Gel‐entrapment of perfluorocarbons: A fluorine‐19 NMR spectroscopic method for monitoring oxygen concentration in cell perfusion systems

Abstract

Oxygenation is a major determinant of the physiological state of cultured cells. ¹⁹F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T₁) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T₁) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 ± 0.15 × 10⁻³ s⁻¹μM⁻¹ and an intercept of 0.50 ± 0.04 s⁻¹. During perfusion with medium equilibrated with 95%/5% O²/CO², changes in PFC T₁s indicate that the average oxygen concentration was reduced from 894 ± 102 μM in the absence of cells to 476 ± 65 μM and 475 ± 50 μM in the presence of 0.7 × 10⁸ EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 μl of FTPA/ml of gel had no effect on the energy status of the cells as indicated by ³¹P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T₁ of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/RO cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate. The average PFC 1/T₁s predicted using different literature values for volume and oxygen consumption− 1.10 ± 0.10 and 1.28 ± 0.36 s⁻¹−agreed well with the experimentally measured value-1.104 ± 0.004 s⁻¹. Thus, the model is a suitable tool for calculation of oxygen consumption rates from PFC T₁s in well-oxygenated cell perfusion systems.