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.
