3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Yeonhee Kim,Padmavathy Rajagopalan,Christophe Egles

doi:10.1371/journal.pone.0015456

Yeonhee Kim, Padmavathy Rajagopalan + Show 1 more

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https://doi.org/10.1371/journal.pone.0015456

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Journal: PloS one	Publication Date: Nov 12, 2010
Citations: 123	License type: CC BY 4.0

Affiliation: Virginia Tech, Center for Systems Biology

Abstract

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.

Highlights

The liver is one of the largest organs in our bodies
In order to determine whether rat liver sinusoidal endothelial cells (LSECs) (rLSECs) would adhere to the underlying polyelectrolyte multilayer (PEM), they were incubated with a red-fluorescent non-toxic membrane permeable dye prior to seeding
Images taken over a period of twelve days demonstrated that rLSECs where only adherent when a chitosan-Hyaluronic acid (HA) PEM was present (Figure 1)

Summary

Introduction

The liver is one of the largest organs in our bodies It performs a multitude of functions such as metabolism, detoxification, and mediation of the body’s complex defense mechanisms. Two of the most commonly observed cell types in the liver are hepatocytes and liver sinusoidal endothelial cells (LSECs). Together, they account for more than 80% of the liver’s mass. LSECs exhibit characteristics that are distinct from endothelial cells that line other blood vessels. They participate in metabolic activities, exhibit fenestrae [1,2] and are often the initial target of hepatic toxicants [3]. LSECs function as a scavenger system in the liver by removing waste macromolecules and play a vital role in the balance of lipids, cholesterol and vitamins [4,5,6,7,8,9,10]

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