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Abstract Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell‐based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano‐ and microfibers (≈200–1000 nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3‐J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome‐P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34‐fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell‐ECM interactions in the human liver.
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Engineering Liver Extracellular Matrix Nanofibers to Functionally Mature iPSC-derived Liver Cells
Drug-induced liver injury (DILI) remains a leading cause of drug attrition and acute liver failures, partly due to the inadequacy of animal models to accurately predict human clinical outcomes, which necessitates the utilization of in vitro models of the human liver. However, primary human hepatocytes (PHHs) are in short supply for routine drug screening. In contrast, induced pluripotent stem cells (iPSCs)-derived hepatocyte-like cells (HLCs) are a nearly unlimited cell source but display a fetal-like (versus adult-like) phenotype when differentiated using conventional protocols on tissue culture plastic or glass adsorbed with 2D extracellular matrix (ECM) proteins. Electrospinning can produce porous nanoscale 3D fibers that have a large surface area and present a high density of receptor ligands to modulate cell phenotype. However, the application of electrospinning to generate 3D liver-derived ECM substrates for HLC differentiation remains unexplored. Therefore, here we developed methods to a) electrospin nanofibers of different porosities and diameters using porcine liver ECM (PLECM) with or without type I collagen and b) use these fibers to determine functional modulation in iPSC-derived HLCs while using PHHs as a control cell type relative to conventional adsorbed ECM substrates.
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- Award ID(s):
- 1933552
- PAR ID:
- 10382011
- Date Published:
- Journal Name:
- Annual Meeting of the Biomedical Engineering Society
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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