citations

                    Evaluating cannabidiol-induced liver injury with and without valproate using a three-dimensional human hepatocyte spheroid model
                

Authors:

Jessica L Beers , Shalon L Harvey , Raeanne M Lanphier , Blake R Rushing , Susan L McRitchie , Susan J Sumner , Klarissa D Jackson

In:

Source: Toxicol In Vitro
Publication Date: (2025)
Issue: 109: 106126

Research Area:

Gastroenterology

Regenerative medicine

Toxicology

Drug Discovery

Cells used in publication:

Hepatocyte, human: Species: human; Tissue Origin: liver

Culture Media:

Hepatocyte Culture Medium

Experiment

2.2. Biological reagents

Primary human hepatocytes were thawed rapidly at 37 °C and suspended in hepatocyte thawing medium (Lonza catalog no. MCHT50), then centrifuged at 100 g for 10 min. Supernatant was removed, and hepatocytes were re-suspended in approximately 5 mL of hepatocyte maintenance media (Lonza catalog no. CC-3198) supplemented with 10% fetal bovine serum (Fisher catalog no. 10-082-147) and Lonza Singlequots^TM supplements (transferrin, ascorbic acid, human epidermal growth factor, insulin, hydrocortisone, and bovine serum albumin), with the exception of gentamicin and amphotericin B, which was replaced with 100 U/mL penicillin and 100 µg/mL streptomycin (Fisher catalog no. 15-140-122). To determine optimal seeding density, both 1500 cells/well and 3000 cells/well were initially tested in 96-well plates. Spheroids generated from 1500 cells/well showed improved morphology over time and with repeated media changes compared to 3000 cells/well; thus, 1500 cells/well was chosen for generating the spheroids described in this study (Bell et al., 2016).

2.3. Cell culture

Abstract

Cannabidiol (CBD) and valproate (VPA) are anti-epileptic medications commonly co-prescribed to treat seizures due to Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex in children. Clinical trial data have demonstrated that CBD carries a risk for severe hepatotoxicity that is greatly increased when prescribed with VPA through an unknown mechanism. The aim of this study was to investigate CBD-induced liver injury in combination with VPA using an in vitro liver model. Three-dimensional human hepatocyte spheroids are an emerging in vitro system that allows investigation of long-term toxicity. Spheroids derived from primary human hepatocytes were treated with vehicle control, 2-200 µM CBD, 0.5-20 mM VPA, CBD + VPA, and 0.1-10 mM acetaminophen (positive control). After 24 h, 8 days, and 15 days of exposure, spheroids were analyzed for ATP depletion, urea production, and CBD and VPA metabolite generation. Untargeted metabolomic analysis was also conducted. A delayed-onset, dose-dependent hepatotoxicity was observed in spheroids exposed to each drug treatment compared to vehicle control. This study is the first to recapitulate the hepatotoxic drug interaction of CBD and VPA in vitro and demonstrates the utility of human hepatocyte spheroids for toxicity studies. Future work is needed to examine mechanisms of CBD-induced hepatotoxicity with VPA.

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