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Updated: May 6, 2026
Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis
Published on: January 12, 2015
Functional Stratification of Adipose-Derived Stem Cells via Gradient Density Centrifugation: D-ASCs Drive
Jie Long1, Guo Chen2, Zijin Qin2,3
1Department of Medical Cosmetic Center, Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, Hangzhou, 310006, Zhejiang, People's Republic of China.
Objective:
To investigate the functional heterogeneity of adipose-derived stem cells (ASCs) in fat grafting and develop a density-based separation method to isolate distinct ASC subpopulations for improving graft survival.
Methods:
ASCs were isolated from human lipoaspirates and subjected to adipogenic differentiation. A novel Percoll density gradient centrifugation protocol (10-100%, 300 × g, 35 min) was developed to separate ASCs into upper-layer (U-ASCs) and lower-layer (D-ASCs) subpopulations. Their adipogenic and angiogenic potentials were assessed through Oil Red O staining and Matrigel tube formation assays. In vivo functionality was evaluated in nude mice (n = 84) receiving fat grafts supplemented with GFP-labeled U-ASCs, D-ASCs, or unsorted ASCs (n = 28/group), with graft retention, histology, and cellular fate analyzed over 12 weeks.
Results:
Density separation yielded two functionally distinct populations: U-ASCs exhibited 3.2-fold greater lipid accumulation (p < 0.001), while D-ASCs showed 2.7-fold enhanced tube formation (p < 0.01). In vivo, D-ASCs grafts demonstrated 58.3 ± 6.7% retention at 12 weeks versus 32.1 ± 5.2% for U-ASCs (p < 0.001), with significantly reduced oil cysts (p < 0.05) and 2.4-fold higher vascular density (p < 0.001). Immunofluorescence revealed U-ASCs primarily differentiated into adipocytes (GFP+/Perilipin+), whereas D-ASCs integrated into vasculature (GFP+/CD31+).
Conclusion:
ASC subpopulations differentially regulate fat graft outcomes-U-ASCs drive adipogenesis while D-ASCs enhance vascularization. The developed density separation method enables isolation of therapeutic ASC subsets, with D-ASCs showing superior potential for improving graft survival through neovascularization. These findings support precision strategies in cell-assisted lipotransfer.
Level Of Evidence I:
This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
