Harvesting technique affects adipose-derived stem cell yield

Abstract

Background: The success of an autologous fat graft depends in part on its total stromal vascular fraction (SVF) and adipose-derived stem cells (ASCs). However, variations in the yields of ASCs and SVF cells as a result of different harvesting techniques and donor sites are poorly understood.

Objective: To investigate the effects of adipose tissue harvesting technique and donor site on the yield of ASCs and SVF cells.

Methods: Subcutaneous fat tissues from the abdomen, flank, or axilla were harvested from patients of various ages by mechanical liposuction, direct surgical excision, or Coleman's technique with or without centrifugation. Cells were isolated and then analyzed with flow cytometry to determine the yields of total SVF cells and ASCs (CD11b-, CD45-, CD34+, CD90+, D7-FIB+). Differences in ASC and total SVF yields were assessed with one-way analysis of variance. Differentiation experiments were performed to confirm the multilineage potential of cultured SVF cells.

Results: Compared with Coleman's technique without centrifugation, direct excision yielded significantly more ASCs (P < .001) and total SVF cells (P = .007); liposuction yielded significantly fewer ASCs (P < .001) and total SVF cells (P < .05); and Coleman's technique with centrifugation yielded significantly more total SVF cells (P < .005), but not ASCs. The total number of SVF cells in fat harvested from the abdomen was significantly larger than the number in fat harvested from the flank or axilla (P < .05). Cultured SVF cells differentiated to adipocytes, osteocytes, and chondrocytes.

Conclusions: Adipose tissue harvested from the abdomen through direct excision or Coleman's technique with centrifugation was found to yield the most SVF cells and ASCs.

Figure 1.

Representative analysis of stained cells isolated from fresh human adipose tissue. (A) Aqua Dead staining was absent in 31.5% of cells. (B) Most Aqua Live–stained cells (81.1%) did not express both CD11b and CD45. (C) Of the cells that did not express CD11b or CD45, 21.6% expressed both CD34 and D7-FIB. (D) Of the live CD11b−, CD45−, CD34+, D7-FIB+ cells, 95.4% also expressed CD90.

Figure 2.

Quantitative analysis of total stromal vascular fraction (SVF) cell and adipose-derived stem cell (ASC) yields of adipose tissues harvested from the flank by means of different harvesting techniques. Error bars show the standard error of the mean (SEM) and Ns indicate the numbers of samples. Compared with Coleman's technique without centrifugation, direct excision and Coleman's technique with centrifugation both yielded significantly more total SVF cells. Compared with liposuction, Coleman's technique without centrifugation yielded significantly more total SVF cells. The number of ASCs obtained utilizing direct excision was significantly higher than the numbers of ASCs obtained from blood–oil waste or utilizing liposuction or Coleman's technique with or without centrifugation. The numbers of total SVF cells and ASCs obtained from blood–oil waste were similar to those obtained utilizing liposuction.

Figure 3.

Quantitative comparison of total stromal vascular fraction (SVF) cell and adipose-derived stem cell (ASC) yields of fat tissue harvested from different locations. Error bars show the standard error of the mean (SEM), and Ns indicate the numbers of samples. Coleman's technique was utilized to isolate the cells. The number of total SVF cells obtained from adipose tissue harvested from the abdomen was significantly higher than the numbers of total SVF cells obtained from adipose tissue harvested from the flank or axilla.

Figure 4.

Characteristics of cultured adipose-derived stem cells (ASCs). Flow cytometry analyses revealed that the cultured cells were (A) CD11b− (0.73%), (B) CD45− (0.03%), (C) CD34− (0.79%), (D) CD90+ (99.82%), and (E) D7FIB+ (96.7%). (F) At third passage, the cells had a spindle-like morphology. (G-I) Differentiation of ASCs to adipocytes (G and J, Oil Red O staining), osteocytes (H and K, Alizarin red S staining), and chondrocytes (I and L, Safranin O staining) in vitro. (J-L) ASCs cultured in basic medium served as controls.