15502-m-pleumeekers

presence of 1% Bovine Serum Albumin (BSA; PAA Laboratories Gmbh, Cölbe, Germany) in LG- DMEM. After 60 minutes of enzymatic digestion in an orbital shaker, floating adipocytes were separated from the precipitating MSC fraction by centrifugation (10 minutes, 1500 RPM), washed with ‘standard MSC expansion medium’, and filtered through a 100-μm nylon mesh. Before cell seeding, the amount of nucleated cells was calculated using methylene blue, and cell number was calculated with a hemocytometer. The cell suspension was seeded at an initial density of 40,000 cells/cm 2 in ‘standard MSC expansion medium’. All cells were cultured at 37 o C in air containing 5% carbon dioxide. Mediumwas changed twice a week. When cell cultures reached 80% confluence, chondrocytes and MSCs were trypsinized using 0.05% trypsin–EDTA. Chondrocytes were seeded at a 7500 cells/cm 2 and MSCs at a 2300 cells/cm 2 cell density for further expansion to increase cell number. All third-passage (P3) cells which were approaching subconfluence were detached and cultured in a three-dimensional alginate system (as described below) to promote chondrogenesis. In order to determine the proliferation rate of cultured ECs, NCs, ACs, BMSCs and AMSCs, growth kinetics of three donors from each cell source were evaluated in monolayer expansion using the number of population doublings (PD) until subconfluency and the time to reach passage four. Therefore we have calculated the PD/D (Population Doublings per Day) by using the formula: PD/D = (ln (N2/N1) / ln (2))/D; where N1 was the number of cells at the beginning of each passage,N2 the number of cells at subconfluency and D the amount of days to reach passage four. Chondrogenic differentiation For three-dimensional alginate culture, isolated cells from four donors of each cartilage source and six donors from each of the MSC-sources were suspended at a density of 4x10 6 cells/mL in clinical grade 1.1% low viscosity alginate solution dissolved in 0.9% NaCl (Batch MG-004, CellMed, Alzenau, Germany). Afterwards, the cell-alginate mixture was transferred into a 10- mL sterile syringe from which the suspension was slowly passed through a 23-gauge needle to produce drops, which fell into a 102 mM CaCl 2 . Following instantaneous gelation, the beads were allowed to further gelate for a period of 10 minutes in the CaCl 2 solution. After being washed once with 0.9% NaCl and HG-DMEM, the beads were transferred to 24-well plates. Controls were cultured in 150 μL/bead ‘control differentiation medium’ containing serum-free HG-DMEM supplemented with 50 µg/mL gentamycin; 0.5 µg/mL Fungizone; 1 mM sodium pyruvate (Gibco); 40 μg/mL L-proline (Sigma-Aldrich); supplemented Insulin Transferrine Selenium (ITS+ ; B&D Bioscience, Bedford, USA); 10 -7 M dexamethason; and 25 μg/mL L- ascorbic acid 2-phosphate. In the experimental condition (‘chondrogenic differentiation medium’) 10 ng/mL Transforming Growth Factor β1 (TGFβ1; R&D Systems, Minneapolis, USA) was added to induce chondrogenesis. Medium was changed twice a week. After two and five weeks, alginate beads were processed for biochemical or gene-expression analysis as described below. For all in-vitro experiments four donors for the chondrocyte sources and six donors for the MSC sources were used, with at least duplicate samples per analyses for each individual donor. To study in-vivo functionality and stability of cartilage TE constructs after in-vitro cell- culture, larger flat constructs were created from cells of three donors of each cell source as 63 CARTILAGE-FORMING CAPACITY OF SEVERAL CELL SOURCES 4