Hypoxic culture increases expansion efficiency and decreases in senescence. Cells were seeded at 50 cells/cm² and cultured under normoxic and hypoxic conditions. After 12 days of culture, the cells were recovered and reseeded at 50 cells/cm² and cultured under the same conditions. Cell expansion rate (A) and population doubling time (B) for each passage were calculated. Normoxic culture decreases in cell expansion rate and increases in population doubling time as the increase of passage number (Pn: Passage No). (C) Normoxic and hypoxic cells were incorporated with BrdU for 18 hours and then detected by flow cytometry. Hypoxic cells increase in BrdU incorporation rate compared with normoxic cells. (D) Photomicrographs (original magnifications, 10×/0.25 NA dry objective) of normoxic and hypoxic cells were captured using an Olympus IX70 inverted phase contrast/fluorescence microscope equipped with an Olympus DP70 digital camera (Olympus Japan) and analyzed with the Zeiss AxioVision 4.4 software. Hypoxic cells increase in cell size and become large and flat after expansion compared with hypoxic cells. (E) Cells expanded under normoxic and hypoxic conditions were stained with β-galactosidase (β-gal). Normoxic cells increase in the percentage of β-gal expression compared with hypoxic cells. (F) Western blotting and quantification for senescence marker protein-30 (SMP-30). After expansion, normoxic cells decrease in SMP-30 level, while hypoxic cells increase the expression. (G) Real time-PCR (top panel) and quantitative real time-PCR (bottom panel) analysis for markers of senescence. After expansion, the expression of senescence-related genes significantly increases under normoxic conditions compared with hypoxic conditions. Values are mean + SD; *P < .05 and **P < .01 indicate significant variance (independent t test) between normoxia (Nor) and hypoxia (Hyp). Bar = 50 μm.