Isolation of human adipose-derived stromal cells using laser-assisted liposuction and their therapeutic potential in regenerative medicine

Abstract

Harvesting adipose-derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third-generation ultrasound-assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser-assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser-assisted lipoaspirate and suction-assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic nude mice. Although ASCs isolated from suction-assisted lipoaspirate and laser-assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34(+)CD31(-)CD45(-)) in the stromal vascular fraction were all significantly less with laser-assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro-computed tomography revealed significantly more healing with ASCs isolated from suction-assisted lipoaspirate relative to laser-assisted lipoaspirate at the 4-, 6-, and 8-week time points (p < .05). Therefore, as laser-assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction-assisted liposuction is preferable for tissue-engineering purposes.

Keywords: Adipose; Adult stem cells; Laser lipoplasty; Liposuction; Stem cell transplantation; Stromal cells.

Figure 1.

Cell yield, viability, and proliferation. (A): Overall number of cells isolated from adipose tissue harvested via laser-assisted liposuction and suction-assisted liposuction. Laser-assisted liposuction resulted in a lower viable cell yield than suction-assisted liposuction (*, p < .05). Viable cells were identified by trypan blue exclusion. (B): Laser-assisted liposuction was also associated with a lower percentage of cell viability (*, p < .05). (C): Effect of the liposuction technique on the proliferation of adipose-derived stromal cells in vitro was determined by the XTT cell proliferation assay. There were significant differences in absorption values between the proliferation of cells obtained by laser-assisted liposuction and suction-assisted liposuction at 18, 24, 72, 96, 120, 144, and 168 hours (*, p < .05). Abbreviations: OD, optical density; SAL, suction-assisted liposuction.

Figure 2.

Measuring frequency of adipose-derived stromal cell phenotype via flow cytometry. (A): Triple staining was performed using monoclonal antibodies against CD34, CD31, and CD45 to interrogate stromal vascular fraction cells exhibiting an adipose-derived stromal cell (ASC) phenotype. (B): The frequency of cells exhibiting a CD34⁺CD31⁻CD45⁻ phenotype was determined. The difference in ASC frequency between adipose tissue harvested using laser-assisted liposuction and suction-assisted liposuction was significantly different (*, p < .05). Abbreviation: SSC, side scatter.

Figure 3.

Differentiation toward the osteogenic lineage. (A): Osteogenic differentiation of adipose-derived stromal cells (ASCs) isolated from adipose tissue harvested via laser-assisted liposuction and suction-assisted liposuction was demonstrated by alkaline phosphatase staining (top) and alizarin red staining (bottom). Magnification, ×10. (B): Photometric quantification of alizarin red staining showed no significant difference between laser-assisted and suction-assisted liposuction-derived ASCs. (C): Gene expression of early (RUNX2), intermediate (OPN), and late (OCN) osteogenic markers (*, p < 0.05). Abbreviations: OCN, osteocalcin; OPN, osteopontin; RUNX2, runt-related transcription factor 2.

Figure 4.

Differentiation toward the adipogenic lineage. (A): Adipogenic differentiation of adipose-derived stromal cells (ASCs) isolated from adipose tissue harvested via laser-assisted liposuction and suction-assisted liposuction was demonstrated by Oil Red O staining, which demonstrated lipid droplet formation. (B): Photometric quantification of Oil Red O staining showed no significant difference between laser-assisted and suction-assisted liposuction-derived ASCs. (C): Gene expression of PPAR-γ, AP2/FABP4, and LPL (*, p < 0.05). (D): Gene expression of heat shock protein HSPA2 (*, p < 0.05). Abbreviations: AP2, adipocyte protein 2; FABP4, fatty acid binding protein 4; HSPA2, heat shock-related 70-kDa protein 2; LPL, lipoprotein lipase; PPAR-γ, peroxisome proliferator-activated receptor γ.

Figure 5.

Application of adipose-derived stromal cells in calvarial defects. (A): Three-dimensional reconstruction of calvarial defects. Mice were scanned at 2, 4, 6, and 8 weeks following surgery. (B): Quantification of osseous healing by micro-computed tomography revealed significantly more healing with adipose-derived stromal cells isolated from adipose tissue harvested via suction-assisted liposuction relative to laser-assisted liposuction (*, p < .05) at the 4-, 6-, and 8-week time points. (C): Calvarial defects 4 mm in size were allowed to heal for 8 weeks before histological analysis by Movat's pentachrome staining. Pictures were taken in the middle of the defect site. In pentachrome stains, bone appears yellow.