(J-371) Bioprinting of Human Liver Tissues with Endothelial Microvessels for Regenerative Medicine

Introduction:: Given the shortages of donor organs, engineered human liver tissues are urgently needed for cell-based therapy for patients suffering from end-stage liver failure. Bioprinting has emerged as a useful technology to engineer complex in vivo like architectures with precise cell-extracellular matrix (ECM) and cell-cell interactions; however, current bioprinted livers have several drawbacks such as a) the lack of natural 3D printable bioinks that are mechanically and biochemically tunable, b) high shear stress applied on the cells during the printing process, and c) suboptimal cell-cell interactions critical for maintaining the functions of primary human hepatocytes (PHHs). Furthermore, vascularization of multicellular 3D constructs remains a challenge for engineered liver models, limiting oxygen and nutrient supply. We have previously shown that PHHs, when co-cultured with endothelial cells, can maintain a high level of hepatic functions in 3D collagen microtissues generated using high-throughput droplet microfluidics as part of a 3D printable bioink versus the printing of single cell suspensions. Here, we sought to test the novel hypothesis that combining engineered 3D printed human liver tissues with endothelial microvessels formed de novo in an ECM hydrogel supports hepatic functionality and integration with a neovasculature.


Materials and Methods:: PHHs were resuspended in a collagen-I solution at 4^oC and single emulsion droplet microfluidic devices were used to generate ~300 µm diameter collagen-in-oil emulsions (microtissues; Fig. 1A). Following thermal cross-linking (37^oC), microtissues were resuspended in culture media. Liver-derived (liver sinusoidal endothelial cells [LSECs]) were seeded atop the PHH-collagen microtissues for ‘coated’ conditions, then seeded directly into printed gelatin scaffolds (10% gelatin w/v cross-linked with EDC/NHS). After 1 week of culture, human umbilical vein endothelial cells (HUVECs) and normal human lung fibroblasts (NHLFs) were suspended in a solution containing fibrinogen, thrombin, and culture media, seeded atop microtissue-containing gelatin scaffolds, and thermally cross-linked (37^oC). Cultures were placed on a seesaw rocker to apply shear stress to endothelial cells. Hepatic functions (albumin secretion and cytochrome-P450 [CYP] activity) were assessed over time.


Results, Conclusions, and Discussions:: LSEC-coated microtissues of PHH density up to 4.5x10⁶ cells/mL were successfully produced, seeded within bioprinted gelatin scaffolds containing square microwells, overlaid with fibrin gels containing HUVECs and NHLFs, and subsequently cultured for 3+ weeks (Fig. 1B-C). Self-assembled microvessels formed luminal structures (not shown), interacting with LSEC-coated microtissues, and creating a network within the bioprinted scaffolds (Fig. 1D). Applied shear stress (via rocker) on LSEC-coated microtissues in bioprinted scaffolds with and without the microvascular gel promoted ~1.8 and ~3.4-fold, respectively, higher CYP3A4 activity than static scaffolds (Fig. 1E). Additionally, albumin production was stable for 3+ weeks in the bioprinted scaffolds containing microvasculature gel while exposed to shear stress, whereas neither static scaffolds, with or without microvasculature gel, induced albumin production to the same extent (not shown).

We bioprinted for the first-time human liver microtissues generated via droplet microfluidics using a gelatin bioink containing a hydrogel capable of de novo formation of endothelial microvessels. The ability to precisely tune the microtissue dimension, cellular composition, and ECM, while forming a connected network amongst the tissues allowed for higher levels of hepatic function for 3+ weeks. Future efforts are focused on incorporating other liver non-parenchymal cell types and implanting into rodent models with injured livers.




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