Long‐term continuous monitoring of dissolved oxygen in cell culture medium for perfused bioreactors using optical oxygen sensors

Abstract

For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were +/-5.1 mm Hg and -3.8 mm Hg at the bioreactor outlet, and +/- 19 mm Hg and -18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.

MEMS Device to Monitor Biological Oxygen Uptake at Arrays of Single Cells and Small Cell Clusters

“…Therefore, measuring oxygen uptake of cells can provide insight into metabolic processes and overall status of cells. Oxygen sensors using optical detection of dissolved molecular oxygen have been developed to monitor extracellular oxygen concentration as a measure of cell viability in cell culture [24,25]. Oxygenation status of normal and tumor tissue in vivo has been monitored using oxygen-dependent quenching of phosphorescence [26,27].…”

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

“…11 For both probes, the spatial resolution is limited by the size and surface area of the probe head with measurements restricted to a single point. 12 These probes also lack the ability of registering the measurement location. Alternatively, luminescence utilizes fluorescence or phosphorescence to measure the intensity or lifetime of a fluorophore.…”

“…Alternatively, luminescence utilizes fluorescence or phosphorescence to measure the intensity or lifetime of a fluorophore. 12,13 Luminescence applied non-invasively, and used with lifetime rather than intensity, results in measurements that are independent of probe concentration, photobleaching and excitation drifts.…”

An Overview on Bioreactor Design, Prototyping and Process Control for Reproducible Three‐Dimensional Tissue Culture

“…Process control is based on online-sensing of oxygen and pH, and samples of culture supernatant are periodically harvested (at 12 h and 24 h) to this purpose. New approaches of miniaturized fl uorescence-based online sensing for pO 2 , pCO 2 and pH can be implemented [113,114]. In perfusion culture, the pH and defi ned levels of dissolved oxygen, metabolites and factors in the PCS can be adjusted by media and gas exchange.…”