New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells

Integr Biol (Camb). 2015 Nov;7(11):1412-22. doi: 10.1039/c5ib00170f.

Abstract

To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Albumins / chemistry
  • Animals
  • Bile / chemistry
  • Cell Culture Techniques / methods
  • Cell Membrane / metabolism
  • Coculture Techniques
  • Diffusion
  • Dimethylpolysiloxanes / chemistry
  • Endothelial Cells / cytology*
  • Flow Cytometry
  • Hepatocytes / cytology*
  • Hepatocytes / metabolism
  • Humans
  • Immunohistochemistry
  • Liver / pathology*
  • Male
  • Membranes, Artificial
  • Oxygen / chemistry
  • Rats
  • Rats, Wistar
  • Tissue Engineering

Substances

  • Albumins
  • Dimethylpolysiloxanes
  • Membranes, Artificial
  • baysilon
  • Oxygen