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

Gene Expr. 2020 Jun 12;20(1):1-18. doi: 10.3727/105221620X15868728381608. Epub 2020 Apr 14.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 3T3 Cells / cytology*
  • Animals
  • Cell Encapsulation*
  • Cells, Cultured
  • Coculture Techniques / methods*
  • Collagen Type I / chemistry*
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Enzyme Induction / drug effects
  • Gels
  • Gene Expression
  • Hepatic Stellate Cells / cytology*
  • Hepatocytes / cytology*
  • Humans
  • Lab-On-A-Chip Devices
  • Mice
  • Omeprazole / pharmacology
  • Polymerization
  • Rifampin / pharmacology
  • Spheroids, Cellular
  • Tissue Engineering / instrumentation
  • Tissue Engineering / methods*

Substances

  • Collagen Type I
  • Gels
  • Cytochrome P-450 Enzyme System
  • Omeprazole
  • Rifampin