Joris van Dongen

123 tSVF acts anti-inflammatory on chondrocytes in vitro ANNEX: FAT PROCEDURE The lipoaspiratewas harvested from the abdomen and upper legs with a lipo-harvesting cannula (Tulip, Medical Products, San Diego, CA) after distribution of 500 ml modified Klein’s solution (per 500 ml saline, 20 ml of lidocaine, 1% epinephrine 1:100.000 and 2 mLof bicarbonate).The FATprocedurewith a re-usable one-hole fractionator in a closed system was performed with the disposable Arthrex Autologous Conditioned Plasma (ACP) Double Syringe Systems (Arthrex, Inc. ABS-10014, Naples, USA). Disposable ACP double syringes were filled with decanted lipoaspirate and centrifuged at 956xg for 2.5 min. with a swing out rotor centrifuge at room temperature (RT) (Hettich Rotofix 32, benchtop, swing out rotor, Tuttlingen, Germany). After centrifugation, the upper oily fraction was discarded by pulling the inner syringe. Then, the lower aqueous fraction was discarded by opening the red closing cap yielding condensed lipoaspirate. Condensed lipoaspirate was used for mechanical dissociation according to the fractionation of adipose tissue (FAT) procedure. After mechanical dissociation, samples were centrifuged again for 2 min. at 760 xg. This yielded an oily fraction that could be easily removed leaving tSVF in the larger outer ACP double syringe.