Liang Cao1, Feng Xiaoming2, Qiang Zhang3,4, Junbiao Fang5, Chunhua Chu1, Jinlong Lv1, Yuyuan Ma6, Gang Lu6, Kaichuang Yang7, Ruolang Pan8,9. 1. Department of Acupuncture and Moxibustion, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China. 2. Department of Neurosurgery, The First Hospital of Jiaxing, Affiliated hospital of Jiaxing University, Jiaxing, Zhejiang, China. 3. Institute for Cell-Based Drug Development of Zhejiang Province, S-Evans Biosciences, Hangzhou, China. 4. Key Laboratory of Cell-Based Drug and Applied Technology Development in Zhejiang Province, Hangzhou, China. 5. Department of Anesthesiology, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China. 6. Department of Neurosurgery, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China. 7. Department of Neurosurgery, Zhejiang Provincial People's Hospital and People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China. kfemme@zju.edu.cn. 8. Institute for Cell-Based Drug Development of Zhejiang Province, S-Evans Biosciences, Hangzhou, China. ruolang_pan@163.com. 9. Key Laboratory of Cell-Based Drug and Applied Technology Development in Zhejiang Province, Hangzhou, China. ruolang_pan@163.com.
Abstract
BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains heterogeneous cell populations and has enormous potential for clinical therapy. There are two main methods for SVF isolation: enzymatic isolation and mechanical isolation, both of which have shortcomings. In this study, optimized conditions for the isolation of high-quality SVF were established, and applications in fat grafting were evaluated. METHODS: Adipose tissue was chopped into small pieces and then ground into an erosive shape using a syringe. The pieces were digested with 0.15% type II collagenase for 35 min at 37 °C. After centrifugation, the pellets were resuspended in DMEM and passed through a 100-μm strainer. The filtered cells were analyzed by flow cytometry. The fat graft was enriched with isolated SVF and subcutaneously transplanted into nude mice. Three weeks after transplantation, grafts were isolated, and H&E staining, immunocytochemistry, and western blotting were conducted. RESULTS: The harvested SVF cells reached > 2 × 106/ml of adipose tissue within 90 min of operation. The number of CD34+ ADSCs in our SVF pellets was > 6 × 105/ml of adipose tissue, which has the potential for differentiating into osteoblasts, adipocytes, and chondrocytes. Freshly collected adipose tissue is better for SVF isolation, and isolated SVF should also be kept at 4 °C and used as soon as possible. SVF may promote revascularization after fat grafting. The adipose tissue of an SVF co-transplanted group had an integral structure, clear capillaries, and higher VEGF expression. SVF co-transplantation inhibited adipose cell apoptosis. CONCLUSION: Our study provides an efficient procedure for SVF isolation, its application in fat grafting, and possible underlying mechanisms. LEVEL OF EVIDENCE IV: 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 .
BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains heterogeneous cell populations and has enormous potential for clinical therapy. There are two main methods for SVF isolation: enzymatic isolation and mechanical isolation, both of which have shortcomings. In this study, optimized conditions for the isolation of high-quality SVF were established, and applications in fat grafting were evaluated. METHODS: Adipose tissue was chopped into small pieces and then ground into an erosive shape using a syringe. The pieces were digested with 0.15% type II collagenase for 35 min at 37 °C. After centrifugation, the pellets were resuspended in DMEM and passed through a 100-μm strainer. The filtered cells were analyzed by flow cytometry. The fat graft was enriched with isolated SVF and subcutaneously transplanted into nude mice. Three weeks after transplantation, grafts were isolated, and H&E staining, immunocytochemistry, and western blotting were conducted. RESULTS: The harvested SVF cells reached > 2 × 106/ml of adipose tissue within 90 min of operation. The number of CD34+ ADSCs in our SVF pellets was > 6 × 105/ml of adipose tissue, which has the potential for differentiating into osteoblasts, adipocytes, and chondrocytes. Freshly collected adipose tissue is better for SVF isolation, and isolated SVF should also be kept at 4 °C and used as soon as possible. SVF may promote revascularization after fat grafting. The adipose tissue of an SVF co-transplanted group had an integral structure, clear capillaries, and higher VEGF expression. SVF co-transplantation inhibited adipose cell apoptosis. CONCLUSION: Our study provides an efficient procedure for SVF isolation, its application in fat grafting, and possible underlying mechanisms. LEVEL OF EVIDENCE IV: 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 .