Joris van Dongen

73 FAT procedure to obtain SVF for regenerative purposes activation suchmechanical shear. Therefore, only quantification of vWFwas performed on samples that expressed vWF staining, implying the presence of non-activated EC. Perilipins are lipid-coating proteins that protect against lipase action. Therefore, the presence of Perilipin marks intact adipocytes. After mechanical dissociation, adipocyte ‘ghosts’ were present in the FAT-SVF occasionally (Fig. 3). Mainly large adipocytes were visible in the control, whereas only smaller ones in the FAT-SVF. Masson’s Trichrome staining showed more collagen deposition (blue) between the cells (red) in the FAT-SVF as compared to the control (Fig. 3), implying enrichment of extracellular matrix in the FAT-SVF by disrupting adipocytes compared to controls. Mechanical dissociation cell yield Complete mechanical dissociation of adipose tissue was performed intraoperatively within 8 to 10 minutes of extra operating time and resulted in a FAT-SVF with a mean volume of 0.96 ml [range 0.75 to 1.75]. Enzymatic isolation of the FAT-SVF resulted in a mean number of cells of 2.7 x10 6 [+/- 1.1 x10 6 ] per 1 ml (Fig. 4). Enzymatic isolation of 10ml of the control resulted in a mean number of cells of 3.5 x10 5 [+/-5.1 x10 6 ] per 1 ml (p<0.001), a 7.7 times lower number of cells in 1 ml adipose tissue as compared to 1 ml of FAT-SVF (Fig. 4). Three patients were excluded from the study. One patient had an uncountable low number of cells in the FAT-SVF sample. In one FAT-SVF and one control sample of two different patients, we were unable to culture enough cells to reach confluency. Cultured cells had a spindle-shaped fibroblast-like morphology in both samples (Fig. 4, Supplemental Content). There was no visible difference between both groups. Cells cultured frommechanically dissociated adipose tissue harbor ASC characteristics For subset A, a mean of 99.8% [+/-0.2] of the FAT-SVF showed expression of CD90 and no expressions of CD31 and CD45 were shown. For the control, a mean of 99.8% [+/- 0.1] showed expression of CD90 with also no expressions of CD31 and CD45. There was no significant difference between the two groups (P>0.05). For subset B, a mean of 99.8% [+/0.2], 99.0% [+/0.7] and 95.9% [+/- 4.5] of the FAT- SVF showed expression of CD29, CD44 and CD105. For the control, a mean of 98.2% [+/-3.5], 98.5% [+/1.2] and 96.6% [+/2.6] showed expression of CD29, CD44 and CD105, respectively. No significant differences occurred between the two groups (P>0.05). Representative data of CD-surface marker expression is shown in figure 5, Supplemental Content.