Combining a Real-Time In Vitro Cell Viability Assay and RNA Extraction from the Same 3D Spheroids

Although the implementation of 3D cell culture models is growing rapidly, there is an unmet need to establish in vitro assay methods to interrogate larger and more complex 3D structures. We report here the results of using a novel small molecule probe that viable cells convert into a substrate for a shrimp-derived luciferase to generate a signal proportional to the number of viable cells. The detection reagent can be incubated with cells and produce a luminescent signal for days while having minimal effect on the biology of the system. An advantage of this approach is that viable cells remain in culture and be used for multiplexing with a variety of other assay chemistries such as: orthogonal methods of confirming viable cell number, measuring dead cells, gene reporter assays, apoptosis assays, and extraction of high quality RNA from individual 3D spheroids. The results of RNA extraction from the same samples used for viability measurements demonstrate the presence of the real-time cell viability reagent did not have an effect on yield or integrity of the RNA. Multiplexing the real-time assay with other methods provides a more efficient experimental approach compared to evaluating replicate samples from parallel assay plates.