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Updated: Jan 16, 2026
Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells
Published on: June 16, 2019
Measuring Propidium Iodide Brightness as a Function of Viability: A High-Throughput Quantitative Assessment of
Michael G Megaly1, Diane Tobolt1, Bat-Erdene Namsrai1
1Division of Transplantation, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA.
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.
