A cell culture model of high HBV replication in primary human hepatocytes derived from HBV infected humanized mice

Question Primary human hepatocytes (PHHs) freshly derived from donors show higher innate immune responses than undifferentiated hepatoma cell lines but their availability and in vitro infection efficacy are limited. The aim of this study was to establish a model of in vivo infected human hepatocytes that are transferred to cell culture to achieve high levels of HBV replication intermediates in vitro.

Methods uPA SCID beige (USG) mice were transplanted with PHHs and infected with genotype D hepatitis B virions. After at least 12 weeks of infection when nearly all PHHs were stably HBV infected, hepatocytes were isolated by collagenase perfusion followed by repeated low-speed centrifugations. 4x10^5 cells/well were seeded on collagen-coated 24-well plates and cultured for 21 days in Williams’ medium E with DMSO. Interferon-alpha-2a (IFN)/lamivudine treatment or HDV superinfection were started 1d after seeding. RNA and DNA were isolated from the same wells and HBV DNA, HBV pgRNA and ISG mRNA expression/GAPDH+RPL30 in cell lysates as well as HBV DNA, HBeAg and HBsAg in supernatants were determined by RT-PCR and ELISA.

Results The amount of PHHs isolated from USG mice, HBV DNA/cell and relative pgRNA expression remained stable up to 21d after seeding. High amounts of secreted HBV DNA (mean 1.8x10^8 cop/ml), HBsAg (mean 2.2x10^2 IU/ml) and HBeAg (mean 2.5x10^2 S/CO) could be detected (d14). Compared to untreated cells, treatment with 1000 IU/ml IFN resulted in significant reductions of intracellular HBV DNA/cell (p=0.013, 3.5-fold), pgRNA expression (p=0.024, 28.4-fold), HBeAg (p=0.048, 5.9-fold) and HBsAg (p=0.044, 5.3-fold) on day 14. As expected, LAM treatment reduced intracellular HBV DNA levels (d14: p=0.005, 3.9-fold). Vigorous induction of interferon-stimulated genes OAS1 and MxA remained stable in interferon treated cells between d7 and d21 (OAS: p<0.0001, 15.8-fold; MxA: p<0.0001, 55.15-fold; mean mRNA expression on d14 vs baseline). Coinfection of PHHs with genotype 1 HDV resulted in highly detectable intracellular HDV RNA but significant decline of intracellular HBV DNA (p=0.003, 7.31-fold) and relative pgRNA expression (p=0.017, 7.05-fold) on day 14 compared to early infection (d3).

Conclusion PHHs exposed to long-term HBV spreading in chimeric mouse livers can be stably cultured in vitro. Such novel cell culture model allows comprehensive drug and innate immunity studies in highly replicative HBV infected cells.

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021