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Material and Methods 14

Material and Methods 14 2.2.2. WST-1 metabolic assay A WST-I assay (Roche Diagnostics, Mannheim, Germany) was performed to assess the differences in metabolic activity of the cells for the different cell culture conditions. The assay was performed according to the manufacturer’s recommendations. In brief, cells were seeded in quintets for each preparation in 24 well plates at a density of 2,500 cells/cm² and incubated for 24 hours in normal cell culture medium (α-MEM). WST-I reagent was freshly mixed with medium at a ratio of 1:10. 400 µl of this suspension were added to each well, followed by 4 hours of incubation. Subsequently, 100 µL of the reacted suspension from each well were transferred in triplicates into a 96 well plate and absorbance was measured at 450 nm using an ELISA plate reader. The suspension without cells serves as a control. The measurement of these wells was subtracted from all acquired values. The normoxic group was set to 100% and hypoxic values were calculated in relation to the normoxic group. Experiments were performed in three independent runs in quintets. 2.2.3. Morphological changes Morphological changes within long-term culturing of hMSCs were evaluated according to quality parameters of hMSC 69 . 100 cells per passage were examined for cell area, circularity, ferrets diameter and aspect ratio for each culture condition. To prevent bias due to cell division, only single cells, obviously not in cell division were selected from phase contrast images during the entire cum PD time. Using ImageJ (http://rsbweb.nih.gov/ij/) each cell was manually encircled with defined settings for magnification, contrast and brightness. In sum, 3,200 cells were evaluated manually from both groups and all passages. Table 2 shows the criteria for the different subpopulations of hMSCs defined by Haasters et. al. 70 . Tab. 2: Morphological cell criteria of hMSCs. subpopulation abbreviation cell area ferret’s diameter rapidly self-renewing cells RS ≤ 6617 µm 2 ≤ 157 µm spindle shaped cells SS ≤ 6617 µm 2 ≥ 157 µm flattened cells FC ≥ 6617 µm 2 ≥ 157 µm

Material and Methods 15 The definition of Haasters et. al. was based on immortalized single cell picked hMSCs. The application of these values on freshly isolated bone marrow derived hMSCs revealed some disadvantages in respect to flattened cells, which then were mainly represented in the cell population. The ferret’s diameter presents the maximum distance in a geometric object. The aspect ratio, in contrast to the ferret’s diameter, demonstrates the right-angled ratio between the maximum and the minimum diameter and therefore includes an additional information. Due to that fact, an area of 10,000 cm 2 and an aspect ratio of 5 were chosen. 2.2.4. Cell volume measurement Since cells are three dimensional, the two dimensional data provided by the shape description parameters only reflect part of the morphological information. In order to better characterise the cells dimension the cell volume was quantified using a white light confocal microscope (PLµ 2300, Sensofar, Terrassa, Spain). Glass slides were coated with collagen I (Col1), fibronectin (FN) or laminin (LN), all freshly mixed in a concentration of 10 µg/ml in PBS, for at least 24h at 4°C. NUNC plastic microscopy slides served as a standard control. Coated slides were washed once with PBS previous to cell seeding with 1,000 cells/cm 2 in passage 3 and kept unaffected for 48h to ensure complete cell adhesion and spreading. Subsequently, cells were washed with PBS (with Ca 2+ and Mg 2+ ), fixed with 4% PFA for 15 minutes at 37°C and an ascending ethanol row up to 100% was performed in 10% steps for 10 minutes at RT. Final dehydrogenisation was performed by adding electronic grade tetramethylsilane (Sigma-Aldrich, Deisenhofen, Germany) drop wise onto the glass slides. Samples were stored in a desiccator (Duran, Wertheim, Germany) overnight and subsequently coated with 10 nm gold in a sputter coater (Bio-Rad, SCD 040, Munich, Germany). 25 cells per protein and culture condition were imaged using the white light confocal microscopy equipped with 50x/0.8 objective and a black/white camera (XC-HR58, Nikon, Düsseldorf, Germany). 51 sections with a distance of 200 nm were imaged. Data was analyzed using the open-source program Gwyddion (http://gwyddion.net/). 2.2.5. Time-lapse analysis To analyse the influence of the different cell culture conditions on cell migration, we performed time-lapse analysis with bone extracellular matrix protein coated polysty-

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