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In bringing a therapuetic through clinical trials, one of the leading contributors to clinical failure is liver toxicity causing 15% of failures. It is for this reason that there has always been significant interest in developing inexpensive and moderate throughput in vitro models which are capable of predicting clinical drug-induced liver injury (DILI). MatTek has leveraged its decades of experience in developing highly reproducible advanced cell culture models to launch EpiLiver.
Tissue morphology has been characterized by histology and albumin expression has been evaluated by immunohistochemistry and ELISA. The model shows recapitulation of the liver’s structure with 3D columnar hepatocyte tissue formation and hexagonal cellular structure (topical view imaging). Albumin production (immunohistochemistry), and albumin release to the basolateral and apical sides (ELISA) has also been demonstrated.
Figure 1. H&E-stained histological cross-section of the 3D human liver tissue model showing the hepatocyte polarization and stratification (A). Immunohistochemistry of albumin (orange) production is shown in (B). Tissues were grown on collagen coated underlying microporous membrane support (pore diameter= 0.4 um).
Figure 2. Albumin release by the 3D human liver tissue model at different time points of the culture period.
Figure 3. Reduction of albumin release following treatment of the 3D human liver tissue model with different drugs dosed at different concentrations.
Changes in gene expression levels for drug metabolizing enzymes associated with first pass metabolism have also been assessed via qPCR over a period of 23 days. qPCR results demonstrate high level expression of enzymes involved in drug metabolism such as CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, and CYP4A11. Both Phase I and Phase II enzymes were expressed by the differentiated liver tissue model.
Table 1. qPCR Results (RT2 Profiler PCR Array Kit) showing Phase I and II drug metabolizing enzyme gene expression levels by the 3D human liver tissue model.
The utility of the tissue model for liver toxicity studies has been demonstrated by dosing the reconstructed liver tissue with 100 µM of 5 model drugs (SN38, Bosentan, Diclofenac, Fialuridine, and Tolcapone) that are known to have adverse effects on liver in humans. Outcome measurements for liver toxicity include increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) release, two biomarkers with clinical relevance in liver functionality tests. Repeated application of Fialuridine, a drug intended for hepatitis B treatment that was abruptly terminated due to induction of liver failure or causing of severe liver toxicity during clinical trials, showed an increase in ALT and AST levels in a time-dependent manner at days 5 and 7, which is indicative of drug induced liver injury (DILI). The positive control, SN38, a metabolite of the cancer drug Irinotecan, also shows an increase in ALT and AST levels.
Figure 4. Acute exposure of the 3D human liver tissue to drugs known to induce liver toxicity showed increases in ALT and AST release, two biomarkers clinically used for liver function test.
Development of this novel 3D human liver tissue model using primary adult hepatocytes provides researchers the opportunity to screen the potential liver toxicity of candidates with a highly relevant model that are in the drug development pipeline. Such a model will allow formulation scientists to identify adverse effects of therapeutic candidates early in the drug development process. This model will also reduce animal use for experimentation.
We have designed the EpiLiver model to be applicable to a broad range of applications from evaluating drug metabolism (DMPK) to assessing drug induced liver injury which accounts for 15% of preclinical and clinical stage failures.
Drug-Induced Liver Injury (DILI)
The highly relevant EpiLiver model is capable of assessing and predicting in vivo liver toxicity.
Understanding how drugs are metabolized is crucial to understanding the route of administration and any toxicity liabilities associated with metabolism.
LIV-100 EpiLiver kits consist of 24 individual tissues. Each kit contains tissues, culture medium to support experiments for 24 hours, and plasticware. Please contact MatTek for specific kit contents.
Culture: Air-liquid interface/ wet film
Lot numbers: Tissue lots produced each week are assigned a specific lot number. A letter of the alphabet is appended to the end of the lot number to differentiate between individual sets of 24 tissues within a given production lot of tissues. All tissue kits within a production lot are identical in regards to cells, medium, handling, culture conditions, etc.
Shipment: At room temperature on medium-supplemented, agarose gels
Shipment day: Every Monday
Delivery: Tuesday morning via FedEx priority service (US). Outside US: Tuesday-Wednesday depending on location.
Shelf life: Including time in transit, tissues may be stored at room temperature (RT) for up to 2 days. However, the best reproducibility will be obtained if tissues are used consistently on the same day, e.g. Tuesday afternoon or following overnight storage at RT (Wednesday morning).
Length of experiments: Tissue cultures can be continued for ONE (1) MONTH or more with good retention of normal morphology. Tissues must be fed every other day with 5.0 mL of maintenance medium (LIV-100-MM). Cell culture inserts are hanging top 12-well plates (HNG-TOP-12) to allow use of 5.0 mL.
Type: Primary human liver hepatocytes (HLH)
Genetic make-up: Single donor
Derived from: Healthy adult donor
Alternatives: Alternate donors available upon request
Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma
Base medium: Williams E Medium, phenol red free
Growth factors/hormones: Epidermal growth factor and other proprietary factors
Antibiotics: Gentamicin 5 µg/mL, Penicillin/streptomycin (100 U/mL / 100 µg/mL)
Anti-fungal agent: Amphotericin B 0.25 µg/mL
pH Indicator: None
Other additives: Proprietary
Alternatives: Phenol red-free, antibiotic-free, anti-fungal-free medium and tissues are available. Agents are removed at least 3 days prior to shipment.
Visual inspection: All tissues are visually inspected and if physical imperfections are noted, tissues are rejected for shipment.
Sterility: All media used throughout the production process is checked for sterility. Maintenance medium is incubated with and without antibiotics for 1 week and checked for sterility. The agarose gel from the 24-well plate used for shipping is also incubated for 1 week and checked for any sign of contamination.
Screening for pathogens: All cells are screened and are negative for HIV, hepatitis B and hepatitis C using PCR. However, no known test method can offer complete assurance that the cells are pathogen free. Thus, these products and all human derived products should be handled at BSL-2 levels (biosafety level 2) or higher as recommended in the CDC-NIH manual, “Biosafety in microbiological and biomedical laboratories,” 1998. For further assistance, please contact your site Safety Officer or MatTek technical service.
Notification of lot failure: If a tissue lot fails QC or sterility testing, the customer will be notified and the tissues will be replaced without charge. Because our QC and sterility testing is done post-shipment, notification will be made as soon as possible (Under normal circumstances, ET-50 failures will be notified by Thursday 5 p.m.; sterility failures will be notified within 8 days of shipment)
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