Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.

Abstract

Liver cells isolated from newborn rats and seeded on a non-adherent plastic substratum were found to spontaneously re-aggregate and to form, within a few days, spheroidal aggregates that eventually reached a plateaued diameter of 150-175 ~,m. Analyses on frozen sections from these spheroids by immunofluorescence microscopy using antibodies to various cytoskeletal elements and extracellular matrix components revealed a sorting out and a histotypic reorganization of three major cell types. A first type consisted of cells that segragated out on the aggregate surface forming a monolayer cell lining; a second type was identified as hepatocytes that regrouped in small islands often defining a central lumen; and a third group of cells reorganized into bileduct-like structures. This intercellular organization in the aggregates was paralleled by the accumulation of extracellular matrix components (laminin, fibronectin, and collagen) and their deposition following a specific pattern around each cell population structure. Determinations of albumin secretion and tyrosine aminotransferase induction by dexamethasone and glucagon at various times after the initiation of the cultures revealed a maintenance of the hepatocyte-differentiated functions for at least up to 2 mo at the levels measured at 3-5 d. It is concluded that cells dispersed as single cells from newborn rat liver conserve in part the necessary information to reconstruct a proper threedimensional cyto-architecture and that the microenvironment so generated most likely represents a basic requirement for the optimal functioning of these differentiated cells.

Recovery of Mature Hepatocytic Phenotype following Bile Ductular Transdifferentiation of Rat Hepatocytes in Vitro

“…Cultures of hepatocytes in spheroidal aggregates have been shown to be beneficial in the maintenance of differentiated phenotypes of hepatocytes. 17,18 Thus, spheroid formation appeared to be important for the recovery of hepatocytic phenotypes, probably via increasing cell-cell contacts in self-organized three-dimensional structures. Furthermore, the interaction of cells with the components of the Matrigel might play important roles in the recovery partly through the modulation of expression of transcription factors, including HNF-4α, 19 and the activation of integrin-linked kinase that is involved in transmitting integrin signaling to the interior of the cell.…”

“…Furthermore, the interaction of cells with the components of the Matrigel might play important roles in the recovery partly through the modulation of expression of transcription factors, including HNF-4α, 19 and the activation of integrin-linked kinase that is involved in transmitting integrin signaling to the interior of the cell. 20,21 The recovery of albumin expression in transdifferentiated hepatocytes was enhanced by Dex, which has been shown to increase albumin and TAT expression in spheroidal aggregate cultures of newborn rat hepatocytes 18 and decrease CK 19 mRNA expression in monolayer-cultured rat hepatocytes. 7 Although the mechanism of the effect of Dex on hepatocytic phenotypes has not been elucidated, this study suggests that the inhibition of the MEK and JNK signaling pathways may be important.…”

In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates

“…In addition, we wanted to culture hepatocytes as small aggregates, known to maintain many of the cytoarchitectural characteristics found in vivo and to exhibit higher and more prolonged functional activity than hepatocytes cultured in monolayers (25)(26)(27). Special attention was paid to optimal oxygenation of the cells, since oxygen plays an important role in hepatocyte attachment (28) and function (29,30).…”

Spheroid formation and functional restoration of human fetal hepatocytes on poly–amino acid-coated dishes after serial proliferation

“…Spheroid culture is effective in the upregulation of liver-specific functions of primary adult hepatocytes and human hepatoma cell lines (Matsushita et al 1991;Yamashita et al 2001), and in the reorganization of new-born rat hepatocytes (Landry et al 1985). This study investigated the proliferation of normal human fetal hepatocytes in vitro and the restoration of their functions by forming spheroids cultured on a suitable surface of a culture dish modified with poly-L-amino acids.…”