In Vitro Hypoxia Responsiveness Of Fdg And Faza Retention: Influence Of Shaking Versus Stagnant Conditions, Glass Versus Polystyrene Substrata And Cell Number Down-Scaling
Abstract Background. In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose (FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. Methods. Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2h to 21%, 0.5% or 0% O2 and FDG or FAZA was added, followed by cell harvest and analysis of radioactivity 1h (FDG) or 3h (FAZA) after. Experiments were conducted with/without orbital shaking. Results. The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven FAZA, and to some extent FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. Conclusions. Most experiments can be performed appropriately in the absence of shaking and with downscaling of cell number but the use of conventional plasticware is highly problematic for studies on tracers and drugs that are metabolized and retained or activated at low O2 levels.