Joris van Dongen

77 FAT procedure to obtain SVF for regenerative purposes DISCUSSION In this study,we have demonstrated that the FATprocedure is both efficient and reliable intra-operative method to isolate the FAT-SVF from adipose tissue within 8 to 10 min in a simple and reproducible way, suitable for injection: the FAT procedure dissociates lipoaspirate by disrupting adipocytes mechanically and consequently results in a stromal vascular fraction containing culturable ASC as well as small vessels embedded in an extracellular matrix. This FAT-SVF is indicated to increase scar remodeling and might even accelerate wound healing in vivo. ASC derived from the FAT-SVF as well as ASC from the control showed similar expression with regard to the CD-surface markers, had similar like spindle shaped fibroblast-like morphology, the same multipotent differentiation potential and colony formation capacity. Phenotypically, ASC have two main characteristics. First, ASC possess markers such as mesenchymal markers (CD29+, CD44+, CD90+ and CD105+) and endothelial and hematopoietic markers (CD31- and CD45- respectively). 24 ASC freshly isolated from adipose tissue are CD105-, but become CD105+ after culturing. 17 Therefore, the variation of CD105+ cells can be larger than for otherCD surfacemarkers. Furthermore, endothelial cells also express CD105, which is TGF-beta receptor type III, which might influence the FACS results. 17 Second, ASC are able to attach to culture plastic flasks and present as having a spindle shaped fibroblast-like morphology. Functionally, ASC are characterized by their ability to differentiate into adipocytes, osteoblasts and smooth muscle cells. 12,24 Since there are no significant or visual differences in function and phenotype between ASC derived from the SVF and the control, the FAT procedure apparently does not affect the potential of ASC. Furthermore, the FAT procedure results in a FAT-SVF that contains only living cells and significantly more nucleated cells in 1 ml of adipose tissue as compared to non-dissociated lipoaspirate. TheFATprocedure is a reproduciblemethod togenerate consistentvolumes of injectable FAT-SVF. The composition of the FAT-SVF is histologically heterogeneous: some parts consist of small blood vessels mainly, while other parts consists of ECM only. We surmise that the mechanical disruption of the adipose tissue causes a redistribution of softer (extracellular matrix) and harder (microvasculature) component of the FAT-SVF. The heterogeneity, however, could also be a consequence of inter-patient variation. For this study anonymized samples were obtained. Therefore, future clinical trials might reveal variation among patients. Von Willebrand Factor staining was only present in the non-activated endothelial cells in the FAT-SVF. Activated endothelial cells secrete