# Three-Dimensional Culture of Primary Hepatocytes in a Single-Cell Layer on Poly(vinyl alcohol) Nanofibrous Membrane

## TL;DR

This study develops a 3D culture system for liver cells using nanofibrous membranes, supporting long-term cell function and potential use in drug research.

## Contribution

A novel 3D single-cell layer culture system for primary hepatocytes using RGD-PVA nanofibrous membranes is introduced.

## Key findings

- Hepatocytes cultured on RGD-PVA NM formed a single-cell layer and maintained prolonged survival.
- CYP3A4 activity was higher in cocultured hepatocytes on RGD-PVA NM compared to monocultured cells.
- The system supports long-term functionality for drug discovery and toxicity studies.

## Abstract

Primary hepatocyte cultures serve as an ex vivo model of liver physiology. This study aims to employ poly(vinyl alcohol) (PVA) nanofiber membranes (NMs) to establish a three-dimensional (3D) culture system that supports the long-term functionality of primary hepatocytes. Primary hepatocytes were monocultured on a PVA NM or indirectly cocultured with NIH3T3 fibroblasts on a distinct polycaprolactone (PCL) NM layer. Monocultured and cocultured hepatocytes maintained prolonged survival without supplemental growth factors. Cocultured hepatocytes formed larger aggregates composed of cell clusters attached to untreated nanofibers than monocultured cells. However, most primary hepatocytes cultured on NaOH-treated PVA NM and Arg–Gly–Asp (RGD) peptide-blended PVA (RGD-PVA) NM, under monoculture and coculture conditions, formed non-aggregated cells in a single-cell layer. In a bioinert assay, unstimulated dendritic cells were activated on untreated but not NaOH-treated PVA NM. CYP3A4 activity was higher in cocultured cells on RGD-PVA NM with fibroblasts than in monocultured cells on PVA and RGD-PVA NM. Functional hepatocyte cultures were successfully maintained in a 3D single-cell layer on RGD-PVA NM, along with fibroblasts in a layer-by-layer coculture, for a prolonged period. The prolonged culture of hepatocytes in a 3D single-cell layer may facilitate further drug discovery, toxicity studies, and translational liver research.