The choice of ultra‐low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function

Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments.