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KMID : 0356620100250020103
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Journal of Korean Society of Endocrinology
2010 Volume.25 No. 2 p.103 ~ p.109
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Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method
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Kim Min-Kyung
Park Yong-Soon
Woo Jeong-Taek
Rhee Eun-Jung
Park Hee-Soon
Kim Sun-Woo
Suh Kwang-Sik
Kim Won-Jun
Park Se-Eun
Park Cheol-Young
Lee Won-Young
Oh Ki-Won
Park Sung-Woo
Choi Jung-Mook
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Abstract
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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.
Results: 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.
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KEYWORD
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Rat adipose tissue, Adipose-derived stem cell, Stromal vascular fraction, Density gradient, Nycodenz
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