Fisher, Benjamin

Abstract Hepatic spheroids are widely used in bioartificial liver (BAL) systems, hepatotoxicology, and pharmacology due to improved functional stability compared with monolayer cultures. However, many existing methods rely on specialized equipment or static platforms that limit scalability and dynamic culture applications. Primary rat hepatocytes were cultured as three‐dimensional spheroids using a rocker‐based suspension system. Spheroid formation was evaluated across a range of rocking speeds (7–18 cycles per minute [cpm]) and inoculation densities. Spheroid morphology was quantified over time using two‐dimensional imaging. Functional performance was assessed by urea and albumin production on days 1, 4, 7, and 10. Spheroid formation efficiency was evaluated using volumetric modeling. Mean spheroid diameter decreased from 199 ± 41 μm at 7 cpm to 80.2 ± 17.6 μm at 18 cpm. The spheroid area was significantly influenced by both culture day ( p < 0.0001) and rocker speed, with significant differences observed across speeds at all time points ( p < 0.001, days 1–7). Mean spheroid area at 10 cpm was 13,750 ± 9051 μm 2 (day 1), 26,730 ± 8658 μm 2 (day 4), and decreased to 22,526 ± 8705 μm 2 (day 7). Urea production differed across speeds (ANOVA, p = 0.043), with peak performance at 12 cpm (median 3.47 μg/μg DNA) compared to 7 cpm (median 0.11 μg/μg DNA). Albumin production was 1086.8 ng/μg DNA on day 1 and 1534.4 ng/μg DNA on day 10. Urea production was 0.62 μg/μg DNA on day 1 and 1.25 μg/μg DNA on day 10. Median hepatocyte incorporation was 86% (range 73%–116%) by day 4. This rocker‐based method provides a simple, low‐cost, and scalable approach for generating hepatic spheroids with tunable size distributions under dynamic conditions. The system supports progressive functional maturation and is compatible with established scale‐up strategies, supporting its potential for translational applications.