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International Journal of Bioprinting Liver printing: from structure to application

μm), along with relatively low costs, as commercial printers has drawbacks such as high cost, time-consuming, and low
can be directly modified into inkjet bioprinters. 149,150 cell viability. 22,153,154
Matsusaki et al. designed a liver microtissue chip integrated
with over 400 simplified multilayered structures using 4.5. Photocuring-based bioprinting
inkjet printing, co-cultivating HepG2 cells and HUVECs Photocuring-based bioprinting involves continuous
for high-throughput drug screening (Figure 7A). The crosslinking of hydrogels for constructing 3D-printed
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3L (HUVEC/HepG2/HUVEC) structure exhibited higher bodies. This is achieved by locally solidifying hydrogels
hepatocyte activity compared to the 2L (HUVEC/HepG2) within a 2D plane and vertically moving crosslinking layers
and 1L (HepG2) structures due to increased cell–cell to build the final 3D structure. Photocuring-based printing
interactions between the top and bottom layers (Figure 7B). was initially used for scaffolds containing cells, and it was
Jian et al. utilized multicellular droplet-based bioprinting later adapted for bioprinting applications. Depending on the
technology to construct liver organoids with biomimetic modes of light transmission, photocuring-based printing
hepatic lobule structures. The printing process utilized two generally falls into two categories: stereolithography (SLA)
types of bioinks: (i) sodium alginate-HA-RGD containing and digital light processing (DLP). In SLA printing, a
``HepaRG cells” and “Fmoc-Tyr-Lys’’ (Fmoc-YKd)- scanning laser beam is projected point-by-point onto a
liquid photosensitive material to form a solidified layer.
calcium chloride containing HUVECs/hepatic stellate After the first layer is solidified, the platform rises to a
cells. These bioinks retained the printed structure through certain height, followed by crosslinking of the second layer.
in situ gelation facilitated by electrostatic interactions. This process is repeated until a complete 3D structure is
Compared to 2D monolayer cultures, the printed liver printed. In contrast, DLP uses digital mirror devices
organoids exhibited higher cell viability and superior ALB to directly solidify printing layers, rapidly forming 2D
and urea synthesis capabilities. 152
patterns. Ma et al. utilized a DLP-based 3D bioprinting
However, this technology faces significant challenges. system to embed hepatocyte progenitor cells derived from
Due to the low driving pressure of the printheads, it is hiPSCs, along with endothelial and mesenchymal support
challenging to print high-viscosity (>10 cP) materials cells, into a 3D hexagonal hydrogel construct. This setup
and high-density cells, resulting in printed structures facilitates the mimicking of liver lobules to simulate the
with weak mechanical integrity. Moreover, during inkjet cellular arrangement of the liver under physiological
printing, the low droplet placement accuracy and size conditions. Compared to traditional 2D and 3D models,
uniformity may cause mechanical or thermal damage to the three-cell co-culture model constructed by this strategy
cells. These drawbacks limit the widespread application of reported enhanced expression of liver-specific genes
inkjet printing technology. 19 (HNF4α, TTR, and ALB), increased secretion of metabolic
products (ALB and urea), and enhanced CYP induction
4.4. Laser-assisted bioprinting function. Similarly, Zhu et al. employed a DLP-based
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Laser-assisted bioprinting generally consists of two main microscale continuous optical bioprinting technology
techniques: laser-guided direct writing (LGDW) and to construct vascularized liver tissue with complex 3D
laser-induced forward transfer (LIFT). It is a nozzle-free microstructures, observing an alignment between the
printing technology typically composed of three layers printed vascular network and the host vascular system after
from top to bottom: an energy-absorbing layer, a donor transplantation. Wang et al. also utilized GelMA mixed
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layer, and a bioink layer. The bioink layer is suspended at with hyaluronic acid methacrylate (HAMA) for DLP-
the bottom of the donor layer. When a laser pulse is applied based liver bioprinting. Notably, by using hyaluronidase
to the energy-absorbing layer (e.g., titanium or gold), (Hase) to enzymatically digest HAMA molecules, the
the corresponding position of the donor layer below is mechanical properties of the printed constructs could be
vaporized, generating high-pressure bubbles that push the adjusted to match those of physiological tissue. A mixture
bioink onto a collection platform containing biopolymers of 5% GelMA and 1.5% HAMA was used for liver tissue
or cell culture medium. By controlling the z-axis of the construction and subjected to enzymatic digestion (Figure
collection platform, 3D structures can be formed. One 7C). Hepatocytes in the Hase-treated group exhibited
inherent advantage of laser-assisted bioprinting is its better cell viability and metabolic activity, as well as higher
nozzle-free printing method, eliminating issues related ALB and urea production (Figure 7D and E). Grigoryan
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to nozzle-clogging and preventing mechanical damage to and colleagues utilized SLA-based printing equipment to
cells. Therefore, it is suitable for printing high-viscosity prepare fibronectin hydrogel tissues containing aggregates
biological materials and bioinks with high cell densities. of hepatocytes. Compared to tissues containing single
The printing accuracy can reach the level of single-cell cells, the activity of the ALB promoter in aggregate-
droplets (approximately 10 μm in size). However, it also loaded hydrogel tissues was increased by over 60-fold.
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Volume 10 Issue 5 (2024) 133 doi: 10.36922/ijb.3819