Reproducibility remains one of the biggest challenges in HBV infection assays using primary human hepatocytes (PHHs). Infection efficiency can vary considerably between experiments despite consistent hepatocyte sources and standardized protocols. While most attention is placed on the cells themselves, our experience suggests the answer often lies elsewhere.
In theory, an HBV experiment in primary hepatocytes is simple. You take human liver cells, then add virus from a patient, and measure viral entry during the first few days and viral replication approximately two weeks later.
Several research groups have used our hepatocytes for these experiments, and over time a clear pattern emerged. It appeared that when an experiment went wrong, the cells were rarely to blame. In truth, it was the virus.
The virus arrives within the plasma from infected donors, and no two lots are ever the same. Different genotypes, different titers, different freezing conditions, and donor-specific characteristics turn up later as a difference in the number of cells that actually get infected. That is why two labs, following the same protocol on hepatocytes from the same donor, end up with numbers that don’t match. The truth is that such disagreement started long before either of them opened the incubator.
Much of what makes a vial of plasma unpredictable is very easy to miss. Here, qPCR helps quantify the number of viral genome copies in a sample. However, it does not tell you how many of them can infect cells, and that fraction also shifts from donor to donor. Patient plasma also carries the genotypes and resistance variants that circulate in real infections, while the virus grown in a cell line represents a single sequence, adapted to cell culture and unchanged for years. For siRNA, ASO, or entry inhibitor work, that gap is the whole question. Moreover, the infectivity fades faster than DNA does, so long after a lot has stopped infecting properly, qPCR will still return a reassuring number.
These observations motivated us to develop a more standardized approach. We started building a biobank of HBV plasma with the genotype and titer established and validated storage conditions. Alongside the biobank, we provide our inoculation and replication protocols for plateable PHH, which are available as a research service.
We are continuing to expand this collection by adding donors with diverse HBV genotypes and well-characterized viral loads while refining standardized infection workflows for plateable PHHs.
If reproducible HBV infection is part of your research, we’d be happy to discuss how these resources could support your study.
Written by Sofia Smovzh