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International Journal of Bioprinting Three-dimensional bioprinting in toxicological research

4. Recently used two-dimensional (2D) test activity reduce in just few days in culture. Moreover,
systems primary cells derived from different donors can be used
to investigate interindividual differences but they have
The liver plays a key role in drug processing, so investigating lower reproducibility. 2D primary hepatocytes and
its response to different drugs is critical in pharmacokinetics. sandwich cultures dedifferentiate in a couple of days and
In vivo animal models are a usable and convenient tool for lose many of their hepatocyte-specific functions, such as
drug testing, but due to animal welfare considerations and metabolic capacity, transporter expression, and sensitivity
their disadvantages, such as costly experiments and the to toxic effects. Freshly isolated hepatocytes are known
fact that the physiology of animal cannot be recapitulate as the best cell type in resembling the intact liver, though
that of humans, animal models may not be the perfect tool recently, cryopreserved cells have been found to maintain
for drug testing. There are many 2Dand 3D in vitro liver hepatocyte characteristics quite well, so they might be a
models, which are useful for ADME-Tox tests, but each surrogate to freshly prepared hepatocytes and provide
has its own limitations . Frequently applied 2D in vitro a solution for addressing the limited availability of fresh
[23]
methods are using human primary hepatocytes (hNHEPS) liver tissue. The interindividual variability of human
and cell lines, immortalized hepatoma lines, stem cells- hepatocytes often makes the interpretation of preclinical
derived hepatocyte-like cells, liver slices, and microsomes. drug testing challenging [23,35] In pharmacokinetic research,
Modeling the liver architecture is an extremely difficult task using primary hepatocyte cultures from humans and
because the cells have to be polarized, owning functionally other species is the standard practice, which facilitates
distinct membrane domains, expressing the right type and the identification of unique human metabolites and the
number of enzymes, transporters and other proteins, and exploration of proper liver models that can be used in
in contact with blood and bile flow to perform the proper the testing of drugs for human diseases. Important steps
and native tissue-like liver function. Finding the best 3D in drug development are metabolite prediction, biological
liver model is still a challenge; the current methods are barrier modeling, prediction of in vivo pharmacokinetic
mainly suitable for studying the intrinsic hepatic clearance processes, mitochondrial toxicological testing, and
in human in the presence of drugs [23-27] . quantitative in vitro-in vivo extrapolation [1,23,36-39] .
Hepatocyte fractions as well as primary hepatocytes
in two dimensions and in sandwich cultures are the 5. Micropatterned hepatocyte cultures
most widely used models for mechanistic ADME-Tox In the micropatterned co-culture technique, 24- or
studies at the early stage of drug development. 2D models 96-well plates are used. Collagen islands with a diameter
cannot provide a proper structure, but the maintenance of 500 μm are fabricated on these plates at a distance of
cost is relatively low; and the models are easy to handle, 900 – 1200 μm from each other. Collagen islands provide
allows unlimited growth; and can be used with cell lines, a 3D extracellular matrix for primary human hepatocytes,
immortalized hepatoma lines (HepG2, Hep3B, Huh7, anf so the cells are able to form organ-like morphology and
HepaRG) and stem cell-derived hepatocyte-like cells. Other polarity. In this case, the inter-island space is filled with
methods are liver slices, but they have major limitations, supporting J2-3T3 murine fibroblast. The co-culture
such as short-term maintenance and rapid loss of activity. system of primary human liver cells and J2-3T3 mouse
These slices are able to produce albumin and urea, but embryonic fibroblasts is sustainable for 4 – 6 weeks and
they are not suitable for longer tests [32,33] . These models are is suitable for drug testing, representing an easy-to-use,
often used for drug processing studies, but do not show robust and reproducible system formed simultaneously
long-term metabolizing activity and/or cannot express all from fresh or cryopreserved hepatocytes derived from
metabolic enzymes. Furthermore, gene expression in cell multiple donors. Once the co-culture is established, the
lines would change during passaging, showing that their islands can be infected with a virus involved in various
similarity to native tissue would lose with time. Applying liver diseases, making it an excellent model for mimicking
immortalized hepatocyte cell lines, such as HepG2, individual liver diseases (Figure 3) [40-43] .
HepaRG, Huh7, and Fa2N-4, might provide a solution for Khetani et al. found that HepaRG/3T3-J2 co-cultures
these limitations, as these cell lines are well characterized produced higher albumin than mono-cultures after
and less variable, but many of their hepatic functions are 4 weeks of culturing and showed increased sensitivity to
underexpressed or even totally lost in these cells [4,23,34] . drug-mediated CYP induction and hepatotoxicity since

Primary hepatocytes may be the best option among they have more stable albumin and CYP activity . The
[40]
2D models because they contain endogenous enzymes micropatterned hepatocyte cultures are expensive and not
and transporters and are polarized in sandwich suitable for high-throughput screening, which are some of
configuration, but their expression level and functional the drawbacks of this technique.

Volume 9 Issue 2 (2023) 201 https://doi.org/10.18063/ijb.v9i2.663