. Long-Term Continuous Monitoring of Dissolved Oxygen in Cell Culture Medium for Perfused Bioreactors Using Optical Oxygen Sensors Frank G. Gao, 1 Antony S. Jeevarajan, 2 Melody M. Anderson 2 1 Wyle Laboratories, Life Sciences, Systems and Services, 1290 Hercules Drive, Suite 120, Mail Stop BT-37, Houston, Texas 77058 2 NASA/ Johnson Space Center, 2101 NASA Road One, Mail Code SJ, Houston, Texas 77058; telephone: 281-483-4298; fax: 281-483-0402; e-mail: antony.s.jeevarajan @nasa.gov Received 10 September 2003; accepted 22 December 2003 Published online 24 March 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20010 Abstract: For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concen- tration of dissolved oxygen (DO) present in the culture med- ium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was devel- oped 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 sen- sors 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 bio- reactor 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 out- let and 80 – 116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were F 5.1 mm Hg and  3.8 mm Hg at the bioreactor outlet, and F 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. B 2004 Wiley Periodicals, Inc. Keywords: pO 2 (partial pressure of oxygen); dissolved oxygen; optical sensor; tris(4,7-diphenyl-1,10-phenan- throline)ruthenium(II) chloride; cell culture; BHK-21 cells INTRODUCTION While growing mammalian cells in a perfused bioreactor, it is essential to monitor the concentration of dissolved oxygen (DO) present in the medium to ascertain the health of the cells. Continuous measurement of the amount of DO in the cell culture medium under sterile conditions in NASA’s perfused rotating bioreactors requires that the oxygen sensors provide adequate sensitivity, be sterilizable, and nontoxic to the cells. Additionally, long-term cell culture experiments require that the calibration be maintained continuously for several weeks or months. The commonly used oxygen sensors to date are primarily electrochemical sensors based on Clark-type oxygen elec- trodes (Clark, 1959). The Clark electrode suffers from a va- riety of limitations including long-term instability, drifts in calibration, flow dependence, and susceptibility to electrical interferences when used in rotating bioreactors. Optical fiber optode sensors based on luminescence quenching provide a promising alternative to amperometric methods in solving the problems mentioned above. Bergman described the first oxygen sensor based on fluorescence quenching in 1968 (Bergmann, 1968), which was introduced into medicine by Lu ¨bbers and Opitz in 1975 (Lu ¨bbers and Opitz, 1975). The introduction of immobilized indicators was an important landmark in the development of optical sensors for conti- nuous monitoring in biological fluids. Due to its great poten- tial for widespread application, optical sensing has received much attention and very intensive studies in this field have been carried out (Bambot et al. 1994; Choi and Xiao, 1999; Holst et al., 1997; Klimant and Wolfbeis, 1995; Lin et al., 1976; Palaniappan and Kumar, 1993; Trettnak et al., 1995; Wolfbeis et al., 1986; Wolthuis et al., 1992). Optical oxygen sensors are based on the property of molecular oxygen quenching of fluorescence. The lumines- cent probe molecules like tris(2,2V-bipyridyl)ruthenium(II), B 2004 Wiley Periodicals, Inc. Correspondence to: Antony S. Jeevarajan Contract grant sponsor: NASA Contract grant number: NAS9-97114