Drug discovery is a complex and challenging process that requires a significant investment of time, resources, and expertise. One of the key challenges in drug discovery is developing effective assays that can accurately predict the efficacy and safety of potential drug candidates. Liver fibrosis is a critical area of research, given the high prevalence of non-alcoholic fatty liver disease (NAFLD) and the lack of effective treatments for liver fibrosis. At Visikol, we offer a Liver Fibrosis Assay that uses 3D cell culture models incorporating a mixture of hepatocytes or hepatocyte-like cells with nonparenchymal cells (NPCs) to provide the greatest utility. The assay analyzes gene expression via qPCR, allowing for many genes of interest to be evaluated using a single sample. In this blog post, we will provide practical advice for researchers looking to use Visikol’s Liver Fibrosis Assay for drug discovery. We will cover tips and tricks for optimizing the assay, including best practices for plate cells, spheroid formation, treatment, endpoint assays, and analysis. We will also provide guidance on how to interpret the results of the assay and use them to make informed decisions about drug development.
Optimizing the Assay
Our Liver Fibrosis Assay is a powerful tool for drug discovery, but it requires careful optimization to achieve the best results. The first step in optimizing the assay is to carefully plate the cells. For generation of liver spheroids, 1500 cells/well (60% hepatocytes, 40% NPCs) are seeded into ULA U-bottom plates and maintained under standard culture conditions for 14 days to enable spheroid aggregation to a size of approximately 200 μm in diameter. Spheroids are then treated with test compounds, and if test compounds are intended to ameliorate rather than prevent fibrosis, test compounds will be added after induction of fibrosis. Following 1 h of pre-treatment (other timepoints available upon request), fibrosis is induced via addition of 100 ng/mL TGF-β for an additional 72 hours. Test article concentration is maintained through induction of fibrosis. It is important to maintain consistency in the number of cells plated, the plate format, and the quality controls used.
Spheroid formation is another critical step in the assay. The spheroids should be allowed to aggregate for at least 14 days before treatment to ensure that they are of the appropriate size and shape. The spheroids should be monitored regularly to ensure that they are healthy and viable.
Spheroid Treatment
Treatment of the spheroids is another critical step in the assay. The test compounds should be added at the appropriate concentration and timepoint to ensure that they are effective. The assay can be used to test compounds intended to prevent or treat fibrosis. The test compounds can be added after induction of fibrosis, and the assay can be used to assess the efficacy of anti-fibrotic agents.
Endpoint assays are critical for measuring the efficacy of drugs in liver fibrosis assays. The endpoint assays that can be used to measure the efficacy of drugs in liver fibrosis assays include qPCR, ELISA, and immunofluorescent labeling. The choice of endpoint assay will depend on the specific research question being addressed.
Interpreting the Results
Interpreting the results of the Liver Fibrosis Assay requires careful analysis of the data. The assay analyzes gene expression via qPCR, allowing for many genes of interest to be evaluated using a single sample. The results of the assay can be used to identify promising targets for modulation and amelioration of liver disease. Dose response curves and EC50 values for select genes of interest in response to anti-fibrotic agents are provided. The results of the assay can be used to make informed decisions about drug development, including which compounds to pursue and which to discard.
Why Work With Us?
Our Liver Fibrosis Assay uses 3D cell culture models that incorporate a mixture of hepatocytes or hepatocyte-like cells with nonparenchymal cells (NPCs) to provide the greatest utility. The assay analyzes gene expression via qPCR, allowing for many genes of interest to be evaluated using a single sample. We also offer custom drug discovery solutions to researchers who require a more customized approach to their drug discovery projects. Our quality control process that we have in place for all new cell and control compound lots ensure standardization of our liver assays. Additionally, we offer unparalleled techniques for evaluating compounds which modulate the processes along the continuum of NAFLD, including lipid deposition, inflammation, extracellular collagen deposition and fibrosis, and adjacent deleterious processes.
By following the tips and tricks outlined in this blog post, researchers can maximize the potential of the assay and make informed decisions about drug development. To learn more about, please reach out to a member of our team today!