Calibration for water and culture media oxygenation

Fluid oxygenation profile studies for both water and cell culture media (Dulbecco’s Modified Eagle Medium 1 g/L glucose [Gibco 11885-084, Grand Island, New York, USA], 10% FBS, and less than 1% supplement), were performed utilizing a NeoFox Phase Fluorometer (Ocean Optics, Inc., Largo, Florida, USA) fitted with an oxygen sensing probe via an optical cable. The oxygen sensor was inserted into the FABRICA through a custom lid and submerged into the internal inlet within the bioreactor. The NeoFox was connected to a Dell laptop (Dell, Inc., Round Rock, Texas, USA) through a USB cable and measurements were recorded using NeoFox-provided Viewer Software.

The FABRICA bioreactor was placed in a cell culture incubator and acclimated to 37 °C and 8% carbon dioxide (CO₂). Calibration measurements were taken at the FABRICA bioreactor internal inlet. To calibrate the NeoFox Oxygen Sensing Probe, oxygen-free water, oxygen-free culture media, equilibration water and equilibration culture media were prepared. The oxygen-free solutions were prepared using two grams of sodium sulfite in 50 mL of purified water or culture media. Equilibration solutions were produced by bubbling house air in 100 mL of water or culture media for half an hour or more. For each solution, a 33 mL volume was poured into the FABRICA bioreactor and the system was primed for ten minutes prior to calibration at 10 mL/min. The oxygen probe was inserted into the top of the removable lid of the FABRICA bioreactor such that the sensor was positioned at the internal inlet of the FABRICA (Figs. 2 and ​and3).3). Once the readings stabilized, an oxygen measurement was read and used as the zero percent level point for the calibration. The system was emptied and then flushed with 33 mL of Dulbecco’s phosphate-buffered saline (DPBS) for 10 minutes, ensuring that no remnants of the sodium sulfite remained. The system was then emptied again and filled with 33 mL of equilibration solution. Once the measurements had stabilized, an oxygen reading was read and used as the 20.9% oxygen calibration level for the oxygen-sensing probe.

Photo of FABRICA perfusion setup with FABRICA bioreactor, peristaltic pump, oxygenator, and NeoFox oxygenation sensor probe connected by silicone tubing. For the oxygenator-free setup, the oxygenator and the vacuum are removed so that the outlet from the FABRICA goes directly into the pump.