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TECH NOTE

Using High Content Analysis to Study Mechanistic Hepatotoxicity in Cellartis Enhanced hiPS-HEP cells

Overview

Cellartis Enhanced hiPS-HEP cells are a highly homogenous population of induced pluripotent stem (iPS) cell-derived hepatocytes that express drug metabolizing enzymes. The cells are ideal for use in drug studies, toxicity testing, and other applications that demand a highly reproducible platform and a continuous supply of functional human hepatocytes. Here, we show that Cellartis Enhanced hiPS-HEP cells are well-suited for monitoring mechanistic hepatotoxicity using high content analysis (HCA), an imaging-based approach for drug screening.

Results

Control cultures of Cellartis Enhanced hiPS-HEP cells were used for high content analysis (HCA). Five days after plating, the cells were exposed to DMSO for 24 hours. The screening parameters included nuclear staining (Hoechst), plasma membrane breakdown (TOTO-3), mitochondrial membrane potential (MitoTracker), and lysosomal activity (LysoTracker). HCA could easily be conducted on the hiPS-HEP cells in monolayer culture. The hiPS-HEP cells contained many active mitochondria similar to what is observed in human primary hepatocytes.

Monitoring hepatotoxicity with HCA

Amiodarone is a compound known to impair mitochondrial function. Cellartis Enhanced hiPS-HEP cells were exposed to increasing doses of amiodarone (3, 30, and 300 µM) for 24 hours. HCA showed that amiodarone impaired mitochondrial membrane potential in hiPS-HEP cells in a dose-dependent manner. Cell viability was impaired at the highest concentration (300 µM). This response to amiodarone is similar to what is observed for human primary hepatocytes.

Similar response as primary hepatocytes

Cellartis Enhanced hiPS-HEP cells were treated with compounds known to be either nontoxic or toxic with different mechanisms of toxicity (see Table I in Methods). After 24 hours of treatment, HCA was performed. The known nontoxic compounds metformin, pioglitazone, and DMSO did not have a measurable effect on the hiPS-HEP cells for the parameters tested. Changes in mitochondrial membrane potential were observed for amiodarone, nefazodone, and perhexiline as expected. Perhexiline induced lysosomal activity with increasing doses. Visible precipitation was observed for nefazodone at concentrations ≥50 µM, likely decreasing the effective concentration and explaining why the values returned towards normal at the higher doses.

Conclusions

Cellartis Enhanced hiPS-HEP cells have a similar phenotypic response as human primary hepatocytes to the non-toxic and toxic compounds tested. These results indicate that Cellartis Enhanced hiPS-HEP cells are well-suited for imaging-based HCA in monolayer culture, and that the cells recapitulate hepatotoxic responses in a similar manner as primary hepatocytes.

Methods

Cell culture

Cryopreserved Cellartis Enhanced hiPS-HEP cells were thawed and plated in 96-well plates according to recommended protocol. Cells were maintained for five days with a media change every other day prior to the start of the toxicity assay.

Toxicity assay and HCA

hiPS-HEP cells were exposed to hepatotoxic and non-hepatotoxic compounds (Table I) (0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, and 300 µM, all dissolved in 1% DMSO). The cells were incubated with each compound for 24 hours prior to fixation and staining with fluorescent probes (Table II). Cells were monitored by HCA using the Thermo Fisher ArrayScan VTI high content imager, and data was analyzed using the integrated Thermo Fisher BioApplication V4 software. Six images were captured per well, compound, and dose. Data are expressed as % of vehicle control (mean ± SEM, n=6).

Table I. Hepatotoxic and non-hepatotoxic compounds used in this study
Compound Hepatotoxicity (mechanism)
Amiodarone Toxic (mitochondrial impairment)
Nefazodone Toxic (unknown)
Metformin Nontoxic
Pioglitazone Nontoxic
Perhexiline Toxic (lysosomal activity and mitochondrial impairment)
DMSO Nontoxic

Table II. Fluorescent probes used in this study
Probe Read-out
Hoechst Nuclear counts
LysoTracker Green Lysosomal activity
MitoTracker Orange Mitochondrial membrane potential
TOTO-3 Plasma membrane integrity

These results were kindly provided by H. Lundbeck A/S

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