RESOURCES

Abstract
Abstract Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-like cellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural–functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTs in a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200–300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.

Abstract
The spheroid culture system has gained significant attention as an effective in vitro model to mimic the in vivo microenvironment. Even though numerous studies were focused on developing spheroids, the structural organization of encapsulated cells within hydrogels remains a challenge. Allylated gelatin or GelAGE is used as a bioink due to its excellent physicochemical properties. In this study, GelAGE was evaluated for its capacity to induce spontaneous spheroid formation in encapsulated HepG2 cells. GelAGE was synthesized and characterized using 1HNMR spectroscopy and ninhydrin assay. Then the physicochemical and biological attributes of GelAGE hydrogel was examined. The results demonstrate that GelAGE has remarkable ability to induce the encapsulated cells to self-organize into spheroids.

Abstract
Three-dimensional liver bioprinting is an emerging technology in the field of regenerative medicine that aids in the creation of functional tissue constructs that can be used as transplantable organ substitutes. During transplantation, the bioprinted donor liver must be protected from the oxidative stress environment created by various factors during the transplantation procedure, as well as from drug-induced damage from medications taken as part of the post-surgery medication regimen following the procedure. In this study, Silymarin, a flavonoid with the hepatoprotective properties were introduced into the GelMA bioink formulation to protect the bioprinted liver against hepatotoxicity. The concentration of silymarin to be added in GelMA was optimised, bioink properties were evaluated, and HepG2 cells were used to bioprint liver tissue. Carbon tetrachloride (CCl4) was used to induce hepatotoxicity in bioprinted liver, and the effect of this chemical on the metabolic activities of HepG2 cells was studied. The results showed that Silymarin helps with albumin synthesis and shields liver tissue from the damaging effects of CCl4. According to gene expression analysis, CCl4 treatment increased TNF-α and the antioxidant enzyme SOD expression in HepG2 cells while the presence of silymarin protected the bioprinted construct from CCl4-induced damage. Thus, the outcomes demonstrate that the addition of silymarin in GelMA formulation protects liver function in toxic environments.

Abstract
Three-dimensional (3D) liver bioprinting is a promising technique for creating 3D liver models that can be used for in vitro drug testing, hepatotoxicity studies, and transplantation. The functional performance of 3D bioprinted liver constructs are limited by the lack of cell–cell interactions, which calls for the creation of bioprinted tissue constructs with high cell densities. This study reports the fabrication of 3D bioprinted liver constructs using a novel photocrosslinkable gelatin methacrylamide (GelMA)-based bioink formulation. However, the formation of excess free radicals during photoinitiation poses a challenge, particularly during photocrosslinking of large constructs with high cell densities. Hence, we designed a bioink formulation comprising the base polymer GelMA loaded with an antioxidant cocktail containing vitamin C (L-ascorbic acid (AA)) and vitamin E (α-tocopherol (α-Toc)). We confirmed that the combination of antioxidants loaded in GelMA enhanced the ability to scavenge intracellular reactive oxygen species formed during photocrosslinking. The GelMA formulation was evaluated for biocompatibility in vitro and in vivo. These results demonstrated that the bioink had adequate rheological characteristics and was biocompatible. Furthermore, when compared to bioprinted constructs with lower cell density, high-density primary rat hepatocyte constructs demonstrated improved cell-cell interactions and liver-specific functions like albumin and urea secretion, which increased 5-fold and 2.5-fold, respectively.

Abstract
Three dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.

Abstract
Designing printable bioinks for 3D bioprinting capable of supporting cellular viability with post-printing functionality remains challenging. Native ECM offers several physical, chemical, and biological cues that are difficult to restore using only a single component. Herein, we have optimized a multicomponent-based bioink formulation comprising alginate (ALG), gelatin (GEL), diethylaminoethyl cellulose (DCEL) and fibrinogen (FIB), termed as ALG-GEL-DCEL-FIB bioink for potential application in bioprinting and biofabrication of skin tissue equivalents. The designed formulation was extensively studied for its printability, physico-chemical, rheological, and biocompatibility properties. Excellent printability, shape fidelity and cell-laden tissue equivalent printing were established using the RegenHu 3D Discovery Bioprinter. The human primary fibroblast and keratinocyte-laden bioprinted constructs exhibited good cell viability. Long term culture of 4 weeks comprising 5 days of air-liquid-interphase followed by 21 days of submerged culture produced biomimetic tissue histology in the ALG-GEL-DCEL-FIB bioink printed constructs. Specific epidermal-dermal marker expressions proving functionality were evident in immunohistochemical, biochemical and gene expression analysis. The ALG-GEL-DCEL-FIB bioink may be explored further for potential biofabrication and therapeutic applications.

Abstract
Methacrylated gelatin (GelMA) in the form of methacryloyl, methacrylate, and methacrylamide is an established and widely accepted photocrosslinkable bioink, for three dimensional bioprinting of various tissues. One of the limitations of photocrosslinkable bioinks is the inability to control the free radicals generated by photoinitiators and ultraviolet (UV) rays. The presence of excess free radicals compromises the viability and functionality of cells during crosslinking. In this study, ascorbic acid, a known free radical scavenger (FRS) molecule, was introduced into the GelMA bioink formulation to protect the cell viability, proliferation, and tissue functions of 3D bioprinted parenchymal liver constructs. The concentration of FRS in the bioink was optimized and used for 3D bioprinting of HepG2 cells. The results confirmed that the inclusion of 3.4 mM FRS in the GelMA bioink formulation nullified the excess ROS formed inside the cells. Furthermore, the optimized GelMA formulation containing FRS preserved and improved the cell activity, albumin, and urea synthesis in the 3D construct over 7 days in culture. In the future, this concept could be implemented in the biofabrication of large liver constructs that require multiple or longer durations of UV irradiation.

Abstract
Layer-by-layer additive manufacturing process has evolved into three-dimensional (3D) “bio-printing” as a means of constructing cell-laden functional tissue equivalents. The process typically involves the mixing of cells of interest with an appropriate hydrogel, termed as “bioink”, followed by printing and tissue maturation. An ideal bioink should have adequate mechanical, rheological, and biological features of the target tissues. However, native extracellular matrix (ECM) is made of an intricate milieu of soluble and non-soluble extracellular factors, and mimicking such a composition is challenging. To this end, here we report the formulation of a multi-component bioink composed of gelatin and alginate -based scaffolding material, as well as a platelet-rich plasma (PRP) suspension, which mimics the insoluble and soluble factors of native ECM respectively. Briefly, sodium alginate was subjected to controlled oxidation to yield alginate dialdehyde (ADA), and was mixed with gelatin and PRP in various volume ratios in the presence of borax. The formulation was systematically characterized for its gelation time, swelling, and water uptake, as well as its morphological, chemical, and rheological properties; furthermore, blood- and cytocompatibility were assessed as per ISO 10993 (International Organization for Standardization). Printability, shape fidelity, and cell-laden printing was evaluated using the RegenHU 3D Discovery bioprinter. The results indicated the successful development of ADA–gelatin–PRP based bioink for 3D bioprinting and biofabrication applications.