AbstractThe intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma‐derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT‐PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon‐stimulated genes [ISG]) to interferon‐α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well‐differentiated ICOs using spinoculation, with time and donor‐dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor‐dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon‐α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor‐dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals.