Non-destructive viability assessment of cancer cell spheroids using dynamic optical coherence tomography with trypan blue validation.

3D cell cultures are widely used in biomedical research for the recapitulation of microenvironments. Viability assessment and monitoring of these intricate conformations remain an open problem as standard cell viability protocols based on colorimetry or microscopy are not directly applicable to intact 3D samples. Optical coherence tomography (OCT) has been explored extensively for subsurface structural and quasi-functional analysis of 3D cell cultures and tissue. Recent studies of dynamic OCT as a source of cellular contrast have found qualitative associations with necrosis in cell spheroids, suggesting potential as a viability marker. We present empirical and validated evidence for dynamic OCT as a quantitative indicator of cell viability in 3D cultures. We analysed over 240 MCF-7 cancer cell spheroids with dynamic OCT and corresponding viability measurements using the trypan blue exclusion assay. Significant effects of common reagents dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) on OCT readouts were noted. We proposed a regression-based OCT brightness normalisation technique that removed reagent-induced OCT intensity biases and helped improve correspondence to the viability assay. These results offer a quantitative biological foundation for further advances of dynamic OCT as a novel non-invasive modality for 3D culture monitoring.