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Kim, Park, Park, Choi, Kim, Park, Rhee, Park, Lee, Oh, Park, Kim, Suh, and Woo: Isolation of Density Enrichment Fraction of Adipose-Derived Stem Cells from Stromal Vascular Fraction by Gradient Centrifugation Method
Original Article
Endocrinology and Metabolism

Endocrinology and Metabolism 2010; 25(2): 103-109.

Published online: 30 June 2010

DOI: https://doi.org/10.3803/EnM.2010.25.2.103

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 Park1, Jung Mook Choi1, Won Jun Kim2, Se Eun Park2, Eun Jung Rhee2, Cheol-Young Park2, Won Young Lee2, Ki Won Oh2, Sung Woo Park2, Sun Woo Kim2, Kwang Sik Suh3, Jeong Taek Woo3

Department of Food and Nutrition, Hanyang University, Seoul, Korea.

1Diabetes Research Institute, Kangbuk Samsung Medical Center, Seoul, Korea.

2Department of Internal Medicine, Kangbuk Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

4Department of Endocrinology-Metabolism, College of Medicine, Kyunghee University, Seoul, Korea.

Corresponding author: Cheol-Young Park. Department of Internal Medicine, Kangbuk Samsung Medical Center, 108 Pyeong-dong, Jongno-gu, Seoul 110-746, Korea. Tel: +82.2-2001-2440, Fax: +82.2-2001-1588, cydoctor@chol.com

Received 12 October 2009       Accepted 23 December 2009

Copyright © 2010 Korean Endocrine Society

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.

Keywords: Rat adipose tissue, Adipose-derived stem cell, Stromal vascular fraction, Density gradient, Nycodenz

Figures and Tables

Fig. 1
Amount of adipose tissues is most extensive in subcutaneous, epididymal, mesenteric, and lowest in retroperitoneal adipose tissue. Data is expressed as mean ± SE.
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Fig. 2
Cell numbers of undifferentiated SVFs were estimated by trypan blue staining. A. Mesenteric adipose tissue had the most abundant cells than other adipose depots (MAT > EAT > RAT > SAT). B. SVFs extracted to fraction 3 (Fx-3) had highest cell numbers than other fractions.
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Fig. 3
Post-confluent ASCs were cultured in adipogenic medium. On day 7 of adipogenic induction, lipid vacuoles were observed within the ASCs, and a significant difference was detected in the number, size and distribution patterns of the lipid vacuoles (magnification, × 200).
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Fig. 4
Comparisons in the differentiation were examined by Oil red O stain for adipogenesis. The retroperitoneal and subcutaneous adipose tissue derived stem cells had the highest efficiency in adipogenic differentiation whereas the mesenteric adipose tissue dereived stem cells did not differentiate (magnification, × 200).
enm-25-103-g004
Fig. 5
After Oil red O staining, the optical density of Oil red O-positive cells was assessed by ELISA reader. The retroperitoneal adipose tissue derived stem cells had the highest efficiency in adipogenic differentiation. The adipogenic differentiation of fraction 1 or 2 was higher compared to that of the unfractionated cells (control) and other fractions. Data are expressed as mean ± SE.
enm-25-103-g005

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