Full-Time Physioxic Cell Culture and Handling Improves MSC Proliferation Over Hypoxic Pre-Conditioning in Vitro

Cells cultured and handled in traditional room air experience a dramatic shift in oxygen levels when administered in vivo. It has been reported that hypoxic pre‐conditioning of mesenchymal stem/stromal cells (MSC) in culture can change cell function, increasing cell survival in vivo after administration, changing cell migration and modulating cytokine and exosome secretion patterns. These treatments place cells in low oxygen atmospheres for lengths of time from 15 minutes to 36 hours, sometimes with a period of reoxygenation in room air conditions before administration, risking oxidative stress. Here, we compare hypoxic pre‐conditioning to full‐time physioxic conditions with the null hypothesis that in vitro culture conditions make no difference to MSC morphology and proliferation rates after the cells are introduced to physioxic conditions as if administered. MSC cultured and handled under completely controlled conditions in the Xvivo System were split into two groups: (1) cells cultured under full‐time physioxia (5%O2), and (2) cells cultured under supraphysioxic room air incubator conditions (18%O2) and then pre‐conditioned in low oxygen (1% O2) conditions for 24 hours before reoxygenation at supraphysioxic room air incubator oxygen (18% O2) for 24 hours. The cells were then exposed to venous blood oxygen levels (5% O2) as if injected in vivo . Using an immersion oxygen probe, we recorded vessel headspace and pericellular medium oxygen levels under each regimen. Using the PHI Holomonitor M4, we recorded holographic time‐lapse images of the cells for changes in cell morphology or proliferation rates. In looking at cell culture oxygen change kinetics, we found that sharp changes in atmospheric oxygen levels were followed by much slower oxygen changes in the vessel headspace, and pericellular oxygen. MSC were actually at low oxygen conditions for far less time than the pre‐conditioning period. Hypoxic pre‐conditioning after incubation at 18%O2 increased the numbers of cells that failed to divide at physioxic oxygen levels, disproving the null hypothesis. We concluded that full‐time physioxic conditions for cell handling as well as incubation may be more conducive to MSC proliferation than hypoxic pre‐conditioning. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .