Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheetsin vitro

Abstract

Cell sheet stratification technology has been used for reconstituting highly functional three-dimensional (3D) hepatic tissues in vitro. Triple-layered hepatic tissues with a hepatocyte-specific polarity were fabricated by sandwiching a hepatocyte sheet (Hep sheet) between two endothelial cell (EC) sheets. The morphological and functional characteristics of the triple-layered hepatic construct (EC-Hep-EC) were evaluated and compared with those of a double-layered hepatic construct with a single EC sheet (Hep-EC) and a Hep sheet only. Transmission electron microscope (TEM) observations revealed that the extracellular matrix was observed to be deposited in the space between the ECs and hepatocytes on both the upper and lower sides of the hepatocytes in the EC-Hep-EC construct. Immunohistochemistry with basolateral (CD147) and apical [multidrug resistance-associated protein (MRP2)] membrane polarity markers clearly showed the recovery of in vivo-like hepatocyte polarization in the EC-Hep-EC group. In addition, hepatocyte-specific functions, including albumin secretion, ammonia removal and the induction of cytochrome P450, were also highly preserved. The presented technology for stratifying multiple cell sheets was simple in operation and successfully reproduced both the heterotypic/homotypic cell-cell and cell-matrix interactions with the inherent hepatocyte configurations, thus closely mimicking the in vivo environment. The triple-layered 3D hepatic constructs could therefore be valuable as a new experiment tool for drug-screening tests, an implantable tissue model for cell-based therapies and an efficient culture platform for bioartificial liver devices. Copyright © 2015 John Wiley & Sons, Ltd.

Spontaneous hepatocyte migration towards an endothelial cell tube network

“…Primary hepatocyte isolation was performed using a modified collagenase perfusion method from donor rats in accordance with previous reports (Kim et al, ). The isolated cells were filtered through a cotton mesh membrane and 45‐μm stainless mesh and then purified 3 times via centrifugation at 50 g for 2 min each.…”

“…However, due to the shortage of donors, alternative treatments are needed. Hepatic tissue engineering has been established as a promising alternative to liver transplantation (Kim, Utoh, Ohashi, Kikuchi, & Okano, ; Lu et al, ). However, there remain unresolved issues before this type of treatment can be widely introduced in clinical practice (Zhang et al, ).…”

Architecture in 3D cell culture: An essential feature for in vitro toxicology

“…An essential step to illustrate the role of protein location on cell phenotype was to demonstrate that forcing the relocation of the protein, instead of changing its expression level, was sufficient to alter the differentiation stage of cells (Lelièvre et al, 1998). Structural proteins such as CTCF, NuMA and lamins organize chromatin (Abad et al, 2007; Dechat et al, 2008; Kim et al, 2015), and by doing so they might redistribute when the phenotype of cells changes. Proteins like NuMA and splicing factor speckle SC35 have been used with high-content analysis of nuclear morphometric descriptors to identify changes in differentiation stages, notably of stem cells, before standard markers are modified (Liu et al, 2010; Vega et al 2015; Vega et al, 2017.…”

“…Normal tissues are used for assessing the impact of exogenous factors (e.g., drugs, cosmetic materials, pollutants, oxidative environments) on cellular functions and the risk of carcinogenesis (Kim et al, 2015). Whereas tumors are used to assess mechanisms of toxicity that could help improve anticancer treatments (Katt et al, 2016).…”

Shunts, channels and lipoprotein endosomal traffic: a new model of cholesterol homeostasis in the hepatocyte

“…The hepatocyte is polarized having a basolateral surface in contact with the blood supply and an apical surface connected to the bile canalicula. It is very difficult to reproduce polarized hepatocytes in culture, but this has been achieved in a few studies [ 42- 45 ] . …”