Peptide-based scaffolds for the culture and maintenance of primary human hepatocytes

Douglas Macpherson,Yaron Bram,Jiwoon Park,Robert E Schwartz

doi:10.1038/s41598-021-86016-5

Douglas Macpherson, Yaron Bram + Show 2 more

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https://doi.org/10.1038/s41598-021-86016-5

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Journal: Scientific Reports	Publication Date: Mar 24, 2021
Citations: 26	License type: open-access

Affiliation: Cornell University

Abstract

We report here the use of a nanofibrous hydrogel as a 3D scaffold for the culture and maintenance of functional primary human hepatocytes. The system is based on the cooperative assembly of a fiber-forming peptide component, fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF), and the integrin-binding functional peptide ligand, Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) into a nanofibrous gel at physiological pH. This Fmoc-FF/RGD hydrogel was formulated to provide a biomimetic microenvironment with some critical features such as mechanical properties and nanofiber morphology, which were optimized to support hepatocyte culture. The material was shown to support maintenance and function of encapsulated primary human hepatocytes as indicated by actin staining, qRT-PCR, and functional cytochrome P450 assays. The designed gel was shown to outperform Matrigel in cytochrome P450 functional assays. The hydrogel may prove useful for liver development and disease models, as well as providing insights into the design of future implantable scaffolds for the regeneration of liver tissue in patients with liver disease.

Highlights

We report here the use of a nanofibrous hydrogel as a 3D scaffold for the culture and maintenance of functional primary human hepatocytes
Primary human hepatocytes were utilized to evaluate the 3D culture method in the hydrogel system originally developed by Zhou et al for the culture of human dermal fibroblasts[27]
Consistent with the actin-phalloidin staining, cell viability is low for hepatocyte aggregates cultured in Fmoc-FF hydrogels suggesting that this gel composition cannot support survival of the human hepatocytes (Fig. 2E,F)

Summary

Introduction

We report here the use of a nanofibrous hydrogel as a 3D scaffold for the culture and maintenance of functional primary human hepatocytes. The system is based on the cooperative assembly of a fiber-forming peptide component, fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF), and the integrin-binding functional peptide ligand, Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) into a nanofibrous gel at physiological pH This Fmoc-FF/RGD hydrogel was formulated to provide a biomimetic microenvironment with some critical features such as mechanical properties and nanofiber morphology, which were optimized to support hepatocyte culture. We endeavored to use a three-dimensional cell culture environment that more closely mimics the in vivo hepatic microenvironment over traditional two-dimensional tissue-culture plastic models or current 3-D models[5,6,7] Previous work in this area has focused on the use of synthetic polymers[8] or animal-derived materials such as collagen[9,10] or M atrigel[9]. With regard to the culture of hepatocytes, peptide-based hydrogels based on the sequence RADA-1610,22,23 have been previously utilized

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