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 x 10(-3) s-1 microM-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 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids

“…[17][18][19] Perfusate was prewarmed to 40°C and gassed with 95%O 2 /5%CO 2 in a large reservoir and recirculated through the sample from bottom to top at a rate of 6 ml/ min; the spheroids tumbled gently within the sample volume during the experiment. The temperature within the cell chamber (35°C) was regulated by the spectrometer temperature control unit and monitored continuously using a fiber optic temperature sensor (Luxtron, Mountain View CA).…”

31p nme spectroscopic study of the effects of γ‐irradiation on rif‐1 tumor cells perfused in vitro

“…The cell density of RIF-1 cells in the alginate beads is 5 X lo7 cells/cm3, one-half the cell density in the viable rim of typical multicellular tumor spheroids ( 12,13). For EMT6 / Ro cells in an identical alginate preparation, we have estimated the magnitude of oxygen gradients within the beads using an I9F NMR method (14). These calculations indicate that greater than 96% of the EMT6 /Ro cells experience oxygen tensions greater than 20 pM, sufficient to saturate both the radiobiological oxygen effect ( 1 5 ) and the K, for mitochondria1 oxygen consumption (0.25 pM in ascites tumor cells, Ref.…”

Monitoring of dissolved oxygen and cellular bioenergetics within a pancreatic substitute

“…Specifically, the inverse of the T 1 relaxation time of specific perfluorinated chemical groups are known to vary linearly with the DO concentration surrounding the emulsion (Fan et al, 2002;McGovern et al, 1993;Noth et al, 2004;Reid et al, 1985;Shukla et al, 1996) 19 F chemical shift imaging, long acquisition times are required, which are a caveat to in vivo applications due to the long anesthesia times required of experimental animals. To overcome this limitation, one can acquire a single DO concentration from the entire construct, representing a volume-average measurement of the oxygen profile within the volume-of-interest (VOI); a mathematical model can then be used to assess the cellular and oxygen spatial profiles within the construct, thus allowing for rapid measurements in vivo (Gross et al, 2007).…”