03550nas a2200613   4500000000100000000000100001008004100002260001500043653001800058653002000076653004300096653002500139653002100164653003500185653003700220653001600257653001100273653000900284653001600293653002300309653003200332653003000364653002500394653002400419653001700443653001900460100002200479700002400501700002300525700002300548700001600571700001500587700002200602700001500624700002000639700001700659700002000676700001600696700002100712700002400733700002500757700001700782700001800799700002400817700001500841700001600856700002000872700002900892245012100921300001201042490000801054520186001062022001402922       2018                            d  c2018-04-0110aCell Survival10aCells, Cultured10aChemical and Drug Induced Liver Injury10aCoculture Techniques10acryopreservation10aCytochrome P-450 Enzyme System10aDose-Response Relationship, Drug10aHepatocytes10aHumans10aMale10aMiddle Aged10aModels, Biological10aPharmaceutical Preparations10aPredictive Value of Tests10aPrimary Cell Culture10aSpheroids, Cellular10aTime Factors10aToxicity Tests1 aCatherine C. Bell1 aAnita C. A. Dankers1 aVolker M. Lauschke1 aRowena Sison-Young1 aRoz Jenkins1 aCliff Rowe1 aChris E. Goldring1 aKevin Park1 aSophie L. Regan1 aTracy Walker1 aChris Schofield1 aAudrey Baze1 aAlison J. Foster1 aDominic P. Williams1 aAmy W. M. van de Ven1 aFrank Jacobs1 aJos van Houdt1 aTuula Lähteenmäki1 aJan Snoeys1 aSatu Juhila1 aLysiane Richert1 aMagnus Ingelman-Sundberg00aComparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study  a655-6660 v1623 aPrimary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.  a1096-0929