Joris van Dongen

108 Chapter 5 acid 2-phosphate, 1% Pen / Strep, and 10ng/mL TGF- β 2 (302-B2; R&D Systems). The cells were cultured for 4 weeks in chondrogenic medium and the medium was changed 3 times per week. After 28 days of culture, pellets were formalin-fixed and paraffin embedded. Subsequently, 5 μm sections were stained with 0.125% safranin-O (Merck, counterstained withWeigert’s hematoxylin (Klinipath) and 0.4% fast green (Merck)) to stain deposited proteoglycans. Enzymatic isolation of chondrocytes Chondrocytes were isolated from donor cartilage of various human patients after total knee arthroplasty allowed by the local ethical committee of the UMC Utrecht (TC-Bio protocolnumber 15-092). Cartilage was minced and tissue fragments were subjected to sequential treatments first with DMEM supplemented with 1% FBS, 100 U/ml of penicillin, 100 m g/ml of streptomycin, and 2.5% (w/v) of pronase E (Sigma, St. Louis, MO) for 1 h, then with DMEM supplemented with 25% FBS, 100 U/ml of penicillin, 100 mg/ ml of streptomycin and 0.125% (w/v) of collagenase (CLS-2; Worthington, Lakewood, NJ) for 16 h at 37° C. Cells were filtered through a 70-mm cell strainer (BD Biosciences, San Diego, CA) and washed. Chondrocytes were expanded to passage 2 in T175 flasks in expansion medium. Functional analysis of coculture of tSVF-derived cells and chondrocytes Freshly isolated tSVF-derived cells and passage 2 chondrocytes were cultured in various ratio’s in pellets. A total of 250.000 cells, consisting of 0, 25, 50, 75 and 100% nucleated tSVF-derived cells and 100, 75, 50, 25 and 0% chondrocytes, were cultured in 1 mL DMEM (Gibco) supplemented with 2% insulin-transferrin-selenium (ITS)-X (Invitrogen), 50 µg/mL ascorbate-2-phosphate (Sigma), 2% human serum albumin (Sanquin) and penicillin / streptomycin (100 U/mL, 100 µg/mL), which was refreshed every 2-3 days at 37°C at 5% CO2. Immediately after pelleting and after 21 days of culture, RNA was isolated from 3 pellets for RT real-time PCR to investigate the ratio between the cells.After 28 days, 3 pelletswere papain digested for biochemical analyses. Cell ratio analysis of chondrocytes and tSVF-derived cells cocultures Cocultures of tSVF-derived cells from female donors and chondrocytes from male donors (n = 3 in triplicates) in various ratios were cultured for 21 days. After 21 days, the expression of the Y-chromosomal gene lysine demethylase 5D (KDM5D) gene was measured. 18S was used as housekeeping gene. Total RNA was isolated using Trizol (Invitrogen) as described by the manufacturer. Total RNA (500 ng) was reverse transcribed using the high-capacity cDNA Reverse Transcription kit (Thermo