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36W0300Metabolic and Hepatotoxic Multiplex Assay in 3D HepaRG Model

Lindsey Ott1, Karthik Ramachandran1, Lisa Stehno-Bittel1,21Likarda LLC, Kansas City, KS; 2School of Health Professions, University of Kansas Medical Center, Kansas City, KS

PURPOSE

METHODS

3D cell culture models have gained attention as a drugdiscovery platform to predictively evaluate therapeuticcompound efficacy and toxicity. Current multicellular spheroidgeneration methods are limited by labor intensive and lowthroughput processes or reliance on biomaterial matrices thathave lot-to-lot variation. We have developed a custom-designedmicromold plate that creates uniform cellular spheroids in ahigh-throughput process with an ideal diameter for oxygendiffusion.1,2 Our plate can be utilized with many cell types orscreening methods (e.g., biochemical assays to phenotypic orhigh content screening).3,4

CONCLUSIONS

REFERENCES

We have established that our custom spheroid-generating plates can produce 3D HepaRG spheroids thatpossess liver characteristics and can be utilized in toxicity andmetabolism assays. Four assays were combined into a novelhigh throughput multiplex that provided predictive data on DILIand CYP inducing compounds. Our results demonstrate that our3D liver spheroid system is a promising cell-based method toevaluate liver metabolism and toxicity for early stage drugdiscovery.

1. Williams SJ, Schwasinger-Schmidt T, Zamierowski D, Stehno-Bittel L. (2012) Diffusioninto human islets is limited to molecules below 10 kDa. Tissue and Cell. 44(5):332-341.2. MacGregor RR, Williams SJ, Tong PY, Kover K, Moore WV, Stehno-Bittel, L. (2006)Small rat islets are superior to large islets in vitro function and in transplantation outcomes.Am J. Physiol Endocrinol. Metabl. 295(5):E771-9.3. Ramachandran K, Williams SJ, Huang HH, Novikova L, Stehno-Bittel L. (2013)Engineering islets for improved performance by optimized reaggregation in a micromold.Tiss Eng. 19(5):604-612.4. Ramachandran K, Peng X, Bokvist K, Stehno-Bittel L. (2014) Assessment ofreaggregated human pancreatic islets for secondary drug screening. Br. J. Pharm.171(12):3010-3022.5. Gustafsson F, Foster A, Sarda S, Bridgland-Taylor M, Kenna JG. (2014) A CorrelationBetween the In Vitro Drug Toxicity of Drugs to Cell Lines That Express Human P450s andTheir Propensity to Cause Liver Injury in Humans. Toxicol. Sci. 137(1):189-211.

Figure 1: Microplate design.A) The version 1 plate design allows easy cluster retrieval forassays completed in standard microwell plates.3 B) Version 2 platedesign for complete in-well assays.

A BFigure 3: Cell Morphology and Functional Markers.HepaRG cells (Biopredic International) were cultured in the micromold plates (Fig. 1) and produced uniformspheroids. Photomicrographs (on the left) illustrate cell morphology and immunostaining of liver-specificmarkers (albumin = Alb, CYP3A4, MRP2). Graphs (on the right) illustrate liver-specific functions. A) Albuminsecretion detected by ELISA. 3D cultures secreted more albumin over time, while albumin decreased in 2Dcultures over time. B-C) Basal CYP3A4 and CYP1A activity detected by P450-Glo kit and EROD assay. 3Dcultures possessed more active CYP enzymes than 2D cultures.

Figure 2: Multiplex protocol.The multiplex assays were performed with an automated liquidhandling system and were executed at 24 hour, 3 day, and 7 daytime points (n=4). Viability (ATP-content; Promega CellTiter-Glo),cytotoxicity (lactate dehydrogenase (LDH) production; PromegaCytotox-ONE Homogeneous Membrane Integrity), and liverenzyme assays (CYP3A4; Promega P450-Glo Luc-IPA; andCYP1A; ethoxyresorufin-O-deethylase (EROD) assay) wereincluded in the multiplex.

ü Proprietary plate generates 150 clusters perwell in a 384–well format

A

B

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2D 24 hr

2D7 day

3D24 hr

3D7 day

Alb (g) CYP3a4 (r) MRP2 (g)

ü Comparison of 2D and 3D HepaRG cells tested with known liver toxins—3Dmore sensitive to toxins than 2D

ü HTS multiplex assay evaluates toxicity andmetabolism from a single well

ü HepaRG spheroids maintain 3D shape and express liver-specific markers

B

C

ü Cells tested with known CYP inducers—3Dspheroids are metabolically competent

Figure 5: Liver Enzyme Activity Induction.A) Known CYP enzyme inducers were tested on 2D and 3D HepaRGcells for 24 hours and 7 days. Enzyme activity was determined usingP450-Glo and the EROD assay. The EC50 and Emax values wereobtained from dose-response curves fit with a one-site hyperbolafunction. To be classified as an inducer, the Emax values had to begreater than 1.5-fold induction (Red). B-C) Representative graphs ofdose-response curves for a CYP1A inducer (Omeprazole) in 2D (B)and 3D (C) at 7 days. D) Rank order of inducer compound potency.

DA

Figure 4: Liver Toxicity.A) Known liver toxins were tested on 2D and 3DHepaRG cells for 24 hours and 7 days. Viabilitywas determined using a CellTiter-Glo kit. TheIC50 values were obtained from the dose-response curve fit with a 4PL curve and theMargin of Safety was calculated. B-C) DILISeverity Category5 and a 30XMOS thresholdwere used to determine True Positive (toxic;Red) and True Negative (nontoxic; Green)compounds. D) Representative graph of dose-response curves for compounds in 3D at 7 days.

A

D

B

C