Cryopreserved Primary Human Hepatocyte Infection

Primary human hepatocyte infection in this study followed published methods [34 (link)]. Briefly, cryopreserved primary human hepatocytes (BioIVT, Westbury, NY, USA) were thawed two days prior to infection and seeded with 1.8 × 10³ cells per well in a 384-well collagen-coated plate (Greiner Bio-One). Hepatocyte culture was maintained with daily media change with customized InVitroGro HI medium without dexamethasone (BioIVT) supplemented with 5% human serum (Interstate Blood Bank, Memphis, TN, USA), 1:100 dilution of penicillin/streptomycin/neomycin antibiotic mixture (Gibco, Hampton, NH, USA), and 10 μg/mL gentamicin (Gibco). 1 × 10³ sporozoites were inoculated into the hepatocyte culture, and the plate was centrifuged at 250 g, at room temperature, and for 5 min. Hepatocyte cultures were kept at 38 °C with 5% CO₂ in an incubator (Panasonic, Kadoma, Osaka, Japan).