Optically-tuned bioresins for the ultra-fast volumetric bioprinting of hepatic organoid-laden biofactories


Nunez Bernal, Paulina (Dept. of Orthopaedics, University Medical Center Utrecht )


Introduction: Developing more predictive in vitro platforms for biomedical research remains a major challenge in tissue engineering. 3D bioprinting allows patterning of cell-laden biomaterials into hierarchical structures. Volumetric bioprinting (VBP) is a novel light-based approach that tackles challenges posed by conventional approaches, through the layer-less biofabrication of viable and highly complex cell-laden structures at unprecedented speeds[1]. Given the requirement of high cell densities to create functional tissue mimics, strategies to overcome the light-scattering effect of intracellular organelles are needed to resolve high-resolution prints. Herein, an optically-engineered bioresin was developed to pattern morphologically-undisrupted organoids into complex centimeter-scale assemblies. Patient-derived human hepatic organoid-laden constructs were printed to create advanced in vitro models that capture salient features of the liver involved in systemic homeostasis and detoxification.
Methods: 405nm light back-filtered projections of a 3D object are directed onto a volume of cell-laden bio-resin (gelatin methacryloyl with visible-light photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate), to selectively crosslink the hydrogel in a spatially-controlled fashion. The resolution of VBP in the presence of hepatic cell line (HepG2) or patient-derived hepatic organoids was enhanced through the addition of refractive index-matching compound iodixanol. These optically-tuned resins were used to print high hepatic organoid densities (up to 107cells/mL). Viability and metabolic activity of bioprinted organoids was evaluated, as well as hepatic differentiation capacity of VBP-prints (hepatic markers, albumin secretion, and cytochrome activity) compared to extrusion bioprinted (EB) constructs. Finally, cell-laden, mathematically-derived architectures with different structural properties were printed at high resolution and cultured under dynamic perfusion to evaluate organoid metabolism of ammonia.
Results: VBP-printed constructs were fabricated in tens of seconds, achieving previously unattained resolutions (41.5±2.9μm positive and 104.0±5.5μm negative features). The concentration of iodixanol was optimized to match the refractive index of intracellular components of both HepG2s and organoids, resulting in a significant resolution enhancement of cell-laden constructs (50.5±6.0μm). Hepatic organoids ranging from 100μm to 1mm in diameter were successfully printed via VBP with high accuracy. Compared to EB-printed structures, where shear forces resulted in organoid fragmentation and lower viability (73.2±1.2%), VBP-printed organoids exhibited high viability (93.3±1.4%), maintained their morphology and displayed apicobasal polarity post-printing. Complex gyroid-like structures with different pore architectures printed within 16-20s were integrated in a fluidic system and exhibited differences in permeability and surface-area-to-volume ratio. This resulted in enhanced rates of ammonia metabolism (33.5±5.8-24.3±1.4nmol mgtotal protein-1) compared to static controls (12.7±0.3nmol mgtotal protein-1), as well as shape-dependent changes in metabolism.
Conclusion: This study demonstrated the contactless bioprinting of complex and labile biological structures (hepatic organoids) via VBP. Through a refractive index-matching approach, an optically-tuned gelMA resin enabled high-resolution printing of cell-laden structures. Organoids exhibited high viability and hepatic differentiation capacity post-printing. Furthermore, the dynamic culture of convoluted VBP-printed structures was demonstrated through architectures that could modulate organoid function in a shape-dependent fashion. The combination of organoid technology with the ultra-fast printing times and freedom of design offered by VBP shows promise for the development of new predictive platforms for in vitro disease modeling and drug screening.
[1] Bernal, P.N. et al., Adv. Mater. 1, 1904209 (2019).


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