Designing a Multicellular Organotypic 3D Liver Model with a Detachable, Nanoscale Polymeric Space of Disse

Abstract

The design of in vitro models that mimic the stratified multicellular hepatic microenvironment continues to be challenging. Although several in vitro hepatic cultures have been shown to exhibit liver functions, their physiological relevance is limited due to significant deviation from in vivo cellular composition. We report the assembly of a novel three-dimensional (3D) organotypic liver model incorporating three different cell types (hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells) and a polymeric interface that mimics the Space of Disse. The nanoscale interface is detachable, optically transparent, derived from self-assembled polyelectrolyte multilayers, and exhibits a Young's modulus similar to in vivo values for liver tissue. Only the 3D liver models simultaneously maintain hepatic phenotype and elicit proliferation, while achieving cellular ratios found in vivo. The nanoscale detachable polymeric interfaces can be modulated to mimic basement membranes that exhibit a wide range of physical properties. This facile approach offers a versatile new avenue in the assembly of engineered tissues. These results demonstrate the ability of the tri-cellular 3D cultures to serve as an organotypic hepatic model that elicits proliferation and maintenance of phenotype and in vivo-like cellular ratios.

A microfluidically perfused three dimensional human liver model

“…Larkin et al reported a more complex model of rat LSEC and Kupffer cells separated by a chitosan-hyaluronic acid polymer layer from collagen embedded primary rat hepatocytes. Under static culture conditions the hepatocytes maintained their phenotype without signs of dedifferentiation [62]. A biodegradable chitosan membrane was also used by Salerno et al for co-culture of human primary hepatocytes with HUVEC, where beneficial effects of the co-culture on hepatocyte albumin production, urea synthesis and drug biotransformation again was reported [63].…”

A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture

“…In this technique, alternating layers of cationic and anionic polymers are deposited via electrostatic attraction on charged surfaces or on top of cells. LBL has been used with natural and synthetic 31 polyelectrolytes to cover hepatocytes in plate culture 32–35 , and as a seeding layer for hepatocyte co-cultures in microfluidic devices 36,37 . Here, we report the deposition of an ultrathin, pure collagen nanolayer on hepatocytes in microfluidic devices using the LBL technique with methylated and succinylated collagens.…”

The hepatocyte proteome in organotypic rat liver models and the influence of the local microenvironment

“…Media was exchanged daily in all models, with constructs cultured for 72 hours. While previous studies have shown that longer culture periods promote increases in hepatic functions, such as albumin and urea synthesis as well as CYP activity [30], we chose to evaluate an early time point in order to establish baseline proteomic measurements in the liver models. Hepatocytes and PEMs were subsequently separated and subjected to processing to peptides that were amenable to bottom-up proteomic analyses.

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