Local determination of oxygenation and cell vitality in multicellular spheroids by microelectrodes and photometry

Abstract

Multicellular spheroids are nearly spherical aggregates of cultured tumor cells. They develop radial proliferation gradients and are used as models to study radiation as well as drug effects for improving therapeutic strategies. As the knowledge of oxygenation as well as cell vitality in the spheroids is of importance to understand antiproliferative measures methods have to be applied which are able to resolve the three-dimensional architecture of the spheroids without being destructive. Microelectrodes sensitive to oxygen or ph and specifically designed to record extracellularly symmetrical PO₂- and pH-gradients give information about the relationship between oxygen consumption and lactate production in different depths of the spheroids. The quotient delta PO₂/delta pH can be related to radiation sensitivity and growth rate of the tumor spheroids. Combination of measurements with PO₂-sensitive microelectrodes and photometry of the respiratory chain (Cytochromes, NADH, FAD) give information about the critical PO₂ in different depths of the spheroids or regulation of oxygen consumption by glucose. After staining of tumor cells in spheroids with vital/letal fluorescence dyes for characterisation of areas with proliferative or necrotic cells fluorescence intensity measurements in depths up to 200 mm can be obtained by confocal laser microscopy. Symmetrical distribution of FDA (Fluoresceindiacetate) showed a decline of viably stained cells in the centre at a diameter of 40–100 mm with an appearance of Lucifer Yellow VS stained cells in the centre, indicating starting necrosis.