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

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; R² = 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; R² = 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.