Phenotypic and Cellular Characteristics of a Stromal Vascular Fraction/Extracellular Matrix Gel Prepared Using Mechanical Shear Force on Human Fat

Abstract

The retention of fat-derived grafts remains a challenge for regenerative medicine. Fat aspirates from patients undergoing liposuction were prepared into standard Coleman fat grafts or further isolated using mechanical shear force to prepare a stromal vascular fraction (SVF)/extracellular matrix (ECM) gel. The retention rate of the SVF/ECM gel was significantly higher than that of the Coleman fat at 3, 14, 28, and 60 days following transplantation on the backs of nude mice. The viscosity of the fat was directly proportional to the shearing force. Although the mechanical isolation did not affect the total number of cells, it significantly decreased the number of living cells. Flow cytometry showed a greater number of mesenchymal stem cells, supra-adventitial (SA)-adipose stromal cells (ASCs), and adipose-derived stem cells but a lower number of endothelial progenitor cells in the SVF/ECM gel than in the Coleman fat. Thus, mechanical isolation of fat can increase the pluripotency of adipocytes, which can improve graft retention in cell therapy.

Keywords: Coleman fat; SVF/ECM gel; flow cytometry; shear force; stem cells; tissue regeneration.

FIGURE 1

Preparation process of SVF/ECM gel and Coleman fat. Coleman fat was obtained using centrifugation of fat aspirates. The upper oil and fat layers were retained by removing the lower water layer, and the mixture was pushed to form the enmeshed fat. After centrifugation, the SVF/ECM gel was obtained. The whole preparation involves pure physical action, without the use of any foreign additives.

FIGURE 2

Comparison of the general characteristics and retention rates of fat grafts. (A) Experimental design. (B) General observation of the two types of fat grafts at 3 and 60 days after transplantation. (C) Changes in fat retention rate. *P < 0.001.

FIGURE 3

Histological changes in the transplanted fat. (A) Hematoxylin and eosin staining on day 60 after transplantation showing that the SVF/ECM gel formed a mature lobular fat structure (right), whereas some oil droplets remained in the Coleman fat grafts (left); scale bars = 200 μm. (B) Fluorescence staining showing that 3 days after transplantation, the adipocytes in the SVF/ECM gel grafts were necrotic, and the negative area of perilipin was significantly increased (lower left), compared to the Coleman fat grafts (upper left); 60 days after transplantation, a large number of new adipocytes with positive perilipin staining were found in the SVF/ECM gel grafts (lower right), with many negative areas (upper right) of perilipin in the Coleman fat grafts; scale bars = 100 μm.

FIGURE 4

Relationship between shear force (line) and kinematic viscosity (columns) of different fat types. The SVF/ECM gel was obtained by mechanical isolation of the initial Coleman fat through the application of shear force.

FIGURE 5

Condensation rate of the fat samples and the total number and density of SVF mixed cells. General observation (upper left) and statistical analysis (upper right) of the volume changes in the two types of fat samples with the same initial volume during the preparation of Coleman fat and SVF/ECM gel. *P < 0.05.

FIGURE 6

SVF cell activity of the fat grafts. The apoptosis of SVF cells was detected using flow cytometry with Annexin V/PI double staining in Coleman fat and the SVF/ECM gel. *P < 0.05.

FIGURE 7

Flow cytometry for cell surface markers of SVF cells from the two fat types. The oscillogram represents the phenotypes of SVF cells isolated from the Coleman fat group (left) and SVF/ECM gel group (right). *P < 0.05.

FIGURE 8

Flow cytometry of the proportions of stem cell subsets in the suspension of SVF cells from the two fat grafts. ASC, adipose-derived stem cell; EPS, endothelial progenitor cell. *P < 0.05.