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In vitro human liver models are indispensable for compound metabolism/toxicity screening, disease modeling, and regenerative medicine. While induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) mitigate the sourcing limitations with primary human hepatocytes (PHHs), their functional maturity is rate-limiting for application use. During development, immature hepatoblasts interact with different nonparenchymal cell (NPC) types, such as mesenchyme and endothelia, in a spatiotemporal manner to progress through functional maturation. Modeling such interactions in vitro is critical to elucidate the key regulators of iHep maturation. Here, we utilized high-throughput droplet microfluidics to encapsulate iHeps within monodisperse collagen I microgels (Ø ~ 250 μm), which were coated with NPCs to generate ‘microtissues’ placed within microwells in multiwell plates. Embryonic fibroblasts and liver sinusoidal endothelial cells (LSECs) induced the highest level of iHep maturation over 4+ weeks of culture compared to adult hepatic stellate cells (myofibroblastic), liver portal fibroblasts, dermal fibroblasts, and human umbilical vein endothelial cells. Combining iHep microtissues in plates with Transwell inserts containing different NPC types enabled the modeling of dynamic heterotypic signaling on iHep maturation; introducing embryonic fibroblast signaling first, followed by LSECs, led to the highest iHep maturation. Unique cytokine secretion profiles were detected across the top-performing microtissue configurations; stromal-derived factor-1 alpha was validated as one factor that enhanced iHep maturation. Lastly, gene expression patterns and regulatory networks showed adult PHH-like maturation in LSEC/iHep microtissues compared to iHep-only microtissues. Overall, microtissues are useful for elucidating the microenvironmental determinants of iHep maturation and for future use in downstream applications. 

Human liver models that are three-dimensional (3D) in architecture are indispensable for compound metabolism/toxicity screening, to model liver diseases for drug discovery, and for cell-based therapies; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months. Therefore, here we determined how microscale 3D collagen I presentation and fibroblast interaction affect the longevity of PHHs. High-throughput droplet microfluidics was utilized to generate reproducibly sized (∼300-μm diameter) microtissues containing PHHs encapsulated in collagen I ± supportive fibroblasts, namely, 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as controls. Hepatic functions and gene expression were subsequently measured for up to 6 weeks. We found that microtissues placed within multiwell plates rescued PHH functions at 2- to 30-fold higher levels than spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Importantly, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically relevant manner to drug-mediated cytochrome P450 induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display high hepatic functions for 6+ weeks and are useful for assessing drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find utility for modeling liver diseases and as building blocks for cell-based therapies.