Internally crosslinked alginate-based bioinks for the fabrication of in vitro hepatic tissue models

Abstract

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry, in vitro, of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of different a priori and a posteriori analyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 days, underlining the suitability of Hep3Gel to support mid/long-term cultures.

Microscale tissue engineering of liver lobule models: advancements and applications

“…The entire structure is further surrounded by an outer ring of endothelial cells ( Kang et al, 2020 ) ( Figure 3A ). Other types of structures, when compared to the model described above, exhibit slight variations, including Concentric fan-shaped structures composed of hepatocytes spaced by endothelial cells ( Ma et al, 2016 ; Hong et al, 2021 ) ( Figures 3B, E ); Hepatocytes and supporting cells arranged parallel along the fan-shaped skeleton ( Khati et al, 2022 ) ( Figures 3C, D ); Supporting cells surrounding hepatocytes in a circular pattern ( Wu et al, 2020 ) ( Figure 3F ); Hepatocytes arranged in a circular pattern with a cross in the center ( Guagliano et al, 2023 ) ( Figure 3G ); and Skeletonized triangles composed of hepatocytes spaced by skeletonized triangular endothelial cells ( Janani et al, 2022 ) ( Figure 3H ). By summarizing the impact of the aforementioned structural designs on the functionality of liver lobule models, we regretfully observe that these designs predominantly emphasize enhancing the viability and functionality of hepatocytes (e.g., albumin secretion, urea synthesis) rather than focusing on achieving metabolic zonation and vascular flow in liver lobule models ( Table 1 ).…”

Bioinspired and Photo-Clickable Thiol-Ene Bioinks for the Extrusion Bioprinting of Mechanically Tunable 3D Skin Models

“…In the context of extrusion bioprinting, ionic gelation can be used to assist the deposition of mechanically robust filaments via internal or external crosslinking. While internal gelation requires the use of an insoluble calcium salt (e.g., CaCO 3 or CaSO 4 ) and a pH decrease (e.g., D-glucono-δ-lactone) to promote the controlled exposure of cations throughout the polymer network, external gelation involves the diffusion of the multivalent cations from the outer region of the polymer, being characterized by a faster sol-gel transition [34,35]. External gelation has been applied to tune bioink rheology through pre-crosslinking, yielding a printable formulation, as well as for the bath bioprinting of ionically crosslinkable bioinks, promoting instantaneous filament crosslinking and stabilization [13,36].…”