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International Journal of Bioprinting Three-dimensional bioprinting in toxicological research

linking bio-resin by moving the beam. The polymerization The skin is also responsible for defense mechanisms,
through two-photon absorption allows direct printing and as the first barrier of the body, it is impacted by
into negative-tone photoresist, while reverse imprint many harmful agents that cause irritation, corrosion,
is formed in positive-tone photoresist [111-115] . During or sensitization. Thus, it could not be excluded from
printing, the laser power has to be kept in a range between toxicity studies. Because skin has multiple layers, cell
polymerization threshold and burning threshold to types and appendages, creating native tissue-like models
achieve fine structure formation without material damage. is difficult [123-127] . Abaci et al. demonstrated a human
The printed microstructure could be extracted from skin model with a perfusable vascular network using
bio-resin by ethanol washing to remove unpolymerized primary and iPSC-derived endothelial cells [128] . They
negative photoresist. Its features facilitate high-spatial built a micropatterned vasculature layer using sacrificial
resolution, since two-photon absorption only occurs in hydrogel, and dermal fibroblasts suspended in collagen
the focal point, termed submicron-size voxel. Another type I were seeded around the sacrificial layer. Following
great property of this technique is that the near-infrared the formation of the dermal region, keratinocytes
laser is able to penetrate deeply into the photoresist and were added to form an epithelial layer. This construct
to print in three dimensions. Its drawbacks include long was used for in vitro perfusion experiments or in vivo
printing time and lack of capability to print scaffolds for grafting. Their results demonstrated that micropatterned
soft tissues [10,80,90,94,102-105,111-115] . vascularization enabled the development of complex
human skin equivalents that are graftable and suitable
8.4. Recent achievements in 3D tissue bioprinting for drug toxicity testing. Min et al. used human dermal
At present, the 3D structure of the nephron is too complex fibroblasts, epidermal keratinocytes, and epidermal
to be bioprinted as a complete unit. Therefore, researchers melanocytes to create a full-thickness pigmented skin
must select the segment most affected by renal toxicity, model [129] . The printed tissue showed pigmented clusters
and the proximal tubule segment is commonly selected for consisting of melanocytes and keratinocytes and active
this purpose [116-118] Homan et al. developed a tubule-like melanin production confirmed by histological staining.
structure with proximal tube epithelial cells and sacrificial Ng et al. developed a two-step bioprinting method
hydrogel for forming a tube that is suitable for investigating to produce a pigmented human skin construct and
the mechanism of drug-induced tubule damage [119] . They produced a biomimetic dermal region out of different
bioprinted a tubular structure with gelatin-fibrin hydrogel densities of human fibroblasts and collagen, resulting
on the outside and liquefiable Pluronic F147 on the inside, in a hierarchical porous structure [130] . To achieve native
which was removed at the end of the process. Thus, they tissue-like structure, they printed human keratinocytes
created a vascularized construct with a fully epithelialized, and melanocytes in a well-designed pattern onto
perfusable channel, and albumin uptake, cyclosporin the dermal layer. This technique allows the presence
A-induced nephrotoxicity and polarized epithelium could of melanin units as well as the creation of suitable
be observed. A few years later, Lin et al. developed a model microenvironment. The epidermal region resembled
with two perfusable channels using sacrificial hydrogel [120] . native skin in terms of melanin granules distribution and
In this kidney construct, they seeded proximal tubule presence of biomarkers, such as HMB-45, K1, K6, and
epithelial cells into one of the two tubules and glomerular collagen type VII.
microvascular endothelial cells into the other one. They Hong and Song developed a gelatin-alginate based
observed an active tubular-vascular exchange, albumin HepG2 3D model [131] . They used gelatin and sodium
uptake, and glucose reabsorption. Lawlor et al. showed alginate in phosphate-buffered saline at a concentration of
a high-throughput, self-organized kidney organoid 40% (w/v) sterilized by UV light. The hydrogel-cell mixture,
system made up of pluripotent stem cells, that is suitable consisting of 10% gelatin, 4% alginate, and HepG2 cells,
for nephrotoxicity testing using extrusion-based was printed as spheroid structures in mini-well dishes.
bioprinting [121] . This method makes it possible to produce They observed that this 3D model could mimic the organ
organoids 15 – 20 times faster, with high reproducibility, complexity better than the 2D models and is suitable for
than the manually made organoids. King et al. created a hepatotoxicity tests [131] . Kizawa et al. created a bioprinted
co-culture of renal fibroblast and human umbilical vein liver tissue, which consists of primary hepatocytes, using
endothelial cells (HUVEC) in 50:50 ratio, dispensed scaffold-free technology of Cyfuse Biomedical. This model
Novogel and bioprinted onto a transwell insert [122] . is suitable for drug testing since it retains drug transporter
They created an epithelial layer capable of producing proteins and metabolic enzymes expression [132] . Ma et al.
large amount of extracellular matrix and maintaining a reported their 3D-bioprinted tri-culture, developed by
functional renin-angiotensin system and barrier. digital light processing-based method. Their liver model,

Volume 9 Issue 2 (2023) 208 https://doi.org/10.18063/ijb.v9i2.663