Sustained hyperoxic exposure decreases cell viability and reduces cell growth in pancreatic beta cell lines
Nicholas Price1, Craig S Weber1, Carola G Davila1, Charles W Putnam1, Klearchos K Papas1.
1Department of Surgery, University of Arizona, Tucson, AZ, United States
Background. Immunoprotective encapsulation devices hold promise for enabling cell-based insulin replacement therapies without immunosuppression, for example with human islets or stem cell–derived β cells. However, for the encapsulation device to be of reasonable size, cell densities within the device must be high; this contributes to a hypoxic environment within the device. Supplemental oxygen to an encapsulation device would reduce the highly detrimental effects of low oxygen on the cells residing within it. However, hyperoxia can also be toxic; this study investigates the effects of various oxygen concentrations on pancreatic β-cell lines in order to identify a safe oxygen exposure level.
Methods. Pancreatic β-cell lines were exposed to normoxic or hyperoxic culture conditions (40, 60, and 95%) for 7 days, after which viability was assessed by oxygen consumption rate (OCR) and with acridine orange staining. Cell counts were performed after nuclear DNA staining with DAPI.
Results. In hyperoxia of only 40% O2, MIN6 cells showed reduced OCR (P<0.0001) compared to normoxic controls with toxic effects being shown in as little as 24 hours. INS 832 and bTC3 cell lines, however, showed reduced OCR only under far higher hyperoxic conditions (95% O2) compared to normoxic controls (P<0.0001). 60% O2 culture had a reduction in cell growth in all cell lines compared to 21% normoxic culture and 40% O2 culture over the course of 7 days. 95% O2 culture had a more pronounced reduction in cell growth compared to 21% normoxic culture over the course of 7 days (P<0.0001).
Conclusions. For cell-based insulin replacement in a reasonably sized encapsulation device to be applied as a treatment for Type 1 diabetes, supplemental oxygen delivery will be required to support β cells and other insulin secreting cell lines. Our studies demonstrate that sustained exposure to excessive hyperoxia of more metabolically active pancreatic β-cell lines compromises cell viability and cell growth, suggesting that the range of permissible hyperoxia may be more limited than previously anticipated, especially for rapidly dividing cells.
We would like to thank all of the members of the Papas Laboratory at the Institute for Cellular Transplantation. Partial funding from Procyon Technologies LLC.
