Co-culture of Marmoset Hepatocytes

Cryopreserved marmoset hepatocytes were obtained from Bioreclamation IVT Inc. (Westbury, NY). The co-culture model consists of a mixture of marmoset hepatocytes and non-parenchymal mouse embryonic fibroblast 3T3-J2 cells (CCL-92, ATCC, Manassas, VA) were built as previously described^{36 (link),55 (link)}. Briefly, cryopreserved hepatocytes were thawed and centrifuged at 150 × g for 10 min and then the cells were re-suspended in Hµrel plating medium™ (Visikol Inc., Hampton, NJ). Hepatocyte number and cell viability were assessed using trypan blue exclusion. 3T3-J2 cells were cultured in normal DMEM medium (10% FBS, 200 U/mL penicillin/streptomycin) at 37 °C with 5% CO₂. Hepatocytes were seeded at a density of 30,000 cells in each well of a 96-well plate, respectively. 3T3-J2 cells were added the next day at 15,000 per well of 96-well plate, respectively. HµREL™−96 SACC-PMH are distributed by the Visikol, Inc. (Hampton, NJ). Cells were maintained in 100 μl Hµrel maintenance medium™ (Visikol, Inc., Hampton, NJ). Medium was replaced every 2 days. The cells were co-cultured at 37 °C in a 5% CO₂ for 10 days prior to HBV infections.