Joris van Dongen

78 Chapter 3 their vWF and respond therefore negative to the vWF staining. The ratio between activated and non-activated endothelial cells, however, remains unclear. Quantification of the number of adipocytes as compared to the surface area of FAT-SVF proved methodologically to be impossible. When all adipocytes are mechanically disrupted, the FAT-SVF disintegrates into small unquantifiable parts. When a few adipocytes are still intact, bigger parts of FAT-SVF are visible. In this way only quantification of the bigger parts of FAT-SVF, containing more adipocytes, is possible. So therefore, the number of adipocytes counted is higher as compared to the real number of adipocytes. Interestingly, our method to disrupt adipose tissue yielded an injectable FAT-SVF that contains all the prerequisites for tissue regeneration: ASC, microvasculature and supporting extracellular matrix. Compared to conventional or automated dissociation methods of adipose tissue, the FAT procedure is a faster and more cost-effective method to produce a FAT-SVF. 25 The dissociation device is small and of simple design i.e. even in disposable format can be produced cost effective, in particular in large quantities. The short time necessary for the mechanical dissociation procedure enables the surgeon to use the FAT-SVF during surgery with a minimal delay of operation time. It also likely contributes to the high vitality of cells that are retained in the FAT-SVF. As opposed to enzymatic digestion, where ASC need to survive in an ischemic environment due to the lengthy isolation procedure. Also the use of collagenase or non-autologous material potentially renders enzymatic digestion more sensitive for (bacterial) contamination. Furthermore, enzymatic isolation results in a SVF comprised of a suspension of individual, non- connected cells with a total lack of tissue structure. In general, retention rates of single cell injections (i.e. ASC) are rather low. 26 With mechanical dissociation, the extracellular matrix is retained in the FAT-SVF, which probably functions as a ‘microvascularized’ scaffold for the ASC thus keeping a matrix for tissue integrity. In this way, it can be expected to increase cell survival after injection, thus reducing the otherwise low reported retention rates. The interaction between cells and growth factors in FAT-SVF is expected to increase the regenerative potential in cell-based therapy as compared to the single use of ASC. For instance, the combined application of pericytes and endothelial cells has been shown to augment angiogenesis compared to their single use. 27 Additionally, ASC enhance angiogenesis through secreted growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor(s) (FGF) and hepatocyte growth factor (HGF) 28,29 , in particular under hypoxia. 20 ASC conditioned medium, which is rich in VEGF, FGFs and HGF, both increases the proliferation rate of endothelial cells, while suppressing apoptosis, in particular under hypoxia. 29 Endothelial cell proliferation and