Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel

Abstract

Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into threedimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.

In situ tissue engineering with synthetic self-assembling peptide nanofiber scaffolds, PuraMatrix, for mucosal regeneration in the rat middle-ear

“…8 Moreover, these peptides have the motif arginine-alanineaspartic acid (RAD), which is similar to the ubiquitous integrin receptor-binding site arginine-glycine-aspartic acid (RGD), which has been studied in the context of cell attachment. 8,11,24 At present, several clinical trials of cell therapies using PuraMatrix have been reported for cartilage, 25,26 myocardial, 27 neural, [28][29][30][31] and hepatic 32,33 regeneration, indicating the potential usefulness of PuraMatrix.…”

Two-component protein-engineered physical hydrogels for cell encapsulation

“…In these systems, cells are mixed with precursors in the solution phase then exposed to a change in pH, temperature, or ionic concentration to induce a gel phase transition. Common triggers include temperature sweeps from 4 to 37°C for collagen (12) and Matrigel (13), pH shifts from Ϸ2.5 to 7.4 for PuraMatrix (13) and leucine-zipper systems (5), and cation concentration increases from 20 to 200 mM for alginate (12,14) and peptide amphiphiles (8).…”

New Models for the In Vitro Study of Liver Toxicity: 3D Culture Systems and the Role of Bioreactors

“…Examples of the sophisticated models that, for various purposes, have been obtained from primary hepatocytes, are those generated by using bio-degradable nano-structured substrates (Kim et al, 1998), heat-sensitive polymers (Ohashi et al, 2007), various 3D matrices (Fiegel et al, 2008;ZQ. Feng et al, 2010;Ghaedi et al, 2012), or synthetic self-assembling hydrogels (Wang et al, 2008).…”