ABSTRACT Background Three‐dimensional (3D) hepatocyte spheroids better recapitulate liver microenvironments than monolayers, but robust, high‐throughput viability assessment remains challenging because of diffusion limits and stain penetration. Objective To evaluate a simple imaging‐based approach that quantifies spheroid viability by measuring acridine orange (AO) and propidium iodide (PI) fluorescence and computing a PI brightness‐to‐area ratio. Methods Rat hepatocyte spheroids were formed on a rocker, stained with AO/PI, and imaged on an inverted fluorescence microscope with fixed exposure settings. We prepared mixtures representing nominal 0%, 30%, 50%, 70%, and 100% viable spheroids and analyzed the relationship between viability and the PI brightness‐to‐area ratio. Statistical analyses included one‐way ANOVA with Tukey's HSD and simple linear regression with diagnostic checks. A urea/DNA functional assay as well as a small blinded study both served as an orthogonal validation. Results The PI brightness‐to‐area ratio decreased with increasing percent viable cells (Pearson r = −0.99; R 2 = 0.98; p = 1.81 × 10 −4 ). Residuals were approximately normal and homoscedastic. Urea/DNA strongly and positively correlated with viability ( r = 1.00; p = 1.10 × 10 −4 ; R 2 = 1.00) with regression equation: y = 9.049444 × 10 −6 x + 3.036814 × 10 −5 . All blinded studies were within 10% of established viability; average difference between mean observer estimates and ground truth was +1.7 percentage points (range −3.7 to +10). Conclusions A fixed‐setting AO/PI imaging workflow yields a rapid, accessible proxy for hepatocyte spheroid viability that correlates with a functional readout. This approach is well‐suited to high‐throughput screening and method optimization.
Megaly et al. (Wed,) studied this question.