医药卫生引用本文复制引用
王俊怡,程明亮,金银鹏,李洪超,孟凌玉,李莉,王晓今,周荣,陈成伟,傅青春..超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件[J].中国组织工程研究,2017,21(17):2631-2637,7.基金项目
南京军区医学创新重大课题(14ZX01) （14ZX01）
中国肝炎防治基金—天晴肝病研究基金项目(TQGB20150104) the Medical Innovation Project of the Nanjing Military Region, No. 14ZX01 （TQGB20150104）
China Hepatitis Prevention and Treatment Foundation-Tian Qing Liver Research Fund Project, No. TQGB20150104 （）
