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Introduction
Traditionally, chemical toxicity testing relied heavily on the use of various animal models to
extrapolate potentially toxic events in humans [1]. Such approaches have facilitated the
evaluation of specific toxicological endpoints, including immunotoxicity, genotoxicity,
reproductive toxicity, and various others [2]. However, toxicological assessment of chemicals
is moving progressively toward an in vitro approach for reasons relating to improved
efficiency, scientific relevancy, and cost [3]. Furthermore, in vitro strategies provide a far
more ethical approach to toxicity testing, which remains a major limitation of animal-based
experiments [3]. Implying potential human toxicity events from in vitro based chemical
exposure is an especially difficult task, however recent advancements in in vitro toxicity
testing techniques may encourage increased application of such approaches [4]. In this
answer, I will critically discuss traditional in vitro techniques, along with recent
developments in in vitro approaches and the crucial strengths and limitations of novel
strategies compared to traditional strategies.

Traditional Toxicity Testing Approaches

Cell culture systems describe the dispersion of cells in a synthetic environment which acts as
an appropriate surface for optimal cell growth, through providing a sufficient nutrient supply
and suitable environmental conditions [5]. Over the years, the use of various cell culture
systems have increasingly facilitated the identification of potentially toxic events in response
to novel chemicals [6], with 2-dimensional (2D) cell culture techniques been adopted in
chemical assessments since the early 1900s [7]. Notably, 3-dimensional (3D) techniques were
first applied in the 1970s [8]. In 2D cultures, cell colonies develop as a monolayer present in
a culture flask or petri dish, supported by a plastic structure [9], whereas 3D cultures may be
prepared as suspension models, cultures in concentrated medium, or as cultures on a scaffold
[10]. Traditional cell culture techniques, which typically utilise immortalised human cancer
cell lines, have several advantages in toxicity testing, including a low cost and basic
maintenance protocol; Furthermore, such approaches may be applied for the assessment of
various toxicity types, as immortalised cancer cell lines demonstrate powerful toxicological
predictivity. Two common examples where such traditional approaches are applicable
include in vitro tests for genotoxicity and cytotoxicity: Traditional in vitro tests for
genotoxicity utilise micronuclei assays in immortalised mammalian (‘IVGT’) cells cultured
in suspension or in monolayers [11, 12], while traditional tests for cytotoxicity typically
utilise both cell survival and cell death assays in Balb/c 3T3 cells cultured for formation of
monolayers [13]. Notably, despite their various advantages and toxicological applications,
such conventional approaches are commonly over-shadowed by various limitations (figure
1), which tend to emerge primarily from the cells utilised in these approaches [14]. Human
tumour cells have long acted as the main source of immortalised cells for conventional in
vitro toxicology techniques, however, these cells vary dramatically to living human cells in
terms of genomic stability and thus physiology [14]. Consequently, the traditional models fail
to recapitulate (accurately mimic?) the cellular microenvironment in healthy organisms, thus
generating concerns for how representative these assays are for an average response in

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healthy humans [3]. Therefore, although traditional cell culture-based in vitro approaches
provide a cost effective and simplistic strategy for detecting chemical toxicity, such
approaches are largely overshadowed by inadequate physiological relevance [10].
Furthermore, the impact of such limitations combined with the urgent need for alternative,
more ethical approaches to animal-based experiments has led to the emergence of various
novel in vitro approaches for toxicity testing.

Figure 1: Common limitations of conventional in vitro cell culture approaches [14].

Novel Toxicity Testing Approaches

There are various novel in vitro cell culture strategies which seek to overcome the various
limitations of traditional cell culture approaches in compound toxicity profiling, and these
strategies appear to be significantly more physiologically relevant [14]. Such strategies
include, likely the most promising approach; the use of organ-on-chip technologies, as well
as the application of stem cell-derived human cells in 2D cultures and novel 3D cell culture
models, such as organoid models [14].

Organ-on-chip technologies
One major development in toxicology profiling which seeks to surmount the general
consensus that cell culture techniques are too basic and physiologically irrelevant is the
‘organ on a chip’ model, which describes the next generation of 3D cell culture models [4].
These organs-on-chips (OoCs) have been shown to better represent the microstructure, as
well as biochemical functionalities and diverse mechanical properties of healthy organisms
[15]. Such models incorporate complex microfluidic technologies into actively dividing cells,
cultured in 3D devices generated by microfabrication techniques, to evaluate human
physiology and toxicology in an organ-specific manner [15]. Furthermore, such models
facilitate the development of specific in vitro disease models. Traditionally, the crucial
targeted tissues for chemical toxicity profiling include the liver, heart, kidney, vasculature,
and brain [16]. Conventional in vitro approaches utilised in assessing toxicity in such organs

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generally apply high-throughput, but basic cell culture assays, which are incapable of
mimicking a dynamic systemic response to a chemical [16].

OoCs may provide advantages over traditional in vitro approaches for assessing organ-
specific toxicities by facilitating modelling of intricate human responses in well-regulated in
vitro systems which may permit model organ interface [17], and additionally, may be
designed for specialised contexts of use [18]. Therefore, single-tissue OoCs promote
alternative approaches for the assessment of chemical toxicity in several human tissues. For
example, hepatic cell monolayer cultures inadequately mimic drug responses in human
hepatocytes [19]. Furthermore, rapid dedifferentiation of cultured cells in these 2D liver
models restricts efficiency in assessing both short- and long-term exposure effects and
systemic toxicity effects [19]. However, a novel 3D liver OoC system capable of maintaining
healthy liver cell cultures for over 28 days, and better simulating the in vivo liver
microenvironment through incorporating immune components and haemodynamic flow, may
provide a more efficient approach in assessing liver toxicity compared to classical in vitro
approaches [20]. Nonetheless, liver-on-a-chip systems still face a number of major limitations
compared to traditional cell-culture approaches; For example, the most commonly used liver-
on-a-chip system is made of the silicone rubber, poly(dimethylsiloxane) (PDMS), largely due
to the fact that it is cheap and easy to manufacture, however, this material has recently been
shown to absorb small hydrophobic molecules, which may include numerous
pharmacological agents [21]. Such a feature is undesirable in toxicity profiling as it could
potentially skew results, particularly in trials dealing with hydrophobic molecules such as
micelle delivered small molecules. Furthermore, despite their proposed status of having an
improved longevity compared to conventional cell culture techniques, the lifetime of liver-
on-a-chip systems has been shown to rarely exceed 2 weeks [22]. Furthermore, various heart-
on-a-chip systems have also been produced. Such systems, compared to traditional strategies,
better model the complexity of cardiomyocytes, as well as other cardiac cells such as
fibroblasts and endothelial cells [16]. Novel heart on a chip systems are considerably more
efficient in assessing cardiotoxicity than traditional cell culture approaches as they efficiently
model human responses to cell insult, and exhibit properly aligned sarcomeres as well as
synchronised beating rhythmicity [23]. Furthermore, in comparison with classical cell culture
models, the application of microfluidic chips in cardiotoxicity profiling facilitates the
collection of greater morphometric and electrophysiological data [24]. OoC systems
representative of the nephron and proximal tubules, have been utilised to model
nephrotoxicity profiling readouts including filtration and reabsorption [25]. Notably, in a
recent study, a kidney-on-a-chip system was utilised to determine the role of the cholesterol
biosynthesis pathway in poly-myxin B-associated nephrotoxicity [26]. In doing so, it was
demonstrated that OoCs may not only be applied to toxicity profiling of novel compounds,
but also to further comprehend toxicological pathways of already-approved compounds,
which remain poorly understood due to inadequacies of traditional in vitro toxicological
assessment strategies used in the past. Notably, it has recently emerged that it may be
advantageous to use lung-on-a-chip models rather than conventional 2D and 3D cell culture
models for assessing the toxicity of inhaled compounds due to their ability to recapitulate the

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air-liquid interface of human alveoli [27]. One study has demonstrated that compared to lung-
on-a-chip models, past cell culture models underestimated the toxic impact of air pollution on
our bodies [28].

OoC systems clearly demonstrate significant advantages over traditional cell culture
techniques in novel compound toxicity profiling in vitro, however, there remains a number of
associated limitations compared to conventional in vitro approaches. The shortcomings of
such technology include the discussed organ specific limitations as well as a number of
general limitations, including challenges in standardisation and scale up preventing high
throughput screening [16], the need for external bulky equipment as well as common
undesirable surface effects [29]. Although OoC technologies are relatively new and are still
restricted in use due to a number of associated limitations, their potential to predict
toxicological effects of novel compounds may have a profound impact on the strategies used
for toxicity profiling in drug discovery in the near future.

Stem cell-derived human cells.

The application of human pluripotent stem cell-derived models may act as another efficient
alternative to overcome some of the major limitations of traditional in vitro toxicity models,
such as genetic variations present in immortalised cancerous cell lines [30], limited
differentiation capacity, limited source availability, and various ethical concerns; Human
pluripotent stem cells (hPSCs) have the ability to transform/differentiate into various cell
types in the body, and thus offer appropriate and efficient toxicology screening systems for
specific human tissues [31]. Considering that hPSCs may be derived from already
differentiated cells and that modern technologies can induce genome modifications, hPSCs
may be utilised for chemical toxicity screening in both disease specific and healthy systems
[31]. Importantly, these approaches have demonstrated significant potential for the in vitro
assessment of compound cardiotoxicity, as well as compound hepatotoxicity and
neurotoxicity.

hPSC-derived cardiomyocytes, differentiated from either human embryonic stem cells

(hESCs) or induced pluripotent stem cells (iPSCs), may be utilised effectively as in vitro
models for assessing novel compound cardiotoxicity [31]. Traditional cardiotoxicity profiling
has been based around in vitro electrophysiology tests typically performed on immortalised
animal-derived cardiomyocytes to evaluate the risk of arrhythmia. Notably, although the use
of animal cells are valuable in basic toxicological studies, their poor differentiation and
significant physiological differences considerably restrict the translation of findings to
healthy humans [32]. Furthermore, such approaches depend on the application of cell lines
manipulated to express single ion channels [33]. Considering in vivo cardiac action potential
requires the organisation of multiple ion channels, these assays act as poor predictors of
arrhythmia risk [34]. However, novel stem cell techniques may now be utilised to generate

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large supplies of cardiomyocytes which express multiple ion channel genes and thus, more
efficiently mimic in vivo cardiac electrophysiology and exhibit significant potential for in
vitro high-throughput screening approaches [34]. Notably, although cardiomyocytes, along
with many other human stem cell-derived cells, were first derived from embryonic stem cells;
there were major concerns regarding the ethics of such an approach [31]. Considering this,
new strategies to develop cells for in vitro toxicity profiling were developed, and are focused
around the application of induced pluripotent stem cells (hiPSCs) (figure 2) [31]. Although
approaches utilising hiPSCs have demonstrated significant potential in identifying more
physiologically relevant toxicity endpoints; to date, high-throughput screening approaches
have been largely overshadowed by the maturation state of hPSC-derived cardiomyocytes
[35]. These derived cells typically exhibit a fetal or neonatal phenotype, which may be
appropriate for developmental toxicity studies, however, considerably limits their usefulness
compared to traditional approaches in the assessment of general cardiotoxicity [35]. Notably,
a recent study has demonstrated that increasing energy transfer mechanisms in neonatal
cardiomyocytes may facilitate an improved maturation state, and thus allow for improved,
more physiologically relevant cardiotoxicity assessment in vitro [36].

Figure 2: Various strategies for the derivation of cardiomyocytes from sematic cells in culture. The induction of
pluripotency and differentiation into cardiomyocytes is the most well established approach [36].

Furthermore, there are major limitations of current in vitro approaches utilised in the
assessment of novel compound hepatotoxicity and neurotoxicity. In recent years, the
emergence of drug-induced liver injury has been the most common reason for regulatory
approved pharmaceuticals to be withdrawn from the market [37]. Although the use of
traditional 2D hepatotoxicity cell culture methods, which utilise immortalised human hepatic
cell lines (HepG2 and HepaRG), as well as primary human hepatocytes, are capable of
predicting general hepatotoxicity, the shortage of applicable human liver tissue along with the
genomic instability of immortalised cancer cell lines limit the efficacy of such tests [31].
There is evidence to suggest that the development and application of differentiation
procedures may allow for the various limitations and poor predictability of conventional in
vitro hepatotoxicity techniques to be overcome. Such an approach may facilitate the
generation of genetically stable and functional hPSC-derived human hepato-like cells
(HLCs), which have the potential to revolutionise in vitro hepatotoxicity testing [38].
However, similar to hPSC-derived cardiomyocytes, such novel approaches typically generate

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immature HLCs with inconsistent differentiation efficacy [39]. Therefore, one major
shortcoming of this approach is that stable and functionally reliable HLCs, that may be
utilised for hepatotoxicity screening, have yet to be derived [31]. Finally, the development of
novel in vitro neurotoxicity assessment approaches poses a significant challenge considering
the numerous limitations of current models. Most notably, current neurotoxicity in vitro
models lack blood brain barrier (BBB) function and the ability to assess major neurotoxicity
endpoints [40]. Crucially, the use of hPSCs in neurotoxicology in vitro approaches may solve
some of the major problems associated with current models, including the lack of BBB
function [31]. Notably, various protocols for hPSC differentiation into several neuronal cell
types have already been established, including protocols for dopaminergic [41] and
cholinergic neurons [42]. However, similar to hPSC-derived cardiomyocytes and HLCs,
these differentiated neural cell types typically exhibit a fetal phenotype [31]. Despite this,
multiple hPSC-derived neurotoxicity models have already been utilised for the validation of
established neurotoxic agents [43].

hPSC-derived 3D models (Organoids)

Although the application of specific hPSC-derived cells for toxicity profiling demonstrates
significant potential, especially compared to traditional in vitro approaches, these models are
often inadequate, particularly when modelling of whole human organs and their specialised
functions are required [31]. Therefore, one major challenge of in vitro toxicology is the
development of hPSC-derived 3D organoids which better mimic in vivo systematic
responses. 3D organoid systems are defined as in vitro tissue models which consist of an
assortment of self-organised, organ-specific cell types assembled from pluripotent stem cells
or various primary progenitor cells [44]. Notably, organoids gave been generated with the use
of both embryonic stem cells and hiPSCs [45]. The chosen cell type typically differentiates
through either directed or undirected differentiation in a hydrogen matrix , and through gene-
encoded self-organisation, cells with varying adhesion receptors, accumulate to generate a 3D
structure, generally representing the functional unit a tissue of interest (figure 3) [45]. These
3D models generally produce tissue-specific molecules and exhibit limited organ function.
Furthermore, they may be maintained in culture for several months when cocultured with
specific pathway activators [46]. Therefore, compared to traditional cell culture models,
organoids provide long lasting, more physiologically relevant systems for in vitro
toxicological studies.

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Figure 3: Schematic illustrating the various pathways for which organoids are derived from hPSCs [45].

Human organoid models have been generated for various tissue types, including the liver
[47], brain [48], lungs [49], and kidney [50], and hence, such models demonstrate
considerable potential for improved in vitro toxicity approaches, however, the potential of 3D
organoid models remains to be overshadowed by the numerous limitations associated with
their use [45]. For example, although organoids typically exhibit some mature tissue
functions, these 3D structures generally represent an early developmental stage [45].
Therefore, such models are only suitable for developmental toxicology profiling of novel
compounds. Furthermore, even their application in developmental toxicology studies is often
inefficient compared to traditional methods due to variable reproducibility in relation to
organoid size, differentiation stage and cellular composition [31]. Finally, organoids often
only consist of one or two different cell types, and thus, fail to effectively represent complete
tissue organisation [51]. Consequently, organoid models generally fail to provide an efficient
and more physiologically relevant alternative to current in vitro models utilised for toxicity
profiling.

Conclusion
Despite their cost-effective and simplistic nature, traditional cell culture approaches have
largely prohibited the progression of toxicology assessment from animal based approaches to
more ethical in vitro approaches. This is primarily a consequence of traditional approaches
typically applying immortalised cancer cell lines, which inadequately represent the
physiology and response of healthy human cells. However, several novel in vitro strategies,
including the utilisation of hPSC-derived cells, 3D organoids, and organ-on-chip models,
have demonstrated considerable potential in overcoming some of major limitations of
traditional cell culture models. However, these novel approaches remain largely inefficient in
toxicology assessment due a number of associated limitations, including inadequate stem cell
differentiation in organoid and hPSC-derived models, as well as various organ-specific
limitations in organ-on-chip models. It is thought that the overcoming of these limitations
will encourage increased application of in vitro approaches for novel compound toxicology
assessment.

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