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International Journal of Bioprinting New challenges in liver tissue engineering

to print for recreating the liver microenvironment is the 6.2. Disease modeling using bioprinting
liver lobule, and the majority of studies dedicated to build 6.2.1. DILI
this structure have used extrusion bioprinting because the Bioprinting can also be used to assess drug-induced
resulting constructs are more viable than when using the hepatotoxicity, as it allows reproducible high-throughput
other techniques. The designs have a central hollow zone generation of 3D structures. Different systems with
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with printed endothelial cells, with radiating endothelial different materials in combination with hepatic cells have
cells separated from radiating hepatocytes, and a final been proposed to build 3D platforms for this purpose
surrounding structure with endothelial cells. These (see Table 2).
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structures combine the ECM comprising the ink together
with the cells, which are desirably homogeneous, to better Generally, different materials are combined to optimize
mimic the in vivo situation. bioprinting, as pure components are difficult to print, as in
the case of Pluronic, a material widely used for extrusion
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In extrusion-based bioprinting, bioink viscosity bioprinting due to its good printability and shape fidelity,
is one of the main parameters to be controlled for although, despite these advantages, it has rather poor
successful printing, which depends on characteristics mechanical properties that compromise its applicability. A
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such as the polymer concentration, polymer molecular combination of Pluronic with Alg enhances the mechanical
weight, polydispersity index, cell density, and processing properties of the mixture, a method used by the authors to
temperature. Highly viscous materials produce stable compare the material in supporting HepG2 cell line viability
structures after leaving the nozzle, although they can also and metabolic activity with monolayer cultures, using the
produce high shear stresses in the print head, limiting cell
survival. Low-viscosity materials are gentle on cells, but platform for high-throughput screening of hepatotoxic
printing pressure and filament shape are difficult to control. compounds. Compared to monolayer culture, cell viability
Shear thinning materials can be used to obtain a balance was significantly reduced when the 3D liver models were
between cell survival and shape fidelity. These materials exposed to a dose of acetaminophen, demonstrating the
reduce their viscosity in the presence of shear forces and printed model’s prediction of hepatotoxicity. In the case of
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facilitate bioink deposition without compromising the cells. Col, there were some difficulties in bioprinting. This is
Shape fidelity is guaranteed in this type of material because a low-viscous liquid when dissolved in acidic conditions;
filament viscosity is recovered after exiting the nozzle. however, when neutralized, it begins to assemble into
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Khati et al. developed a shear thinning bioink based on fibers, forming a physical hydrogel that compromises its
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liver dECM, Gel, and PEG that enabled the extrusion of bioprinting. The combination of methacrylated Col with
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dECM at body temperature. The nozzle diameter also thiolated HA produced a printable material with relevant
influences cell viability; lower pressure is required to components in liver ECM. When PHHs were bioprinted,
produce large-diameter filament than small diameters, and they maintained hepatocyte functionality, which
the cells demonstrated good viability after being exposed however declined when the systems were incubated with
to lower shear stresses. Hydrogel crosslinking needs to be acetaminophen, demonstrating the adequate response to
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fast enough to prevent the hydrogels from spreading out so hepatotoxic drugs. Another approach used a combination
that the filament keeps its shape. This better shape fidelity of Alg and Gel as bioink to construct a hepatic tissue model
can be achieved in hydrogels which have undergone a high based on encapsulated HLC derived from iPSC. The
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crosslinking degree. Shape fidelity also depends on the cells within the 3D-printed model had significantly better
hydrogels’ swelling properties after printing. A low degree hepatic functionality than the monolayer cultures and non-
of swelling, which can be regulated by the crosslinking printed sandwich-cultured models. The 3D-printed model
degree of the hydrogel, is required to maintain the shape was used to evaluate acetaminophen’s hepatotoxicity;
of the filament. the cells in the 3D-bioprinted model showed stronger
As bioprinting allows the automatic deposition of resistance to the drug than the sandwich-cultured model.
hydrogels and cells onto platforms with a precise and A three-component mixture is sometimes necessary
desired shape in a very short time, it has allowed the to increase printability, retain shape after printing, and
design of advanced 3D hepatotoxicity platforms to test improve cell viability, as in the case of the 3D liver model
the effect of multiple drugs in 3D-cultured hepatic cells. proposed by Schmidt et al. in which Alg, Gel, and Matrigel
This field has been extended to the automatic preparation were mixed to print scaffolds containing HepaRG cells.
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of 3D platforms to simulate in vitro liver diseases to allow The system allowed the culture of HepaRG cells for long
for the mechanistical exploration of these diseases and the periods, which would allow chronic treatments with
development of new drugs for treatment. Both of these toxic compounds such as albumin, suggesting that these
applications are summarized in the following section. hydrogels are suitable for repeated-dose assays.

Volume 10 Issue 3 (2024) 126 doi: 10.36922/ijb.2706