A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy

Abstract

We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.

Phosphorous metabolites and steady-state energetics of transformed fibroblasts during three-dimensional growth

“…As a result, metabolic hypoxia could be detected in the 31 P-NMR spectra, whereas the radiobio- logical hypoxia would be invisible. Previous work with spheroids does not support this hypothesis (20,21). However, in the spheroid types investigated previously, metabolic hypoxia was accompanied by necrotic cell death, which probably contributes nothing to the 31 P spectrum (27,81).…”

“…Cells dissociated from spheroids by mild trypsinization (0.125-0.25% trypsin) and mechanical means were counted with a standard particle counter (Coulter, Hialeah, FL) equipped with a pulse height analyzer (Nucleus). Acellular debris was excluded from the cell count by determining a region of interest for the cell volume as described previously (20). Cell volume distributions of Ն10,000 cells per cell population were used to calculate the average cellular volume based on calibration of the system with polystyrene microspheres.…”

“…Temperature, PO2, and extracellular pH in the chamber were monitored continuously during one experimental series as described previously (20). Measurements were carried out at 37 Ϯ 0.5°, 20% O2, and a pH of 7.3 Ϯ 0.1 in supplemented phosphate-free medium.…”

“…Peak height analysis was carried out with Bruker analysis software (Bruker Instruments, Billerica, MA). Peak heights in a spectrum within a given experiment were referenced to the corresponding height of the MDA peak (25 mM; 16.4 l) in the spectrum (20,21).…”

“…Multicellular spheroids represent a well-established in vitro model for investigation of the interrelationship among growth/proliferation kinetics, viability, and energetic state of tumor cells (23,39,53). An NMR system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy has been established in our laboratory (20). Based on this technical development, a few 31 P-NMR studies have focused on the phenomenon of the development of a quiescent cell population in the inner viable cell rim of spheroids and the emergence of necrosis.…”