Introduction: The evaluation of drug's cytotoxicity is a crucial step in the development of new pharmacological compounds. 31P NMR can be a tool for toxicological screening, as it enables the study of drugs' cytotoxicity and their effect on cell energy metabolism in a real-time, in a non- invasive and non-destructive way. This paper details a step-by-step protocol to implement a bioreactor system able to maintain cell viability during NMR acquisitions, at high cell densities and for several hours, enabling toxicological evaluation of pharmacological compounds in living cells.
Method: HeLa cells were immobilized in agarose gel threads and continuously perfused with oxygenated medium inside a 5 mm NMR tube. Signals corresponding to intracellular high-energy phosphorous compounds were continuously monitored by 31P NMR to assess cell energy levels, intracellular pH and intracellular free Mg2+ concentrations ([Mg2+]f) under control and in the presence of two different cytotoxic drugs, calix-NH2 or 5-fluorouracil (5-FU).
Results: The bioreactor system was effective in maintaining cell energy levels as well as intracellular pH and [Mg2+]f along time, with a good 31P NMR signal to noise ratio. Calix-NH2 and 5-FU decreased cell energy levels by 35% and 39%, respectively, with a negligible increase in intracellular [Mg2+]f, and without affecting intracellular pH.
Discussion: The immobilization and perfusion system here detailed, along with 31P NMR, is useful in toxicological evaluation of new pharmacological compounds, enabling the continuous assessment of drugs' effect on energy levels, intracellular pH and [Mg2+]f in intact cells, for several hours without compromising cell viability.
Keywords: (31)P NMR spectroscopy; 5-fluorouracil; Bioreactor; Calixarene; Cytotoxicity.
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