Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models

Abstract

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.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro

“…Those spheroids, often called microtissues, displayed pronounced glycogen storage and albumin synthesis capabilities as well as expression of CYP1A2, CYP2C9 and CYP3A4. Maintenance of cell polarity could be shown by MRP2 and BSEP expression [ 64 , 65 , 66 ]. Viability of the spheroids could be maintained for up to 7 weeks in one of the studies, but albumin secretion declined from day 28 on.…”

“…Viability of the spheroids could be maintained for up to 7 weeks in one of the studies, but albumin secretion declined from day 28 on. Interestingly, CYP1A1/2 , CYP2C9 and CYP3A4/5 transcript levels were higher in those microtissues than in clinical liver specimens or pHHs upon long-term culture [ 65 , 66 ]. However, on protein level, only CYP2B6 and UGT1A1 levels remained stable for 7 weeks of culture.…”

Latest impact of engineered human liver platforms on drug development

“…(f) A high-throughput droplet microfluidic device for the generation of 3D liver microtissues. 85 Left to right: Hepatocytes are suspended in collagen solution and perfused through the microfluidic device at 4 °C (to keep collagen from polymerizing) with an oil stream; since oil and water do not mix, the collagen + cells form spherical droplets. These so-called microtissues are collected, heated at 37 °C to promote collagen polymerization and cell encapsulation, oil is drained, and microtissues are resuspended in culture medium within microwells in static or fluidic plates/devices.…”

“…For instance, a high-throughput droplet microfluidic technology was used to encapsulate PHHs within ECM microgels, which were subsequently “coated” with 3T3-J2 fibroblasts or HSCs [ Fig. 3(f) ]; 85 the PHH/fibroblast “microtissues” displayed high levels of liver functions for 6+ weeks in vitro and functionally outperformed self-assembled spheroids and macrogels of the same cellular composition. These microtissues displayed clinically relevant CYP induction with prototypical compounds and could distinguish troglitazone toxicity from that of its non-hepatotoxic structural analog drug, rosiglitazone.…”

Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine

“…Therefore, in a recent study, high-throughput droplet microfluidics was utilized to generate uniform microtissues with PHHs encapsulated in collagen I with supportive fibroblasts (3T3-J2 or HSC). 71 These microtissues displayed high hepatic functions for 6þ weeks and were found to be useful for assessing drugmediated cytochrome-P450 (CYP) induction and hepatotoxicity. Such microtissues are now being adapted to include other liver cell types and to study the pathogenesis of diseases such as NAFLD and fibrosis.…”