Measuring Propidium Iodide Brightness as a Function of Viability: A High‐Throughput Quantitative Assessment of Hepatic Spheroids

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.