2011). in cells cultured under normoxia. To conclude, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies. for 5?min. Pelleted cells were resuspended and cultured for 14C21?days in LG-DMEM containing 1 insulin-transferrin-selenium (ITS; Rabbit Polyclonal to KCY Life technologies-Gibco), 1?mM sodium pyruvate (Life Technologies-Gibco), 0.1?M dexamethasone, 397?g/mL ascorbate-2-phosphate, and 10?ng/mL transforming growth factor-1 (R&D Systems, Minneapolis, MN, USA). Chondrogenic induction was evaluated at 80?% confluence by staining with toluidine blue to detect extracellular accumulation of chondrocyte matrix (Sigma-Aldrich). Culture of BM-MSCs under hypoxic and normoxia BM-MSCs derived from five donors (P5 D1 MSC, P5 D2 MSC, P5 D3 MSC, P2 D4 MSC, and P5 D5 MSC) were maintained under normoxia (37?C, 5?% CO2, 95?% air) for 7?days and then divided into two groups, a normoxia group and a hypoxia group (37?C, 1?% O2, 5?% CO2, and 94?%?N2). Cells were plated at a density of 1000 cells/cm2 and placed in a normoxia or a hypoxia chamber. Cells were observed on day 7 of culture using a phase contrast microscope (Olympus CK40, Melville, NY, USA). Cells were harvested using 0.05?% trypsin/EDTA, incubated with 4?% trypan blue solution, and counted using a hemocytometer (Marienfeld, German). Cells in each group were counted and subcultured once per week for 2?weeks. Among MSCs derived from different donors, donor 1 (D1) MSCs were Colchicine counted and passaged under normoxia or hypoxia once per week for 8?weeks. Cell growth was assessed by counting cumulative Colchicine cell numbers each week following initial plating at a density of 1000 cells/cm2. Cumulative cell numbers were counted for 8?weeks in four independent experiments. At each passage, the number of cell divisions was calculated using the following formula: number of cell divisions?=?Log2(is the final number of cells after 7?days of incubation. Apoptosis assay Colchicine by flow cytometry Apoptosis assays were performed using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis antibody (BD Bioscience) according to the manufacturers instructions. Briefly, BM-MSCs initially plated at 1000 cells/cm2 were maintained for 7? days under normoxia or hypoxia and then subcultured once per week. After 2?weeks, cells were collected and resuspended in binding buffer. Annexin V-FITC and propidium iodide (PI) were added, and the reaction was incubated in the dark for 15?min. The fluorescence intensity of the cells was evaluated by flow cytometry (BD FACSVerse?), and the data were analyzed using the BD FACSuite? software. RNA extraction and RT-PCR analysis Total RNA was isolated from BM-MSCs cultured under hypoxic or normoxia using an RNeasy kit (Lifethechnology-Ambion, Carlsbad, CA, USA) and was used as a substrate for the QuantiTect Reverse Transcription Kit according to the manufacturers instructions (Qiagen, Valencia, CA, USA). The cDNAs were amplified by PCR using the primers shown in Table Colchicine ?Table1.1. The band intensity of each PCR product was measured using NIH image/ImageJ and normalized against that of GAPDH mRNA. Table 1 Primer sequences used for RT-PCR octamer-binding transcription factor 4, Kruppel-like factor 4, v-myc avian myelocytomatosis viral oncogene homolog, C-C motif chemokine ligand 2, interleukin 6, C-X-C motif chemokine 9, C-X-C motif chemokine 10, C-X-C motif chemokine receptor 4, C-X-C motif chemokine receptor 7, glyceraldehyde-3-phosphate dehydrogenase aForward (F) and reverse (R) primers used to detect mRNA expression of the indicated targets Cell size measurements BM-MSCs initially plated at a density of 1000 cells/cm2 were maintained for 7?days under normoxia or hypoxia and then subcultured once per week. After 6?weeks, cells were collected and resuspended in FACS buffer (BD Bioscience). Cell size was measured by flow cytometry (BD FACSVerse?), and the data were analyzed using BD FACSuite? software. FSC-A parameters of the software were used for cell size measurements, as recommended by BD (see BD FACService TECHNOTES, Customer Focused Solutions, Vol. 9 No. 4 October, 2004; Shapiro 2003). Quantitative SA–galactosidase assay The cells were cultured at a density of 4??103 cells/cm2 in 6-well plates containing media. The cells were fixed with 4?% paraformaldehyde in PBS, washed with PBS, and then stained using an senescence-associated (SA) -gal staining kit (Cell BioLabs, San Diego, CA, USA) for 10?h in an incubator chamber at 37?C in the dark. Positive cells were counted and results were expressed as the mean percentage of SA–gal-positive cells among total cells. Statistical analysis Data are expressed as the mean??standard deviation. Statistical significance of test. Results Characteristics of.