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...