Joris van Dongen

106 Chapter 5 knee OA in human subjects demonstrated tSVF is also safe to use. 32 The purpose of this study was to investigate the immunomodulatory and pro-regenerative effect of mechanically derived tSVF on inflammatory chondrocytes in vitro. MATERIAL AND METHODS Lipoharvesting and the FAT procedure Lipoaspirate was collected from healthy female patients between 18 and 65 years of age. The FAT procedure was performed in a standardized way as published earlier (Annex). 15,26,33 Enzymatic Dissociation of tSVF Samples of tSVF were washed with phosphate buffered saline (PBS) three times. After washing, 0.1% collagenase A in PBS/1% bovine serum albumin (BSA) was added as dissociation medium. Samples were stirred for 1.5 hour in a 37°C water bath. Cells were resuspended in erythrocyte lysisbuffer (thermoFisher) and incubated for 5 min. Samples were centrifuged at 8 °C, 600 xg for 10 min. and resuspended in expansion medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% Penicillin/Streptomycin). Viable cells were counted with trypan blue in a Bürker Turk counting chamber. Cells collected from enzymatically processed tSVF samples are henceforth referred to as 'tSVF-derived cells'. Flow cytometry to determine cell composition of tSVF tSVF-derived cells i.e. from the one-hole re-usable fractionator (n=12) were analysed for CD surface marker expression 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, USxA) 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 analyse the samples.