(I-359) 3D Co-axial Bioprinting of Pre-vascularized Macro Hepatic Tissue Modules using Light-activated Decellularized Extracellular Matrix-based Bioinks

Introduction:: Liver tissue engineering aims to produce implantable and functional tissue constructs that meet the clinical demands for liver regeneration. The current challenge is to create functional liver implants that provide enough metabolic support for the recipient. Typically, 30% of the recipient's liver mass is required to meet their metabolic needs, but current liver implants are too small to accommodate the required number of cells necessary for regeneration [1]. In addition, the production of clinically relevant-sized tissue constructs remains a challenge due to issues with cell survival and vascularization. Tissue assembly has emerged as a promising approach to address these challenges by offering high efficiency and flexibility in producing volumetric tissue constructs that promote cell viability and vascularization. However, given that the dimensions of fabricable tissue modules are restricted in scope from micrometers to millimeters, the achievable size of tissue constructs is only a few cubic centimeters, which falls short of meeting clinical demands [2-4]. Recent breakthroughs in 3D bioprinting technology have opened up new avenues for fabricating intricate and multi-cellular tissue constructs. These advancements have expanded the range of achievable tissue module dimensions, enabling the production of larger and more complex constructs with greater accuracy and reproducibility. This study proposes a multi-material bioprinting system using a light-activated decellularized extracellular matrix (dECM)-based bioinks (dERS) and sacrificial material to produce a centimeter-scale 3D macro hepatic tissue module with a high shape fidelity. To improve the vascularization and functionality of MHM, we also generated pre-vascularized MHM using co-axial nozzles and customized dERS bioinks.

Materials and Methods:: Preparation and characterization of light-activated dECM bioinks
Light-activated liver tissue-derived decellularized extracellular matrix (ldECM)-based bioinks (ldERS) were prepared by physically mixing ldECM and ruthenium/sodium persulfate (Ru/SPS). Similarly, light-activated hybrid dECM-based bioinks (hybrid dERS) were prepared by blending ldECM, vascular tissue-derived dECM (vdECM), and Ru/SPS. All dECM biomaterials were obtained by previously reported protocols with minor modifications. Human hepatocytes and HUVECs were gently encapsulated in pH-adjusted ldERS and hybrid dERS bioink, respectively, with a cell concentration of 1 x 107 cells mL^-1 for cell printing. The efficacy of the developed ldERS was evaluated through mechanical properties testing, rheological analysis, and biocompatibility test. In addition, vascular marker-stained immunofluorescence (IF) imaging was performed to confirm the efficacy of hybrid dERS.

3D bioprinting of macro hepatic tissue modules
First, pluronic F-127 (PF-127) was printed as a sacrificial material, and dERS bioinks were printed in the space between the printed PF-127 lines. Next, PF-127 and dERS bioinks were printed in the same order on top of the previous layer with a 0°/90° stacking pattern. Lastly, the printed constructs were incubated with the cell culture medium overnight, resulting in highly porous constructs by removing the PF-127 framework. The pre-vascularized MHN was printed using co-axial nozzles with HUVECs-encapsulated hybrid dERS in the shell and hepatocytes-encapsulated ldERS in the core. The efficacy of the patterned vasculature was evaluated by gene expression levels. In addition, hepatic maturation-related gene expression levels and albumin secretion were investigated. Finally, the in vitro liver-specific functionality of centimeter-scale pre-vascularized MHM was investigated.


Results, Conclusions, and Discussions:: We formulated biocompatible dERS bioinks that can rapidly induce photo-crosslinking under visible light irradiation, ensuring the structural integrity of the printed constructs during printing. Furthermore, we established a multi-material printing system using the dERS bioinks and sacrificial material, allowing the fabrication of centimeter-scale porous living tissue constructs with high shape fidelity. Moreover, the flexibility of the developed printing strategy was demonstrated by controlling the interconnectivity of the printed construct with the adjustment of the sacrificial framework width. In addition, our bioprinting strategy demonstrated biocompatibility, evidenced by the high cell viability of the printed constructs immediately after printing.

Using the developed bioprinting strategy with co-axial nozzles, we also successfully fabricated hepatic tissue modules with patterned vasculatures. We found that patterned vascular structures in the hepatic tissue modules supported hepatocyte maturation while promoting the maturation and self-assembly of vasculatures. Furthermore, we generated centimeter-scale pre-vascularized MHM that mimicked essential physiological hepatic functions, including albumin secretion and urea synthesis. We observed that the MHM with the patterned vascular structures exhibited greater albumin secretion and maintained its function for a prolonged duration compared to the MHM without the vascular structures.

Taken together, we developed dERS bioinks and a multi-material bioprinting strategy to produce centimeter-scale porous MHM with high shape fidelity. Furthermore, it was confirmed that the patterned structure could improve the vascularization and functionality of MHM. Additional validation tests should be conducted to verify that the MHM could recapitulate the physiological functions of the liver.

In conclusion, our study presents the promising outcomes of a 3D co-axial bioprinting system using dERS bioinks and sacrificial material, enabling the creation of functional and centimeter-scale MHM with patterned vasculatures. Our findings are expected to offer a new approach to developing liver implants that could meet clinical demands beyond the currently largest bioprinted tissue constructs by combining tissue assembly systems.

Acknowledgements (Optional): : This research was supported by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (21A0104L1, Republic of Korea), supported by the Alchemist Project 1415180884 (20012378) funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea).


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[3] Zheng, Zhiqiang, et al. "3D Construction of shape-controllable Tissues through self-Bonding of multicellular microcapsules." ACS applied materials & interfaces 11.26 (2019): 22950-22961.

[4] Mazza, Giuseppe, et al. "Liver tissue engineering: From implantable tissue to whole organ engineering." Hepatology Communications 2.2 (2018): 131-141.