Decellularized Liver Nanofibers Enhance and Stabilize the Long‐Term Functions of Primary Human Hepatocytes In Vitro

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.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening

“…Using these ratios and electrospinning conditions, we were not able to fabricate our hybrid scaffolds, possibly because we used rat liver ECM and also did not solubilize the liver ECM with acetic acid before mixing with the polymer solution. Another recent study by Liu et al fabricated porcine liver ECM-based electrospun scaffolds with rat tail collagen and chitosan (5:2:2) at a 1 mL/h flow rate for the culture of primary human hepatocytes . The fabricated scaffolds in this study were cross-linked with EDC/NHS (ethyl-3-(3-dimethyl aminopropyl)­carbodiimide and N -hydroxysuccinimide).…”

“…Another recent study by Liu et al fabricated porcine liver ECM-based electrospun scaffolds with rat tail collagen and chitosan (5:2:2) at a 1 mL/h flow rate for the culture of primary human hepatocytes. 43 The fabricated scaffolds in this study were cross-linked with EDC/NHS (ethyl-3-(3-dimethyl aminopropyl)carbodiimide and N-hydroxysuccinimide). The hybrid scaffolds fabricated in our study, however, did not require any external cross-linking agent since we did not fragment the matrix proteins before mixing them with the polymer solution before electrospinning.…”

Decellularization Techniques for Tissue Engineering: Towards Replicating Native Extracellular Matrix Architecture in Liver Regeneration

“…These scaffolds were then used to culture Primary Human Hepatocytes (PHH) alone or in combination with non-parenchymal cells such as 3T3-J2 murine embryonic fibroblasts and liver sinusoidal endothelial cells. Cells cultured on the fibrous scaffolds displayed superior urea synthesis, albumin secretion, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities compared with those cultured on conventionally adsorbed 2D ECM controls [154].…”

“…These scaffolds were then used to culture Primary Human Hepatocytes (PHH) alone or in combination with non-parenchymal cells such as 3T3-J2 murine embryonic fibroblasts and liver sinusoidal endothelial cells. Cells cultured on the fibrous scaffolds displayed superior urea synthesis, albumin secretion, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities compared with those cultured on conventionally adsorbed 2D ECM controls [154]. Another study incorporated a decellularized human liver ECM directly into the fibers of an electrospun polylactic acid (PLA) matrix to create a bioactive protein-polymer scaffold to enhance the proliferation of THLE-3 hepatocytes [156].…”

Comparison of Wild-Type and High-risk PNPLA3 variants in a Human Biomimetic Liver Microphysiology System for Metabolic Dysfunction-associated Steatotic Liver Disease Precision Therapy

“…MPS are 3D microfluidic platforms composed of multiple cell types that mimic overall organ structure and provide cell-tocell communication using human primary cells, immortalized cell lines, and iPSCs (39)(40)(41). Recently, multiple human liver MPS have evolved and been implemented to study mechanisms of MASLD pathogenesis and serve as drug testing platforms (40,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60). We have implemented the liver acinus microphysiology system (LAMPS), a structured biomimetic, that is a 3D layered model constructed through a combination of sequential cell layering and cell-to-cell selforganization between 4 key liver cell types including primary hepatocytes and liver sinusoidal endothelial cells (LSECs) as well as cell lines for hepatic stellate cells (the LX-2 cell line) and Kupffer-like cells (THP-1 cell line), and maintained under flow to mimic either Zone 1 or Zone 3 oxygen tensions (61)(62)(63) Our overall strategy towards implementing a precision medicine platform for MASLD has been to first test the MASLD LAMPS model with key primary cells as a benchmark for the next phase using "patient biomimetic twins" (PBTs) produced using patient, induced pluripotent stem cells (iPSCs) (40) (Fig 1A).…”