Joris van Dongen

93 FAT procedure with a disposable one-hole fractionator using flow cytometry. Cells were labelled with the following anti-human monoclonal antibodies: CD31, CD34, CD90, CD105, CD146 (Miltenyi Biotec Bergisch Gladbach, Germany) and CD45 (Biolegend, San Diego, CA, USA) as well as 7-Amino Actinomycin D (Invitrogen, molecular probes, Eugene, OR, USA) to stain for dead cells. Cells were mixed well with the antibodies in FACS buffer (5 mM ethylenediaminetetraacetid acid (EDTA), 1% BSA in PBS) and incubated on ice and in dark for 30 min. Stainings with a single antibody and fluorescence minus one (FMO) were used as controls. A BD FACSCanto II system (BD Biosciences) was used to analyze the samples. Colony formation assay Ten thousand viable cells isolated from tSVF obtained by the one-hole fractionator and the three-hole fractionator (n=3) were seeded (six technical replicates) and cultured for twelve days to assess the colony forming capacity of uncultured cells from a single cell. Afterwards, cells were fixed in 4% formalin and stained with 5% Crystal Violet (Sigma-Aldrich, St. Louis, MO). Colony frequency was calculated as the mean number of colonies / total seeded cells x 100%. Statistical analysis Immunohistochemistry staining were analyzed with the use of ImageJ, version 1.4.3.67 (NIH, USA). 17 Descriptive statistics were used to evaluate α -Smooth Muscle Actin (SMA), cell numbers with the use of Graphpad Prism, version 5.01 (Graph Pad Software Inc., Los Angeles). Data were expressed as mean ± standard deviation (SD). A paired t -test was performed with the use of Graphpad Prism, version 5.01 (Graph Pad Software Inc., Los Angeles). RESULTS Fractionation of adipose tissue obtained by the one-hole fractionator condenses tSVF More α -smooth muscle actin ( α -SMA) expression was observed in tSVF (0.83% ± 0.33) as compared to control lipoaspirate (0.094% ± 0.036) for (p<0.05), indicating a higher number of small vessels in tSVF obtained by the one-hole fractionator (Fig. 1). Control lipoaspirate was rich in adipocytes (perilipin A expression) while tSVF was essentially devoid of adipocytes. This difference indicates that adipocytes were destructed by the fractionator. More collagen was present in tSVF in comparison to control lipoaspirate, which indicates that the fractionation condensed ECM.