In Vitro Toxicology
- Hepatotoxicity Kit
- Service Details
- In Vitro Hepatotoxicity
- Benefits of upcyte® Hepatocytes
- Drug Metabolism Enzyme Inhibition & Induction
- Cytotoxicity & Gene Expression
INDIGO's in vitro toxicology platform was developed to meet the increasing demand for more predictive models for liver toxicity due to the fact that hepatotoxicity remains a major reason for drug withdrawal from pharmaceutical development and clinical use. Consequently, the use of hepatocytes for the early identification of drug candidates that induce acute hepatotoxicity provides a powerful predictive tool that can inform drug development decisions.
A primary application of this platform is to screen chemicals for induction and activity of hepatic drug metabolizing enzymes (DME).
The platform now utilizes upcyte® human liver cells to assess chemical and drug-induced toxicity. Unlike transformed hepatic cell lines, upcyte® hepatocytes have drug metabolism and transport activity comparable to primary hepatocytes. These cells are proliferating and able to correctly identify genotoxicants. The upcyte® hepatocytes combine the characteristics and advantages of primary hepatocytes with the added practical advantage of having access to the same donor cells for use in iterative, large-scale testing over extended periods – making them ideal for drug discovery.
For more information on INDIGO's agreement with upcyte technologies, click here.
For more information on INDIGO's use of upcyte® hepatocytes, visit our Gene Expression page.
In Vitro Screening for Drug-Induced Hepatotoxicity using upcyte® Hepatocytes
The principal application of this assay is to rapidly screen test compounds to identify those that induce acute liver cell toxicity.
This kit includes two aliquots of cryopreserved Luminescent Reporter Hepatocytes (upcte®), donor 10-13, isolated from an adult Caucasian female, that have been further modified to constitutively express the luciferase enzyme. The level of luciferase activity expressed in the cells is dependent on the complex coordination of normal cellular processes, including the coupled rates of energy metabolism, transcription, translation, and the turnover of their respective mRNA and protein macromolecules. The hepatocytes are prepared using INDIGO's proprietary CryoMite™ process, which yields high cell viability post-thaw, and provides the convenience of immediately dispensing cells into assay plates. There is no need for intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup. An overnight culture period allows full recovery of the post-thaw hepatocytes and the establishment of a confluent cell monolayer that is ready to receive the user's test compounds.
In addition to two aliquots of the reporter hepatocytes, the kit provides two cell culture-ready assay plates, optimized Cell Culture Media (CCM) for use in all steps of the assay procedure (cell thawing, seeding, and preparation of the treatment media), luciferase detection reagent, and a reference compound that provides a positive control for hepatotoxicity.
The reagents and materials provided in this assay kit are formatted to allow the user to choose between two alternative assay setups. In one scenario 48 culture wells may be setup at two different times. In the other assay scenario 96 culture wells may be setup at one time.
For more information, view the Technical Manual.
|Product Family||Product Number||Product Description|
|In Vitro Toxicology||ULC1003-48||in vitro Screening for Drug-Induced Hepatotoxicty, 2x 48 Assays in 96-well format|
A primary application of INDIGO's in vitro toxicology platform is to screen chemicals for induction and activity of hepatic drug metabolizing enzymes.
upcyte® hepatocytes exhibit basal CYP1A2, CYP2B6, CYP2C9, and CYP3A4, which are all induced by prototypical inducers. As with primary human hepatocytes, the induction response is donor-dependent; therefore, INDIGO offers a panel of different donors with a range of induction responses.
Utilizing this unique platform, INDIGO's services lab can examine your compound's potential to cause liver toxicity through metabolic activation or by induction of drug metabolism enzymes. upcyte® hepatocytes contain equivalent activity of several cytochrome P450's compared to human hepatocytes, thus making examination of metabolism-dependent toxicity biologically relevant. In addition, upcyte® hepatocytes can strengthen and quantify changes in the expression of target genes regulated by PXR, CAR, AhR, LXRs, LRH-1, PPARs, and Nrf2.
Xenobiotic-induced liver injury is a major cause of human morbidity and mortality. A key reason for this problem is our inability to predict hepatotoxicity at the preclinical stage using currently available model systems. Such models include in vivo animal models and in vitro models based on human-derived liver cells or transformed cell systems. Species differences in xenobiotic disposition and mechanisms of cytotoxicity can make whole animal studies unreliable for extrapolation to human. In addition, whole animal models are costly and of low throughput. Therefore, it is essential to develop in vitro models that are more predictive of hepatotoxicity, particularly those that are based on human or "humanized" component cells.
There are two major limitations to the use of human liver cells or their derivatives. First, there are currently limited sources of fresh human hepatocytes worldwide and, when available, they often suffer from low viability and high batch-to-batch variability. Second, the freezing process used to preserve primary hepatocytes and the transformation process needed to make stable and proliferating cell lines results in changes in cell differentiation, proliferation, and metabolic processes.
upcyte technologies has developed a novel technique which allows for the generation of human hepatocyte cultures with the ability to proliferate while maintaining many differentiated functions.
- upcyte® hepatocytes did not form colonies in soft agar and are not immortalized ancorage-independent cells.
- Confluent cultures expressed liver-specific proteins, produced urea, and stored glycogen.
- CYP activities were low but similar to that in 5-day cultures of primary human hepatocytes. CYP1A2 and CYP3A4 were inducible; moreover, upcyte® hepatocytes predicted the in vivo induction potencies of known CYP3A4 inducers. Placing cells into 3D culture increased their basal CYP2B6 and CYP3A4 basal activities and induction responses.
- Phase 2 activities (UGTs, SULTs, and GSTs) were comparable to activities in freshly isolated hepatocytes.
- upcyte® hepatocytes were markedly more sensitive to the hepatotoxin, α-amanitin, than HepG2 cells. The cytotoxicity of of aflatoxin B1 was decreased in upcyte® hepatocytes by co-incubation with the CYP3A4 inhibitor, ketoconazole. upcyte® hepatocytes differentiated between ten hepatotoxic and eight non-heptatotoxic compounds.
- In conclusion, upcyte® hepatocyte cultures have a differentiated phenotype and exhibit functional phase 1 and 2 activities. These data support the use of upcyte® hepatocytes for CYP induction and cytotoxicity screening.
upcyte® Hepatocyte CYP Inhibition
Cytochrome P450 (CYP) enzymes play a major role in the metabolism of the majority of xenobiotics. Cells that perform reliably in the inhibition assays should have reproducible Phase I and II enzyme activities at levels that allow for a good dynamic range for inhibition. upcyte® hepatocytes have donor-dependent basal enzyme activities and represent a reliable tool for xenobiotic inhibition studies.
upcyte® Hepatocyte CYP Induction
upcyte® hepatocytes exhibit basal CYP1A2, CYP2B6, CYP2C9, and CYP3A4 which are all produced by prototypical inducers. As with primary human hepatocytes, the induction response is donor-dependent; therefore, we offer a panel of different donors with a range of induction responses.
upcyte® Hepatocyte for Cytotoxicity
There is an increasing demand to develop more predictive models for liver toxicity due to the high attrition rate of drugs causing liver damage once they enter the market. Immortalized hepatic cell lines are often used for cytotoxicity screening since they grow continuously, are easily available, and can be standardized across laboratories. However, cell lines tend to have low or lacking Phase I and II activities and/or transporter functions and this can lead to false negative results when assessing drug toxicity.
upcyte® Hepatocyte Gene Expression
Knowing that your compounds regulate the activity of a specific nuclear receptor using INIDGO's kits or services is a great first step in characterizing and prioritizing potential new drugs. However, this is just a beginning to fully understanding the biologic or toxicologic effects that may ensue. Coupling our expertise in gene expression and cell biology with the upcyte® platform, we can examine the effects of your compounds on a coordinated and functional response. Whether via examination of gene expression (qPCR, microarray, or Next Gen Sequencing) or addressing biochemical endpoints, we can help you evaluate whether your compounds have effects on hepatic steatosis, mitochondrial toxicity, or other forms of liver damage. Learn more about INDIGO's Gene Expression services.