Femke Mathot

3 MSC-seeding on processed nerve allografts 47 sterilized using g -irradiation and stored at 4 ° Celsius after processing. Analysis of Cell viability To assess the influence of chemical products used to process the allografts on the MSC- viability and to compare the vulnerability of differentiated MSCs and control MSCs, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assays (CellTiter 96® Aqueous One Solution Cell Proliferation Assay; Promega Corporation, WI, USA) were carried out according to the manufacturer’s protocol. 33 This assay is a colorimetric method to determine the number of viable cells. Living cells in culture have metabolic activity and will convert the added reagent in formazan. Formazan quantity is directly correlated to the amount of 490nm absorbance and can therefore be measured with an Infinite® 200 Pro TECAN Reader (Tecan Trading AG, Switzerland). Undifferentiated and differentiated MSCs (5,000 in 100µL growth medium per well) were transferred to a p-HEMA (Poly 2-hydroxyethyl methacrylate; Sigma-Aldrich Corp., MO, USA) coated 96-well plate, to prevent cells from migrating to the plastic surface of the well. After soaking in growth medium for two hours, 48 2mm-segments of processed nerve allografts were divided among the wells containing the cells. The medium was changed every 72 hours; undifferentiated MSCs were cultured in normal growth medium, differentiated MSCs were cultured in differentiation medium. The metabolic activity of undifferentiated MSCs (undifferentiated MSCs + pHEMA + allograft) was compared to differentiated MSCs (differentiated MSCs + pHEMA + allograft) on four estimated time points (T = 1, 2, 3 and 7 days). The remaining groups represented normal cell viability (pHEMA + undifferentiated MSCs and pHEMA + differentiated MSCs) and negative controls (no cells). For each group, the metabolic activity of three replicates per sample was tested twice on each time point. Colorimetric assays were performed with the Infinite® 200 Pro TECAN Reader (Tecan Trading AG, Switzerland) at an absorbance wavelength of 490nm. The metabolic activity of undifferentiated MSCs and differentiated MSCs in the vicinity of an allograft was expressed as a ratio of the metabolic activity of undifferentiated MSCs and differentiated MSCs without an allograft. The experimental design is shown in table 1 . Table 1. Experimental design experiment 1. MSCs: Mesenchymal Stem Cells MTS assay: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay Group Description Time points N Outcome measurements I Undifferentiated MSCs + allograft T1 = 1 day T2 = 2 days T3 = 3 days T4 = 7 days 3 samples per group for each time point Metabolic activity (MTS assay) II Differentiated MSCs + allograft III Undifferentiated MSCs IV Differentiated MSCs