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Primary human hepatocyte (PHH) models have limited longevity and require high inoculum for HBV infection with minimal spread. We aimed to develop 3D cell culture models to overcome the limitations of existing models and to expand their utility for drug-related studies. Here, we report the establishment of two spheroid models utilizing de novo HBV-infected mouse-passaged (mp)PHH and mpPHH isolated from HBV-infected liver chimeric mice (HBV-mpPHH). Our data demonstrates that our models maintain detectable infection and human albumin levels up to 75 days, and therefore have enhanced longevity compared to existing models. As a proof-of-concept we used our de novo HBV-infected model as a drug-testing platform to validate an HBV capsid assembly modulator (CpAM). We report that we have established two HBV-infected 3D cell culture models and have characterized these models as practical and novel approaches with the potential to enhance the relevance and scope of in vitro HBV studies. • Existing models for the study of HBV infection have limitations and alternative models are needed. • 3D models are particularly needed as they more accurately recapitulate the in vivo environment of infection. • Two novel mouse-passaged (mp) primary human hepatocyte (PHH) 3D models are here reported. • Both models maintain HBV infection and have enhanced longevity compared to existing models. • D e novo HBV-infected mpPHH are sensitive to a capsid assembly modulator , supporting their use as a drug-testing platform. [ABSTRACT FROM AUTHOR]