Isolated primary human hepatocytes in adherent culture are traditionally considered to be the most suitable for in vitro testing. Hepatocytes are relatively simple to use and maintain an intact cellular architecture with complete, undisrupted enzymes and cofactors. Primary hepatocytes, however, display a precipitous decline in phenotypic functions using conventional models such as sandwich cultures with extracellular matrix proteins (i.e. collagen, Matrigel). This severely limits the ability of these model systems to accurately predict clinical outcomes.
Unique HepatoPac Features:
- Liver slices retain in vivo cytoarchitecture, but have limited viability and are not amenable to high-throughput screening (HTS)
- Microsomes are used to identify certain enzymes involved in drug metabolism but lack the gene expression, cellular machinery and longevity required for toxicity testing
- Hepatocarcinoma-derived cell lines and immortalized hepatocytes, while relatively inexpensive, lack the ability to fully mimic all liver functions
- Sandwich cultures and 3D spheroids on ECM gels display a sharp decline in liver functions and are only useful for acute dosing studies, 3-24hrs.
Albumin and urea secretion over the course of two weeks is shown for human micropatterned co-cultures and pure hepatocytes. Error bars represent SEM (n=3). Khetani and Bhatia, Nat Biotech, 26(1), p120-126, 2008.
HepatoPac produces more clinically relevant major metabolites than traditional model systems [Wang, W.W et al.]. Human HepatoPac in 24-well format was incubated with 27 drugs from 4 hours out to 7 days without culture medium change in order to identify major metabolites and slowly generated secondary metabolites. For traditional model systems, the incubation time was limited to 4 hours due to the inherently short lifetime. Supernatants from cultures were subjected to LC-MS/MS. Results showed that HepatoPac produced in total 75% of major circulating metabolites and 67% of secondary metabolites (arising from 2 or more enzymatic reactions) observed in the clinic. Traditional model systems, on the other hand, ranged between 43-53% for production of major metabolites in total and between 25-38% for secondary metabolites. Of the traditional model systems, suspension hepatocytes provided better detection of metabolites than S9 fractions and microsomes; however, HepatoPac, with its ability to conduct longer term incubations, identified 41% more total major metabolites and 76% more secondary major metabolites observed in clinical studies.
HepatoPac improves the sensitivity of toxicity screening as compared to sandwich cultures while maintaining high specificity. Primary human hepatocytes from a single donor were first subjected to 2 to 4 doses over 5 to 9 days in HepatoPac, and then hepatocyte functionality and viability were assessed using standard bulk assays such as ATP levels, mitochondrial dehydrogenase activity (i.e. MTT) and reduced glutathione content. Compounds were called toxic if at least one of the five parameters showed a 20‐50% reduction in signal as compared to the vehicle control in the dose range tested (1‐100*C[max]), whereas compounds were called non‐toxic only if all of the parameters remained within 20% of the vehicle control values. Sensitivity was calculated as the ratio of TP (true-positives with respect to clinical observations) and the sum of FN (false negatives) and TP, while specificity was calculated as the ratio of TN (true‐negatives) and the sum of FP (false positives) and TN. Data for sandwich cultures was obtained from their use in a high content imaging assay; however, focused studies with bulk viability assays revealed that sandwich cultures have sensitivity value of less than 35% when utilized with such assays, instead of high content imaging.
HepatoPac exhibits higher compound uptake as compared to sandwich cultures. Radiolabeled compound (Estradiol 17β‐Glucuronide) was incubated on either HepatoPac (Days 7 to 14 of culture) or sandwich cultures (Days 3-5 of culture) for varying time periods at 37°C prior to lysing the cells and measuring the radioactivity using a scintillation counter. HepatoPac was found to take up compounds at a 4 to 5-fold faster rate than sandwich cultures and saturation of uptake occurred at approximately 5-fold higher levels than sandwich cultures.
Type Title Application Note HepatoPac® A Bioengineered Micro-Liver Platform for Predictive Drug Metabolism and Toxicity Studies. A Predictive, In Vitro Micro-Liver Co-culture System Providing In Vivo Performance Case Study Ability of a Micropatterned Hepatocyte Co-culture System to Generate Major Human Drug Metabolites. Publication Bridging in vitro and in vivo metabolism and transport of faldaprevir in humans using a novel cocultured human hepatocyte system, HepatoPac®.Drug Metab and Dispos . Early Access. December 2013. Publication Meeting the challenge of predicting hepatic clearance of compounds slowly metabolized by cytochrome P450 using a novel hepatocyte model, HepatoPac®Drug Metab and Dispos. Vol. 41(12), p 2024-2032 (2013). Publication Long-Term Stability of Primary Rat Hepatocytes in Micropatterned Co-Cultures.Journal of Biochemical and Molecular Toxicology, Vol. 27(3), p 204-212 (2013). Publication The Use of Micropatterned Co-Cultures to Detect Compounds that Cause Drug induced Liver Injury in Humans.Toxicol. Sci. 132 (1): p 107-117, (2013). Publication Assessment of MicroPatterned Hepatocyte Co-culture System to Generate Metabolites. Drug Metabolism and Disposition, Vol. 38(10), p 1900-1905 (2010).