E-Cadherin Protects Primary Hepatocyte Spheroids from Cell Death by a Caspase-Independent Mechanism

Abstract

Cultivation of primary hepatocytes as spheroids creates an efficient three-dimensional model system for hepatic studies in vitro and as a cell source for a spheroid reservoir bioartificial liver. The mechanism of spheroid formation is poorly understood, as is an explanation for why normal, anchorage-dependent hepatocytes remain viable and do not undergo detachment-induced apoptosis, known as anoikis, when placed in suspension spheroid culture. The purpose of this study was to investigate the role of E-cadherin, a calciumdependent cell adhesion molecule, in the formation and maintenance of hepatocyte spheroids. Hepatocyte spheroids were formed by a novel rocker technique and cultured in suspension for up to 24 h. The dependence of spheroid formation on E-cadherin and calcium was established using an E-cadherin blocking antibody and a calcium chelator. We found that inhibiting E-cadherin prevented cell-cell attachment and spheroid formation, and, surprisingly, E-cadherin inhibition led to hepatocyte death through a caspase-independent mechanism. In conclusion, E-cadherin is required for hepatocyte spheroid formation and may be responsible for protecting hepatocytes from a novel form of caspase-independent cell death.Key words: Hepatocyte spheroids; E-Cadherin; Anoikis; Caspase-independent cell death INTRODUCTIONcules on adjacent cells, is therefore a likely candidate required for spheroid formation and maintenance. E-Cadherin, one of the most important and well-studWe have previously shown that spheroids of primary (nontransformed) rat hepatocytes can be formed rapidly ied cell adhesion proteins, is a known factor involved in anoikis (5). The loss of E-cadherin adhesions has been and efficiently using a rocked suspension technique (2). Under these conditions, freshly isolated rat hepatocytes implicated in the induction of anoikis in tumorigenic epithelial cells (6) and during the normal shedding of the aggregate spontaneously to form spheroids of 60-120 µm diameter by 24 h and viability exceeding 95%. As intestinal epithelial cells at the villus tip (3). Therefore, we asked whether E-cadherin was required for spheroid previously shown by others (1), differentiated hepatocyte functions were better maintained in spheroid culformation and as a necessary step to protect hepatocytes from anoikis cell death. Our findings suggest that Eture. However, cell loss became problematic over extended culturing periods. While culturing hepatocytes in cadherin was required both for spheroid formation and the protection of hepatocytes from cell death under ananchorage-independent conditions has many advantages, little is known about how this cell loss occurred over chorage-independent rocked spheroid conditions. In contrast to previous studies, our results suggest that cell time. Anchorage-independent spheroid cell cultures, such as our novel rocked culturing technique (13), rely death can occur via a caspase-independent mechanism. upon calcium-dependent cell-cell adhesions for survival and function, which occur in the absence of a...

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

“…Hepatocytes in aggregate or spheroid culture have been shown to be able to maintain viability and metabolic functions for a longer period than those in monolayer cultures, possibly due to the better retainment of in vivo hepatic morphological characteristics (33)(34)(35)(36). To further study the hiPSC-HPC aggregates, immunofluorescent stainings were performed on E-cadherin, an epithelial marker that had been shown to protect primary hepatocyte from apoptosis (37), and intracellular albumin, a functional hepatic marker. Aggregates after 7 d of culture were stained positively for both E-cadherin and albumin, demonstrating functional spheroid formation (Fig.…”

Pre-Clinical Assessment of the Potential Intrinsic Hepatotoxicity of Candidate Drugs

“…There are different ways to induce the formation of spheroids including continuously-stirred bioreactors [94], the rocked suspension technique [95] and rotating wall bioreactors [96]. Initial experimentation demonstrated that, between spheroids and monolayers, there was indeed differential toxicity induced by 7 day methotrexate exposure.…”

Isolations and Cultures of Primary Hepatocytes

“…The liver parenchyma is permeated by a collagenous fibrillar network, which is embedded in an extracellular matrix, mainly composed of a mixture of elastin, heparin sulfate proteoglycan and surface adhesion molecules, such as laminin and fibronectin [14]. The extracellular matrix, adhesion glycoproteins, Ca 2+ ions and cell surface receptors play an important role in maintaining cell anchorage, shape, polarity and function [15]. Variations of extracellular matrix content between different species result in the degree of difficulty.…”

“…These kinds of essential factors were determined and supplement in the medium for keeping liver cells vigorous. Moreover, co-culture with other cell types may be responsible for re-establishing cell junctions such as E-cadherin which is required for hepatocyte spheroid formation [28], and protecting hepatocytes from cell death, thus is emerging as a prospective way to cultivate and maintain hepatocytes. Seeding the primary hepatocytes with mesenchymal stem cells [29], liver sinusoidal endothelial cells or umbilical vein endothelial cells [30] has been reported to result in enhanced heterotypic cell-cell interactions to form spheroids in a shorter time, which led to improvements in hepatocyte function and prolongation of the survival time.…”