Application Note

Cells:
Tools:

MetaHeps® Cells
CardioExcyte 96

Investigating DILI using MetaHeps® cells on
Nanion’s CardioExcyte 96
Nanion Technologies GmbH, Munich.
Hepatocyte-like cells kindly provided by MetaHeps®
SummaryResults

Drug Induced Liver Injury (DILI) is the major cause of

The impedance signal recorded on the CardioExcyte

acute liver failure in the USA and Europe and is one of

96 changes as a result of alterations in confluency, cellthe main reasons for regulatory actions and market

contact

withdrawals . Indeed, hepatic toxicity has accountedof adherent

for 15 of the 47 drugs withdrawn from the market in theof toxicity. MH cells from MetaHeps® were grown on

period 1975 - 20072. DILI is classified as intrinsic or dose-

NSP-96 plates and base impedance was monitored

dependent, acetaminophen (paracetamol) being theover time. Paracetamol showed a dose-dependent

most important example of this class3, or idiosyncratic

decrease in base impedance (Figure 1A). This result iswhich is unpredictable and not directly dependent on

directly comparable with a standard toxicity (lactatedose4. A number of factors contribute to an individual’s

dehydrogenase (LDH) release) assay5,6 (Figure 1B).

susceptibility to develop idiosyncratic DILI including

Importantly, the CardioExcyte 96 allows continuous

age, sex, alcohol consumption, drug interactions andmonitoring of base impedance using a single plate

genetic variability4. Although improvements have been

revealing that the maximum effect is reached within themade to cellular and animal models to predict intrinsic

first few hours of exposure. Using the standard toxicity(dose-dependent) DILI, it is almost impossible to predictassay, measurements of LDH in the supernatant andidiosyncratic DILI. Withdrawal of compounds at a latelysate are made only at 24 hour intervals or at several

stage (or postmarketing) due to idiosyncratic DILI is costly

time points requiring several plates.
AB1and can lead to incorrect withdrawal of potentially useful(morphologicalshape)

andconductivity

cells and thereby provides a measurecompounds. Monocyte-derived hepatocyte-like (MH)cells have been developed as a tool to investigate longterm hepatotoxicity, metabolism and drug interactions5.
Furthermore, patient-derived MH cells could provide atool for diagnosis or exclusion of idiosyncratic DILI1,6 andprovide the causative agent in polymedicated patients.
In this study, MH cells (MetaHeps®) were used on theCardioExcyte 96 and changes in impedance, andtherefore confluency, were used as a measure of toxicity.
Intrinsic (dose-dependent) effects of paracetamol could
be identified consistent with other methods of liver injury
detection. Therefore, the CardioExcyte 96 in combination
with patient-specific MH cells provides a novel tool forinvestigating intrinsic and idiosyncratic DILI.

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Figure 1: A Increasing concentrations of paracetamol induce a decreasein base impedance of MH cells which can be monitored continuously.
Tween (2%) induced 100% cell death and was used as a positive control.
B Toxicity measurement of MH cells using an LDH release assay.
Paracetamol showed a dose-dependent effect on toxicity in both assays.

Application Note
In subsequent experiments, the effect of low and intermediate concentrations of paracetamol and washout
was investigated. Figure 2A shows the effect on base
impedance of MH cells after exposure for 24 or 48 hoursof 1 mM paracetamol. The results show that exposure
to low doses of paracetamol causes only transient
effects on toxicity parameters compatible with cellular“adaptation”. This is a phenomenon reported in humans7
andanimals8,paracetamolThiseffecteven

whensubsequent

would

normallyinduce

also

consistent

withisdoses

ofliver

injury8.

toxicity

assaysusing LDH release experiments (Figure 2B).

Figure 3: A Normalized impedance versus time (in hours) after MH cells
were exposed to 5 mM paracetamol for 24 or 48 hours. Cells recovered,indicated by the increase in base impedance, when paracetamol waswashed out after 24 hours but toxicity continued when 5 mM paracetamol
was added again for a further 24 hours. B Toxicity measurement using
LDH release assay also shows that toxicity is reversed upon washout ofparacetamol but continues with a 2nd dose of paracetamol after 24hours.

toxicity whereas continued exposure lead to increased
hepatoxicity. This is important in order to identify
compounds with the potential to cause liver damage
despite discontinuation of treatment.
In summary, the CardioExcyte 96 has the capability
Figure 2: A Normalized impedance versus time (in hours) after MHcells were exposed to 1 mM paracetamol for 24 or 48 hours. Repeat
exposure to paracetamol causes adaptation and only transient toxicity
is observed. B Toxicity measurement using LDH release assay also shows
repeated exposure to low doses of paracetamol cause transient toxicityof hepatocytes.

Figure 3 shows exposure of MH cells to an intermediate
dose (5 mM) of paracetamol on the CardioExcyte 96(A) and LDH release assay (B). In this case, washout ofparacetamol after 24 hours resulted in recovery fromto record multiple samples of hepatocyte-like cells
simultaneously in real-time, a critical feature in monitoring
cytotoxicity. The data recorded on the CardioExcyte 96
is comparable with results derived from the standard
LDH release assay and thus strenghtens the scientific
relevance of impedance-based toxicity assays. A
reliable assay for the detection of DILI is urgently needed
to prevent the inappropriate withdrawal of potentiallylife-saving compounds.
Impedance measurements

References
1. Benesic, A., & Gerbes, A.L. 2015. Dig. Dis. 33: 486-491
2. Stevens, J.L., & Baker, T.K. 2008. Drug Discovery Today. 14(3/4): 162-167
3. McGill, M.R., & Jaeschke, H. 2013. Pharm Res. 30(9): 2174-2187
4. Chalasani, N., & Björnsson, E. 2010. Gastroenterology. 38: 2246–2259
5. Benesic, A., et al., 2012. Laboratory Investigation. 92: 926-936
6. Benesic, A., et al., 2016. Gut. 65: 1555-1563

Impedance measurements were conducted according
to Nanion’s standard procedures for the CardioExcyte
96. MH cells were thawed, seeded on the Sensor Plate at
an appropriate density to provide the desired confluency
and cultured under propriety conditions for 24 hours prior
to exposure to paracetamol. About 2 hours before drug
application the medium was completely removed from

7. Watkins, P.B. et al., 2006. JAMA. 296(1): 87-93

the wells and 200 μl fresh medium was added. All signals

8. Shayiq, R.M. et al., 1999. Hepatol. 29(2): 451-463

were normalized to a group of control measurements

Methods(n=5-11) on the same plate.

Cells
Monocyte-derived hepatocyte-like cells from MetaHeps®
were used. MH cells were derived from healthy donors
and cryopreserved after generation.
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