Lindsey Ott Delivers Well Received Poster Session on Likarda’s New 3d Liver Spheroid System at 2016 AAPS Annual Meeting

By December 13, 2016News & Media

Likarda’s product innovation scientist, Lindsey Ott, Ph.D., was a presenting author for a poster session at the 2016 American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition on November 16 in Denver, CO. Session attendees learned about a promising three-dimensional cell-based method to evaluate liver metabolism and toxicity for early stage drug discovery via Likarda’s 3D liver spheroid system.

New 3D Testing Delivers Promising Results
Likarda’s highly efficient, proprietary microplates enhance the formation of miniature 3D structures that can be used for drug testing, developing cell-based transplantations and regenerative medicine for humans and animals. Our micromolds form and test 200,000 clusters in the same space that traditional cell culture plates test only 384. Likarda’s plates generate uniform cellular spheroids in a high-throughput process with an ideal diameter for oxygen diffusion. Our plate can be used with many cell types or screening methods from biochemical assays to phenotypic or high content screening.

Hepatotoxicity and CYP Induction Assays Using HepaRG Spheroids
Drug induced-liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. Designed for in vitro toxicology, we combined our patented spheroid generating plate with a four assay multiplex that assesses hepatoxicity and drug-drug interactions (i.e., CYP induction). Hepatotoxicity is detected by ATP depletion and cell membrane disruption (i.e., lactate dehydrogenase release). P450 enzyme induction is detected by CYP1A and CYP3A4 enzyme activity assays. The entire system can be performed with automated liquid handling.

Hepatotoxicity and CYP induction prediction was verified with 12 DILI reference compounds and 6 CYP induction compounds. Using a 30x MOS cutoff, the 3D hepatotoxicity assay was more sensitive to liver toxins than in 2D at 24 hour and 7-day exposure times (i.e., 3D: 70% sensitivity; 2D: 50% sensitivity). Using a 1.5-fold induction cutoff, the HepaRG spheroids responded to CYP inducing compounds, proving they are metabolically competent.

Our 3D Liver Spheroid System is a Promising Cell-based Method to Evaluate Liver Metabolism and Toxicity for Early Stage Drug Discovery
In summary, Likarda’s custom spheroid-generating plates can produce 3D HepaRG spheroids that possess liver characteristics and can be used in toxicity and metabolism assays. Four assays were combined into a novel high throughput multiplex that provided predictive data on DILI and CYP inducing compounds. Our results demonstrate that our 3D liver spheroid system is a promising cell-based method to evaluate liver metabolism and toxicity for early stage drug discovery.

Download the Poster “Metabolic and Hepatoxic Multiplex Assay in 3D HepaRG Model”

Download the poster to read more about:

  • In vitro drug testing in 3D vs. 2D cell culture
  • Likarda’s proprietary spheroid microplate
  • Automated multiplexed assay to test liver toxicity and drug-drug interactions
  • Improved sensitivity of 3D cells to liver toxins than 2D cells

Liver Metabolism and Toxicity Assay Protocol

  • Spheroid: Cryopreserved HepaRG cells
  • Analysis Platform: BioTek Cytation 5 plate reader and Hamilton STAR
  • Test Article Conc: 9 point dose response curve with top concentration based on 100x Cmax or solubility limit. 4 replicates per concentration.
  • Time Points: 24 hours, 3 days, 7 days, and 14 days
  • Quality Controls:
    • Negative control (0.5% DMSO (vehicle) and dexamethasone (non-toxin))
    • Positive Controls (benzabromarone (toxin), rifampicin (CYP3A4 inducer) and beta-napthoflavone (CYP1A inducer)
  • Data Delivery: Hepatotoxicity (IC50 and Margin of Safety (MOS)) and CYP Induction (EC50 and Emax)

Likarda has a number of new assays, such three-dimensional cell-based testing, custom designed and in development that circumvents limitations of two-dimensional tests that offer promising results in early stage drug discovery.

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