Discussion

Cells cultured as 3D spheroids provide a more physiologically relevant environment that has promise for being more predictive of in vivo toxicity compared to using standard cell culture models. However, the size and complexity of 3D cultures often present a challenge for existing in vitro cell health assay chemistries that were designed for use with cells grown in suspension or as a monolayer on a plastic surface. There is an unmet need for effective assays to screen for cell health markers that have been verified for use with 3D culture models. The size of the 3D spheroid is a key factor that needs to be considered for effectively extracting the desired marker without destroying the biological activity required to make the measurement. We will present the results of experiments designed to improve performance of assays for 3D spheroids. We have improved the lytic capacity of assay reagents by optimizing the detergent concentration and physical disruption parameters necessary to extract the desired markers. Improved protocols and reagents will be described to measure ATP as a cell viability marker, caspase-3/7 activity as an apoptosis marker and HIF-1 promoter driven expression of luciferase as a hypoxia marker.