Cells:

Application NoteiCell ® Hepatocytes 2.0
Tools:
CardioExcyte 96

Comprehensive impedance-based
hepatotoxicity assay for metabolicallyactive iPSC-derived hepatocytes

AtlaZ

Nanion Technologies GmbH, Munich.
FUJIFILM Cellular Dynamics, Inc., Madison, WI, USA

Summary
Hepatotoxicity and drug-induced liver injury (DILI) are leading

harmful effects of drugs could be evaluated over time inreasons for drug failure to market, leading to approximately

a functional 2D cell-based model without the need for 3D

18% of market withdrawals of drugs in the last decade1.

spheroid formation.

Additionally, only half of drugs with a potential to induce
hepatotoxicity are actually identified during preclinical animalstudies2. This highlights the importance of generating moreadvanced cell-based models and experimental strategies
to enhance the predictivity of these assays. Current existingin vitro models employed to predict DILI mostly focus on
hepatocytes, though primary hepatocytes do not maintaintheir phenotype. iCell® Hepatocytes 2.0 (FUJIFILM CellularDynamics, FCDI) are human iPSC-derived cells with awide variety of basic and functional characteristics whichmake them amenable to applications such as compoundmediated ADME-T and DILI toxicity. In addition to displaying
characteristic hepatocyte morphology, (i.e., polygonal shape,polynucleation and formation of bile canalicular channels),these cells also express liver cell markers, including albumin,
A1AT, and HNF4a, and exhibit basic and induced P450
functions, as observed in primary human hepatocytes. iCell®
Hepatocytes 2.0 maintain morphology, marker expression,ResultsiCell® Hepatocytes 2.0 were recorded on the Nanion´s
impedance systems and changes in base impedance,
showing confluency and toxicity, were used to measure DILI.
The impedance-based continuous and multi-parametric cell
monitoring, an advantage over individual endpoint assays,
was used to investigate the kinetics of cell behavior in realtime. Hepatocytes were cultured for 7 days at confluence,
before the compound addition series started. Cells weretreated according to the manufacturer’s protocol. Acuteand chronic liver toxic effects (dose-dependent) were tested
for 5 relevant compounds: acetaminophen, diclofenac,troglitazone, chlorpromazine, and aflatoxin B1. Compoundcontaining medium was exchanged every 2 days andrecordings were performed during a time-period of 15 to 21days, as depicted in experimental layout (Methods).

and metabolic function in culture over a longer time framecompared to primary human hepatocytes, rendering

100500h0Tox effect [%]150

Troglitazone

acetaminophen A, diclofenac B, troglitazone C
and chlorpromazine D. 0h marks the time when
compounds were added. Values represent mean ±SEM from duplicate values.

02h24h72h
141h
168h
192h

exposure times to 5 concentration additions of500hiCell® Hepatocyte 2.0 at acute and prolonged

DTime [h]150

Chlorpromazine

control 100
1 µM
5 µM
15 µM
5075 µM
150 µM
0100

Figure 1: Real-time impedance monitoring ofcontrol
3 µM
10 µM
30 µM
100 µM
300 µM

0Time [h]CDiclofenac

2h24h
72h
141h
168h
192h

cells, thereby providing a measure of toxicity. Dose-dependent

150

control
100 µM 100
300 µM
1 mM
50
3 mM
10 mM

Time [h]control
1 µM
5 µM15 µM
75 µM
150 µM2h24h72h141h
168h
192hcontact (morphological shape) and conductivity of adherent

Acetaminophen

0h

the impedance systems reveal alterations in confluency, cell

1500h

Combining these cells with the planar gold-film electrodes onBA

2h24h72h
141h
168h
192h

DILI responses in a 2D culture system using impedance.

Tox effect [%]these cells useful for investigations of acute and chronic

Time [h]Figure 2: Cell® Hepatocytes 2.0, cultured in 2D,Tox effect [%]150

showed hepatotoxicity of Aflatoxin B1, known toAflatoxin B1

cause such effects in metabolically active cells.

control
100 nM
300 nM
1 µM
3 µM
30 µM

10050

Thereby the use of impedance in resolving toxic
compound effects on hepatocytes does notalways need a 3D culture setting.

141h
168h
192hhh72242h0h0Time [h]Impedance data showed comparable results to other cellcomplex 3D culture setting is not always necessary.

viability and tox assays, rendering impedance as a reliableReferences

but non-invasive tool. We were able to show dose- andtime-dependent effects for all 5 compounds on iCell®
Hepatocytes 2.0. Figure 1 summarizes the impedancechange over time, after addition of acetaminophen,
diclofenac, troglitazone, and chlorpromazine.
Acetaminophen showed expected dose- and timedependent toxic effects. Diclofenac and troglitazone showed
more pronounced effects after 72h, which was consistent for
all compound concentrations. Low doses of chlorpromazine

1. Onakpova I.J., et al. (2016) BMC Medicine 14: 10
2. Olson H., et al. (2000) Regul. Toxicol. Pharmacol. 32(1): 56-673. Kew, M.C. (2013). J. Gastroint. Liver Dis. 22 (3): 305–310. PMID
24078988

Methods

had a smaller effect over time than higher doses, whichCells

showed full tox effects already after 2h. Additionally, effects

We thank FCDI for providing commercially available iCell®

of aflatoxin B1 were tested (Figure 2). The importance ofHepatocytes 2.0. We thank Dr. Katherine Czysz, Ouissame Mnie-

this compound in hepatotoxicity assay development lies in

Filali and Dr. Kirk Twaroski for their help in experimental design

the fact that this extremely toxic compound must first beand assistance with writing the document.

metabolized into its reactive electrophilic form, aflatoxin
B1-8,9-exo-epoxide by cytochrome p4503. The acute andImpedance and contractility measurements

chronic effects of Aflatoxin B1 were observed in standard 2D

Impedance measurements were conducted according toconditions, indicating that the hepatocytes were metabolicallyNanion’s standard procedures for the CardioExcyte 96 or

active and functional when using this impedance-based, non-AtlaZ. iCell Hepatocytes 2.0 were plated on NSP-96 plates andinvasive assay. The lowest concentration tested did not showmonitored according to protocol described below.

any effect, however after only 24h, aflatoxin B1 showed fulltox-effect starting with 1µM concentration.

Feed with Maintenance Medium
Thaw &
plate cells

Daily feed with
Plating medium

In summary, iCell® Hepatocytes 2.0 in combination withNanion´s impedance systems demonstrated results

Days in culture0

comparable to standardized optical readouts such

1234

Feed & ApplyCompounds

57

Feed every 2-3 days with Medium
containing compounds

101596 well NSP-96 plates with cell density 100k cells

as luminescent cell viability assays, with an important
advantage as a non-invasive, time-resolved experimental

To obtain 2D monolayers in 96-well plates, cells were seeded

approach. iCell® Hepatocytes 2.0 are a reliable source ofwith density of 300k cells/cm2, and recordings were performed

hepatocytes with consistent lot-to-lot performance thatstarting day 15 through day 21. Five concentrations per

maintain hepatic function over long culture periods. Taken

compound were applied in the external media, in technicaltogether, iPSC-derived hepatocytes paired with impedance

duplicates. Two hours before drug application the medium

time-resolved recordings, give a reliable and accurate

was removed from the wells and 200 μl fresh medium washepatotoxicity readout in 96-well format. Additionally, thisadded. Data was analyzed using respective Nanion´s software

assay demonstrated that with the use of impedance inare presented in scatterplots with mean ± SD.

resolving toxic compound effects on hepatocytes in a more

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