Late-stage drug failures remain a significant challenge in the clinical development process, resulting in financial losses and wasted resources. That’s why having a reliable, physiologically relevant in vitro model is crucial for building confidence in a therapeutic candidate before progressing to in vivo and clinical studies.
Primary human hepatocytes (PHHs) have long been an important tool in the early-screening arsenal, with their accurate representation of human liver tissue offering valuable insights into a drug’s pharmacokinetics, efficacy, and potential toxicity. PHHs are also useful for studying a wide variety of liver disease indications — from viral hepatitis and metabolic disorders (such as metabolic associated steatohepatitis and metabolic associated fatty liver disease) to rare genetic diseases including Wilson’s disease, urea cycle disorders, alpha-1 antitrypsin deficiency, and more.
However, the practical limitations of traditionally sourced PHHs — including quality challenges, donor variability, limited stock, and short lifespan in culture— can make them challenging to work with.
Now, a promising alternative source of PHHs has emerged, providing a more cost-effective, high-quality, convenient, and consistent in vitro model.
PHHs have long been regarded as the ‘gold standard’ in in vitro liver cell culture1 due to their ability to closely mimic human liver functions and mechanisms, and thus more reliably predict drug responses.
However, despite their widespread use, PHHs derived directly from humans pose several significant challenges:
Obtaining high-quality PHHs can be difficult, as only a limited number of human tissue samples may be suitable for successful primary hepatocyte isolation. Supply from each donor is also limited, so it is common to need cells from multiple donors across a single study, which require validation to ensure batch consistency.
Furthermore, variations in donor characteristics, the sourcing of the liver tissue (e.g., surgical resections, organ donations, or biopsies), and the methods used to isolate the hepatocytes can affect the quality of the cells, impacting drug responses and study reproducibility.
The complexities of sourcing, the limited supply of donors and suitable samples, and the need for multiple validations across a study can make traditional PHH cell models expensive to use (Table 1), particularly in long-term research.
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Commercial Vendor PHHs Grades and Costs |
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Hepatocyte Grade |
Cost per vial |
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Plateable grade |
$1,400–$1,650 |
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CYP-induction certified |
$1,450–$1,700 |
|
Transporter certified |
$1,650–$2,350 |
Not all traditionally sourced PHHs can successfully adhere to a culture plate. Those that fail to effectively adhere have limited applications, so researchers often need to screen multiple PHH batches to find a suitable lot, extending timelines and increasing costs.
Loss of metabolic function and morphology are commonly observed in PHHs; with PHHs often de-differentiating into mesenchymal phenotypes after just a few days in culture1. PHHs also remain viable for just 1–2 weeks in typical culture. Although advancements such as 3D sandwich cultures extend this lifespan, these methods restrict the type of assays that can be performed, limiting their applications in longer-term research1.
The limitations of PHHs, then, present serious obstacles in in vitro research, introducing variability in experimental outcomes, driving up costs, and potentially slowing down developmental timelines. To address these challenges, researchers need a cell model that retains the advantages of PHHs while overcoming their limitations.
Cryopreserved PXB-cells provide an innovative alternative to PHHs generated from more traditional methods. PXB-cells are ~95% pure primary human hepatocytes freshly prepared on-demand from a proprietary in vivo bioreactor, and closely replicate normal human liver physiology.
Now available in a cryopreserved format, PXB-cells can provide researchers with a convenient, consistent, and accurate in vitro model that offers numerous advantages over traditional PHHs.
As cryopreserved PXB-cells are derived from a proprietary in vivo bioreactor, researchers benefit from consistent donor profiles, minimizing variability between experiments and the need for repeated validations in longer-term studies.
Studies have shown that cryopreserved PXB-cells retain the structure and function of fresh PXB-cells. Post-thaw, they maintain the appropriate morphology and have similar human albumin profiles to fresh PXB-cells (Figure 1).
Additionally, cryopreserved PXB-cells are able to mimic human liver function as effectively as fresh PXB-cells, demonstrating stable cytochrome P450 (CYP) expression and induction and activity of liver transporters (Figures 2 and 3). CYP enzymes can also be induced by Rifampicin, β-Naphthoflavone (BNF), or Phenobarbital in cryopreserved PXB-cells, making them ideal for metabolic studies (Figure 4).
Cryopreserved PXB-cells offer similar quality to fresh PXB-cells post-thaw, meaning that, just like their fresh counterparts, cryopreserved PXB-cells have numerous research applications.
Cryopreserved PXB-cells cells can survive at least 28 days in culture, showing similar levels of human albumin production to fresh PXB-cells over that period (Figure 5).
Unlike freshly isolated cells, cryopreserved PXB-cells can be stored and used on demand, allowing researchers to initiate experiments according to their schedules. Moreover, researchers can easily plate cryopreserved cells in the format that best suits their assay (fresh PXB-cells, on the other hand, are primarily offered in 24- and 96-well plate and T75 flask formats). This flexibility simplifies study design planning and reduces the logistical challenges associated with fresh PHHs.
Cryopreserved PXB-cells can be supplied consistently from the same human donor for several years, helping to reduce costs associated with sourcing and batch variability issues associated with traditional PHHs.
Researchers can also bulk order and store cryopreserved PXB-cells for use as needed, lowering shipping costs. What’s more, in vitro work using cryopreserved PXB-cells can pave the way to studies using humanized liver chimeric mouse models generated with the same human hepatocyte donor. This translatability may reduce the risk of unexpected later-stage failure (i.e., during in vivo studies), providing further cost savings.
Although PHHs derived directly from humans have long been the gold standard for in vitro liver cell studies, their limitations in terms of quality, variability, and flexibility can create significant challenges. PXB-cells — in particular cryopreserved PXB-cells — though, offer an attractive alternative.
With more convenient sourcing and consistent quality, PXB-cells can provide an effective replacement to traditional PHHs, capable of delivering more reproducible results in long term culture. In their cryopreserved format, PXB-cells retain these benefits while offering improved cost-effectiveness and the flexibility to thaw and use the cells as and when needed, as well as allowing researchers to plate them in accordance with their preferred assay.
Contact us to find out more about how cryopreserved PXB-cells can support your research!