Endocrinol Metab.  2010 Jun;25(2):103-109. 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

• Kim MK
• Park YS
• Park HS
• Choi JM
• Kim WJ
• Park SE
• Rhee EJ
• Park CY
• Lee WY
• Oh KW
• Park SW
• Kim SW
• Suh KS
• Woo JT

• KMID: 2169015
• DOI: http://doi.org/10.3803/EnM.2010.25.2.103

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.

Keyword

Rat adipose tissue; Adipose-derived stem cell; Stromal vascular fraction; Density gradient; Nycodenz

MeSH Terms

Adipose Tissue
Animals
Azo Compounds
Centrifugation
Centrifugation, Density Gradient
Humans
Insulin
Intra-Abdominal Fat
Iohexol
Male
Mesenchymal Stromal Cells
Rats
Rats, Sprague-Dawley
Stem Cells
Azo Compounds
Insulin
Iohexol

Figure

• 

  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.

• 

  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.

• 

  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).

• 

  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).

• 

  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.

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