1. High-throughput compatible rat liver microtissues to
assess idiosyncratic toxicological effects
Introduction
Although idiosyncratic adverse drug reactions are rare they represent a significant risk for
healthcare and industry. Liver inflammatory cells, in addition to genetic and environmental
factors, are known to mediate such adverse reactions. So far, inflammation-mediated toxicity
has only been successfully reproduced experimentally in animal models. InSphero’s organotypic
rat liver microtissue (rLi MT) platform provides the first high throughput in vitro model shown to
be capable of detecting inflammation-related idiosyncratic effects. The toxicological effect of
Trovafloxacin and Ranitidine were tested either with or without Kupffer cell stimulation using
LPS. An increased toxicological effect could clearly be seen in the LPS-treated cultures. Predicting
idiosyncratic effects is a serious challenge for the pharmaceutical industry. Here, for the first
time, we show that a robust and affordable in vitro liver model, designed for high throughput
data generation, is capable of reproducing inflammation-mediated idiosyncratic toxicity.
Functional characterization of rLi microtissues
Scaffold-free liver microtissues were produced from primary hepatocytes and non-parenchymal
cells (NPC) comprising Kupffer macrophages and endothelial cells. Prior to MT formation in
hanging drops, hepatocytes and NPCs were mixed in MT re-aggregation medium.
Fig.1 Mitochondrial activity of rat liver
microtissues was stable for 5 weeks in culture,
as shown by stable ATP content over time, in
contrast to the corresponding 2D-sandwich
cultures from the same source (A). To assess
metabolic functionality CYP3A activity was
induced by the addition of 10 µM
Dexamethasone for 48 hours. CYP3A-
inducibility maturated over time and was stable
for 28 days in culture (B). Albumin secretion in
rLi MTs was 4-5x higher than in sandwich
cultures (C). To test functionality of Kupffer-
macrophages, the MTs were stimulated by
Lipopolysaccharides (LPS). Elevated levels of
Interleukin-6 (IL-6) were detected only in the
co-culture MTs (D).
InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 1

2. High-throughput compatible rat liver microtissues to
assess idiosyncratic toxicological effects
Inflammation-mediated toxicity
Inflammation has been shown to decrease the threshold for hepatoxicity of certain drugs. Here
we tested 2 drugs ,Trovafloxin and Ranitidine, both known to induce inflammation-mediated
toxicity. The presence of inflammatory cells in the liver microtissue model allows native-like
inflammation conditions to be reproduced, which, in turn, makes it possible to simulate
idiosyncratic drug toxicity.
Fig.2 Mitochondrial activity (A, C) and
cytotoxicity (B, D) of rLi MTs treated with
Trovafloxacin and Ranitidine. Both drugs
were incubated with and without LPS for
48h. Both end points reflect that the
addition of LPS impacts the toxicological
effects of both drugs as compared to the
DMSO control. .
Idiosyncratic adverse drug reactions affect various tissues, but the liver is one of the prime target
organs. Of the 28 drugs withdrawn from the U.S. market between 1976 and 2005, 6 were
withdrawn due to hepatoxicity. Therefore, model systems to detect adverse drug reactions
during drug development is a prime focus for the pharmaceutical industry. However, current
preclinical testing protocols often fail to identify drugs that cause idiosyncratic adverse drug
reactions, due to inappropriate model systems. Currently, only animal models have the ability to
reflect inflammation-mediated idiosyncratic toxicity. The rLi MT model has demonstrated its
ability to predict inflammation-induced hepatoxicity. This model, designed for high throughput
data generation, is easy to implement in drug testing campaigns for efficient early stage drug
de-risking.
InSphero AG, Zurich, Switzerland, www.insphero.com, sales@insphero.com, Phone +41-44-515049-0 2