
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.21.608997
preprint
1
Article
Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture
Lekkala Vinod Kumar Reddy
Shrestha Sunil
Al Qaryoute Ayah
Dhinoja Sanchi
Acharya Prabha
Raheem Abida
Jagadeeswaran Pudur
Lee Moo-Yeal http://orcid.org/0000-0001-6922-609X

22 8 2024
2024.08.21.608997https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.21.608997
nihpp-2024.08.21.608997.pdf
Abstract

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM + endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.
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