(I-357) Development of self-organized human tissue with perfusable multivascular network for organ engineering

Introduction:: Induced pluripotent stem cell(iPS)-derived organoids represent an attractive alternative for organ transplantation. iPS technology enables a personalised medicine approach that reduces the risk of rejection and improves integration1. However, achieving physiological functionality remains a challenge in iPS-derived models.



Therapeutic potential of iPS-derived implants depends on their biological capacity and compatibility with host systems. At a molecular and cellular level, functionality is determined by organoid maturation and stability. At a system level, size and functional exchange dictates the physiological impact of organoid implants. Both of these aspects are associated with transport. At micro scale, lack of vasculature is one of the main limitations for organoid size2. At macro scale, integration into host vascular systems is required for long term sustainability and therapeutic effect of implanted organoids3.


We present a new platform technology for engineered 3D printed devices able to sustain and integrate scalable and physiologically relevant organoids for therapeutic purposes. Design features include adaptable perfusion architectures to match variable host's input/outputs, organoid/tissue types, mechanical robustness and flexibility to withstand stress and fit into specific locations in the host body. Using high-resolution 3D printing of novel materials, we created biocompatible, permeable, flexible, and robust vascular devices. To demonstrate their effectiveness, we leveraged a genetically engineered liver-like organoid system4. We demonstrate scalable, long term functionality and the impact of vascularization on biological capacity. Current efforts are underway to demonstrate in vivo functionality through direct anastomosis to host blood vessels.

Materials and Methods:: We fabricated a 3D branching vascular network using CAD and 2PP printing with a novel material consisting of urethane acrylate resin and a polyethylene glycol-based porogen (Fig 1a). The device was washed with acetone, post-cured, and transferred to PBS. SEM was used to observe the surface structure of the printed structure (Fig 1b), and external stress was applied using forceps to assess its structural integrity (Fig 1c).



For liver organoid creation, we introduced a genetically engineered pulse of GATA6 expression. Normal organoids were generated by directly using a cell pellet, whereas gel organoids were produced by diluting the cell pellet in fibrin gel at a seeding density of 6 million cells/mL. The urea and albumin production of normal and gel organoids was measured from day 14 to day 42 with varying starting cell populations (Fig 2a, b). For gel organoids, immunostaining with hepatocyte and apoptotic markers was performed (Fig 2c, d).



We designed and 2PP 3D printed a branching network that followed Murray's law to minimize tangential stress (Fig. 3a). The vascular device was incorporated into an in vitro perfusion platform with a media reservoir and a peristaltic pump (Fig 3b). Liver-like gel organoids were introduced into the vascular device, which was placed inside a tissue culture incubator. Albumin and urea levels were measured on day 10 and day 19, respectively (Fig 3c, d).


Results, Conclusions, and Discussions:: We conducted SEM imaging to observe the effect of porogen on the surface of a structure. The results showed that porogen introduced a porous structure with a diameter of 1μm, which could enhance the permeability of the entire network (Fig 1b). We tested the robustness and flexibility of a model with a simple branching network by subjecting it to external stress using forceps. The structure remained undamaged after being exposed to bending and tensile stress, demonstrating its robustness and flexibility (Fig 1c).



We created gel organoids by diluting the cell pellet in fibrin gel and measured urea and production from day 14 to day 33 with varying cell populations (Fig. 2a, b). Gel organoids showed scalability with increasing starting cell populations, while normal organoids did not. However, gel organoid exhibited apoptotic regions in the center, revealed by Cleaved-Caspase 3 immunostaining (Fig. 2c). Although gel organoids expressed hepatocyte marker (HNF4A) (Fig. 2d), this suggests a limitation in scalability.



We embedded liver-like gel organoids in an in vitro perfusion device with a branching vascular network (Fig 3a, b). We observed a three-fold increase in albumin secretion on day 10, and in urea secretion on day 19 from perfused organoids compared to non-perfused ones (Fig. 3c, d). These results indicate that the perfusion device with a branching vascular network can enhance the functionality of liver-like organoids.


In conclusion, our platform technology has successfully produced a high-resolution 3D branching network that is permeable, flexible, and robust. Our in vitro perfusion experiment further demonstrated the efficacy of this technology. Moving forward, we anticipate our technology to be utilized in vivo through the direct anastomosis of vascularized tissue to host blood vessels. Furthermore, our technology has the potential to enable the development of plug-in type supplementary organ units comprising diverse tissue types with therapeutic effects.

Acknowledgements (Optional): : We thank Picower Institute Innovation Fund (PIIF) for funding this project.

References (Optional): : 1. Lancaster, M. A. & Knoblich, J. A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

2. Takebe, T., Zhang, B. & Radisic, M. Synergistic Engineering: Organoids Meet Organs-on-a-Chip. Cell Stem Cell 21, 297–300 (2017).

3. Zhang, B. et al. Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis. Nat. Mater. 15, 669–678 (2016).

4. Velazquez, J. J. et al. Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids. Cell Syst 12, 41–55.e11 (2021).