An open access paper providing a detailed characterization of PXB-cells® was published in late 2020 in PLoS ONE journal. These cells are proven to be a reliable in vitro tool in drug development, where high quality human hepatocytes are frequently required.

Figure 1. PXB-cells 12 days after plating. Yellow arrowheads indicate examples of bile canaliculi
Figure 1. PXB-cells 12 days after plating. Yellow arrowheads indicate examples of bile canaliculi. Image is from PhoenixBio’s in-house data.

During preclinical studies, drug metabolism and toxicity assays are routinely carried out using cryopreserved primary human hepatocytes. In many cases, lower activities of enzymes and transporters diminish the predictive value of results obtained from such studies and do not reliably translate to expected clinical outcomes. Fresh human hepatocytes retain higher metabolic activities, but they cannot be supplied on demand, and reproducible experiments using fresh human hepatocytes from the same donor are not possible. PXB-cells® – fresh human hepatocytes isolated from chimeric mice with highly humanized liver (PXB-mouse®) – retain the benefits of fresh human hepatocytes and allow on demand supply from the same donor for multiple years.

The two-step collagenase perfusion method is used to produce PXB-cells® from 14- to 17-week-old PXB-mice with high replacement indexes. The details of the method, medium components, and medium replacement schedule are described in the paper. In all the experiments, PXB-cells® are found to be highly plateable, showing the matured hepatocyte morphology (Figure 1) for at least three weeks, and formed bile canaliculi between cells at day 7. The average ratio of human hepatocytes to mice hepatocytes leftovers in PXB-cells® increased from about 90% in freshly isolated cells to 98% at day 33.

Figure 2. CYP3A4 activity of PXB-cells measured using midazolam as a substrate and compared to the initial donor's human hepatocytes. Image is from PhoenixBio’s in-house data.
Figure 2. CYP3A4 activity of PXB-cells measured using midazolam as a substrate and compared to the initial donor’s human hepatocytes. Image is from PhoenixBio’s in-house data.

The evaluation results of human albumin secretion, urea synthesis, cytochrome P450 (CYP) activities, and CYP, UGT*, and transporter mRNA expression in PXB-cells® from three different donors confirm their usability and high performance for at least three weeks after isolation. The CYP3A activity was evaluated using midazolam as a substrate for all three donors, but the authors show here just a one donor example compared to the donor hepatocyte activity (Figure 2). Expression patterns of several human hepatic genes (cytochrome P450 1A1, 1A2, 2B6, 2C9, 2D6, 2E1, 3A4, UGT1A1, UGT2B7, BSEP, MRP2, OATP1B1, OATP1B3) in PXB-cells® from three donors are shown in the publication. The mRNA expression levels of 10 of 13 CYP and UGT enzymes and transporters were maintained at >10% of the levels of freshly isolated hepatocytes after 21 days.

Localization of the efflux transporter multidrug resistance-associated protein (MRP2) in PXB-cells® was confirmed by immunohistochemistry and its function was evaluated using MRP2 inhibitor cyclosporin A (CsA) at two concentrations. The transporter activity of sodium-taurocholate co-transporting polypeptide (NTCP) and bile salt export pump (BSEP) in PXB-cells® was verified using DBD** and CsA.

It was shown that the cell density of cultured hepatocytes is one of the most essential factors for the maintenance of liver specific functions in vitro. Comparison of behavior of low- and high-density cultures revealed that high-density culture maintained high expressions of some transcriptional factors (HNF4α, PXR, and FXR), which are likely involved in the high CYP, UGT and transporter gene expressions of PXB-cells®. In the meantime, further experiments involving siRNAs or gene transfer-mediated overexpression of these transcriptional factors into PXB-cells® would be beneficial.

This publication demonstrates that PXB-cells® stably maintain hepatic functions for long periods, using a conventional culture method with collagen-coated plates, so they are a valuable tool for in vitro preclinical studies in drug development.

*UGT – UDP-glucuronosyltransferase

**DBD – N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5 β-cholestan-26-oyl)-2’-aminoethanesulfonate (Tauronor-THCA-24-DBD)