Application Note

Cells: Hepatocytes and LSECs
Tools:
CardioExcyte 96

Toxicity analysis in expanded upcyte® liver
cells on Nanion’s CardioExcyte 96
upcyte Technologies and Nanion Technologies GmbHupcyte

www.upcyte.com ● info@upcyte.com

Summary

Results

Drug induced hepatic toxicity is one of the main reasons

The impedance signal recorded on the CardioExcyte 96

for regulatory actions and market withdrawals in the lastchanges as a result of alterations in confluency, cell

50 years [1]. In Europe and the USA, the major cause

contact (morphological shape) and conductivity ofof acute liver failure is indeed Drug Induced Liver Injury

adherent cells and thereby provides a measure of(DILI) [2]. Current existing in vitro models employed totoxicity. Base impedance of expanded upcyte® LSECs

predict DILI mostly focus on hepatocytes. Other cell types(donor 462), upcyte® hepatocytes (donor 10-03) or co-of the liver, such as liver sinusoidal endothelial cells (LSECs),

cultured cells (donor 462 + 10-03) were cultured for three

are often overlooked. It is thus questionable whether,days at confluence, before test compounds were added

hepatotoxicity can be sufficiently predicted by analyzing(72 h post seeding). Classical liver damaging compounds

hepatocytes only. LSECs are highly specialized endothelial

were tested: acetaminophen, imipramine, tamoxifen

cells forming the hepatic sinusoidal wall. Besides their highand diclofenac. Medium containing these compounds

endocytic potential, LSECs have been demonstrated towas exchanged every 2 days (120 + 168 h post seeding).

markedly contribute to liver homeostasis, immunity, andNormalized impedance values were recorded over 7

may partially explain unexpected hepatotoxicity ofdays (Figure 1A & B, data for tamoxifen and diclofenac

selected drug candidates.

are not shown).

Several reports in the literature have highlighted a highAsensitivity of LSECs towards hepatotoxic drugs [3]. It
has been suggested that LSECs act as an early directtargetforacetaminophen(paracetamol)

inducedtoxicity, causing early swelling and loss of uptake activity
and fenestrations before effects on hepatocytes areobserved [4]. LSECs are further important cellular targets

Bduring sinusoidal obstruction syndrome (a distinctiveand potentially fatal form of hepatic injury that occurs
predominantly, if not only, after drug or toxin exposure).
The use of primary LSECs for comprehensive in vitro studies
is compromised by poor cell yields, rapid dedifferentiation,
contamination with other endothelial cells, and limited
proliferation after isolation [5]. In this study upcyte® LSECs
have been developed as a complementary tool topredict hepatotoxicity, uptake and drug interactions. In
addition to conventional toxicity readouts such as ATPlevels, upcyte® LSECs have been successfully tested using
an impedance based system.
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Figure 1: Real-time impedance monitoring of expanded liver cells
treated with acetaminophen (APAP) or imipramine (IMIP). A Illustrated
concentrations for APAP include 10mM, 3.3 mM, 1.11 mM, and 0.37nM
and B for IMIP 10 mM, 2.5 mM, 0.63 mM and 0.16 mM. Values represent
mean ± SD (dotted lines) from duplicate values.

Application Note
Acetaminophen and imipramine displayed a complete
toxic effect on the liver cells (Figure 2A & B). Observing
the IC50 values, acetaminophen was slightly more toxic

towards LSECs compared to hepatocytes. Imipramine,on the other hand, demonstarted higher toxicity towards
hepatocytes. Diclofenac showed no in vitro toxicity effect on both intracellular ATP levels and impedance,
while tamoxifen induced only partial toxicity.
These

initialresults

show

thatLSECsandhepatocytes respond differently to typical hepatic
compounds
shouldandinclude

futuremoreinvitro

applications

thanonecelltype.

Table 1: Comparison of IC50 values for expanded LSECs, hepatocytes andco-cultured cells analyzed by intracellular ATP levels vs. impedance.
Impedance based IC50 values represent mean values from a single
experiment, while ATP-based IC50 values are summarized fromthree independant experiments, in technical duplicates.
APAP - acetaminophen; imipramine - IMIP.

BA

In summary, upcyte® hepatocytes and LSECs are areliable source of well characterized cells, facilitating
their use in hepatotoxicity. Generation of large batchesof these cells enables standardized, reproducible andcost efficient experimental settings. Classical hepatotoxicdrugs had similar pharmacological IC50 values inimpedance recordings compared to the intracellular ATP
measurements, when applied to these cells (Table 1). This
Figure 2: Impedance based dose response curves for expanded upcyte ®
LSECs and hepatocytes treated with hepatotoxic model compounds. A IC50
curve for acetaminophen (41.15 µM - 10mM) and B imipramine (0.98 µM- 1mM), with respective controls. Data represent mean ± SD summarizedfrom one donor per cell type in technical duplicates.

indicates that the impedance signal of the CardioExcyte
96 is a reliable, non-invasive and accurate measurement
for testing hepatoxicity, with an important advantage oftime-resolved experimental design.

References

Impedance measurements

1. Cubbon, R.M., & Lyon, A.R. 2016. IHJ l68: s77 – s85

Impedance measurements were conducted according

2. Bovelli, D., et al. 2010. Annals of Oncology 21 (Suppl. 5): v277–v282

to Nanion’s standard procedures for the CardioExcyte 96.

3. DeLeve LD., et al. 2013;123:1861–6. doi:10.1172/JCI66025

upcyte® hepatocytes (Donor 10-03) and LSECs (Donor4. McCuskey RS., et al. 2006. Clin Hemorheol Microcirc. 34:5–10462) and their co-culture were plated on NSP-96 plates

5. Elvevold K, Smedsrød B, Martinez I. 2008. Am J Physiol Gastrointest Liverand monitored for 3 days for the base impedance signal
to stabilize. Acetaminophen, imipramine, tamoxifen orPhysiol. 294:G391-400

diclofenac were applied in the external media. AboutMethods

2 hours before drug application the medium wascompletely removed from the wells and 200 μl freshCells

medium was added. 6 concentrations were applied perWe thank upcyte® technologies for providing the cells.
We thank Dr. Timo Johannssen and Dr. Astrid Nörenberg
from upcyte® for performing the experiments andassistance with writing the document.

substance, in technical duplicates.

Cultures weremonitored for 7 days, repeated dosing every 2-3 days.
Cells were then analyzed for intracellular ATP levels or
normalized impedance values.

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