Endocrinol Metab > Volume 25(2); 2010 > Article
Endocrinology and Metabolism 2010;25(2):103-109.
DOI: https://doi.org/10.3803/EnM.2010.25.2.103    Published online June 1, 2010.
Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method.
Min Kyung Kim, Yong Soon Park, Hee Soon Park, Jung Mook Choi, Won Jun Kim, Se Eun Park, Eun Jung Rhee, Cheol Young Park, Won Young Lee, Ki Won Oh, Sung Woo Park, Sun Woo Kim, Kwang Sik Suh, Jeong Taek Woo
1Department of Food and Nutrition, Hanyang University, Seoul, Korea.
2Diabetes Research Institute, Kangbuk Samsung Medical Center, Seoul, Korea.
3Department of Internal Medicine, Kangbuk Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. cydoctor@chol.com
4Department of Endocrinology-Metabolism, College of Medicine, Kyunghee University, Seoul, Korea.
Abstract
BACKGROUND
Adipose tissues include multipotent cells, the same as bone marrow-derived mesenchymal stem cells. The stromal vascular fractions (SVFs) from adipose tissues represent a heterogeneous cell population. The purpose of this study was to isolate and purify adipose-derived stem cells (ASCs) in SVFs by the density gradient method. METHODS: SVFs were extracted from the subcutaneous, epididymal, mesenteric and retroperitoneal adipose tissue of 8 weeks old male Sprague-Dawley rats (n = 15) and these were separated into 4 layers according to a Nycodenz gradient (Fx-1: < 11%, Fx-2: 11-13%, Fx-3: 13-19% and Fx-4: 19-30%). The post-confluent SVFs were cultured in adipogenic medium for 2 days, in insulin medium for 2 days and in 10% fetal bovine serum medium for 5 days. To observe lipid droplets in SVFs, we performed Oil Red O staining. RESLTS: The SVFs' cellular fractions (Fx-1, Fx-2, Fx-3 and Fx-4) were isolated by density gradient centrifugation from the adipose tissues of rats. The SVFs extracted to fraction 3 (Fx-3) had the most abundant cells compared to that of the other fractions. However fraction 1 (Fx-1) or 2 (Fx-2) had a superior ability to make lipid droplets. The adipogenic differentiation of Fx-1 or 2 was higher than that of the unfractionated cells. The SVFs extracted from retroperitoneal adipose tissue had the highest efficiency for adipogenic differentiation, whereas the SVFs from mesenteric adipose tissue did not differentiate. CONCLUSION: This density gradient fractionated method leads to efficient isolation and purification of cells with the characteristics of ASCs.
Key Words: Rat adipose tissue, Adipose-derived stem cell, Stromal vascular fraction, Density gradient, Nycodenz


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