Joris van Dongen

15 General introduction and outline of the thesis bigger vessels as supra-adventitial cells, however, controversy remains to their exact nature and location. 38-41 The discovery of ASCs was a breakthrough in understanding the regenerative capacity conveyed by grafted adipose tissue. In vitro , ASCs showed to be capable of multilineage differentiation potential in adipogenic, osteogenic, chondrogenic and myogenic cell lineages. 33,42 Moreover, ASCs could be cultured upon multiple passages without inducing senescence. The multilineage differentiation capacity and extensive culture period suggested that ASCs are a stem cell-like or progenitor cell type. Latter, several other studies showed the capability of ASCs to differentiate into neuron-like cells 43 , cardiomyocytes 44 , hepatocytes 45 , pancreatic cells 46 and epithelial cells. 47 However, these in vitro proof of concepts might not reflect the ASCs’ physiological behaviour. The multilineage differentiation ability of ASCs has led research groups to believe that the therapeutic effect of ASCs is through differentiation into target cells, while other studies have shown that the plethora of secreted paracrine factors, exosomes and cytokines results in stimulation of important processes regarding facial lipofilling e.g. angiogenesis and skin tissue regeneration. 48-52 Clinical use of ASCs as cell-based therapy is expensive and time-consuming due to enzymatic isolation and culture. 53 Also, cultured ASCs changes significantly in terms of phenotype and function after adherence to tissue culture plastic, while the real phenotype of uncultured ASCs remains unknown. After multiple days of culture, the in vivo phenotype of ASCs emerges to an in vitro phenotype by losing CD34 expression and gaining CD105 expression. 38,54 Functionally, the ASCs secretome changes upon culture. During culture, many aspects affect the secretion of cytokines and growth factor such as hypoxia ( e.g. increased vascular endothelial growth factor secretion) or 3D versus 2D culture ( e.g. upregulation of genes related to cell adhesion and wound healing in 3D culture). 55,56 In this way, relating in vitro experimental data to clinical evidence and vice versa is difficult. Till recently, the Gold standard to isolate SVF containingASCs from adipose tissue is by means of enzymes, requires a laboratory and is thus time-consuming and expensive. 33 Enzymatic digestion of adipose tissue yields a heterogeneous cell suspension containing only cells e.g. ASCs, endothelial cells, smooth muscle cells, pericytes, fibroblasts and immune cells. Due to expensive and time-consuming enzymatic isolation procedures, intra-operativenon-enzymatic ormechanical isolationprocedures increasedpopularity. Mechanical isolation procedures to isolate SVF only use centrifugation and shear stress to disrupt the larger and weaker cells i.e. adipocytes.