Joris van Dongen

52 Chapter 2 found between several studies. To avoid variation bias, isolation procedures should be investigated using identical lipoaspirates in the same study. There are, however, differences between non-enzymatic and enzymatic isolated SVFs on a different level. Non-enzymatic isolation procedures resulted in larger volumes (tSVF) than the resulting pellets (cSVF) after enzymatic intraoperative isolation procedures. Because the final products of both types of isolation procedures are different, the clinical purpose of the use of SVF is an important factor which isolation procedure suits best. In some cases, such as the intra-articular injection of SVF in temporomandibular joints requires very small volumes, whereas the end volume of SVF enriched lipofilling is less relevant. Isolation procedures of SVF of adipose tissue are based on reduction of large volume containing tissue or cells, such as ECM and/or adipocytes to concentrate the stromal vascular fraction. Non-enzymatic isolation of SVF results in a smaller volume of adipose tissue containing intact ECM and cell-cell communications between SVF cells (tSVF), because the shear forces are too low to disrupt cell to cell and cell to ECM adhesions 12,80 . Therefore, the tissue structure of lipoaspirate is still intact in the tSVF. Enzymatic procedures, however, likely result in a single cell cSVF, because enzymes likely disrupt all cell-cell interactions and ECM (Fig. 3) 15 . This is may not happen in the Automated isolation system, GID SVF2, Lipokit system and Multi station, possibly due to insufficient enzymatic digestion 69,70 . Clinical use of tSVF has several advantages over the use of cSVF in different clinical applications of regenerative medicine. It is well known that single cells migrate within 24 hours after application 81 . The ECM, containing a microvasculature structure, might function as a natural scaffold for cells like ASCs and most likely also augments rapid vascularization and reperfusion. This will probably increase cell retention rates after injection and enhance clinical effects. In case of early scar formation, wound healing, or organ fibrosis, tSVF might therefore be more an appropriate therapy, which implicates that non-enzymatic procedures are more suitable as compared to enzymatic isolation procedures. In case of excessive pre-existing scar formation, the ECM in the SVF might not be appropriate and therefore the application of a cSVF or ASCs might be more eligible. ASCs could remodel excessive scar formation by immunomodulation or instruction of resident cells. Characterization of subpopulations in the SVF depends upon selection of appropriate markers. Selection of an insufficient number of markers will give a disfigured image of the actual SVF composition (Fig. 3). SVF of adipose tissue can be divided into two major subpopulations based on the expression of CD45, which is a hematopoietic cell marker: adipose derived (CD45min) and blood derived (CD45pos) 7 . Adipose derived cell populations can be divided into endothelial cells (CD31pos) and stromal