Ultrastructural and functional differentiation of hepatocytes under long‐term culture conditions

Knop, Erich; Bader, Augustinus; Böker, K. H. W.; Pichlmayr, R.; Sewing, Karl–Friedrich

doi:10.1002/ar.1092420307

Cited by 57 publications

(22 citation statements)

References 37 publications

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“…It has been suggested that the lack of dorsal interaction induces changes in cell morphology and cytoskeletal organization through a calcium signaling pathway [14]. In addition, it has been shown that cell phenotype, functionality and physiology can be drastically altered by exciting dorsal and ventral receptors [15][16][17][18]. So, although it can be argued that double-layer cultures might not provide a truly 3D environment, since cells are not isotropically stimulated, the use of this approach permits to engineer a large variety of experimental designs, with different material substrates and coating proteins, in a robust way (Fig.…”

Section: Introductionmentioning

confidence: 99%

Dorsal and Ventral Stimuli in Cell–Material Interactions: Effect on Cell Morphology

et al. 2012

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Cells behave differently between bidimensional (2D) and tridimensional (3D) environments. While most of the in vitro cultures are 2D, most of the in vivo extracellular matrices are 3D, which encourages the development of more relevant culture conditions, seeking to provide more physiological models for biomedicine (e.g., cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. In this study, cells were cultured between two protein coated surfaces (sandwich-like culture). Cells used both dorsal and ventral receptors to adhere and spread, undergoing morphological changes with respect to the 2D control. Combinations of fibronectin and bovine serum albumin on the dorsal and ventral sides led to different cell morphologies, which were quantified from bright field images by calculating the spreading area and circularity. Although the mechanism underlying these differences remains to be clarified, excitation of dorsal receptors by anchorage to extracellular proteins plays a key role on cell behavior. This approach-sandwich-like culture-becomes therefore a versatile method to study cell adhesion in well-defined conditions in a quasi 3D environment.

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Section: Introductionmentioning

confidence: 99%

Dorsal and Ventral Stimuli in Cell–Material Interactions: Effect on Cell Morphology

et al. 2012

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“…7 However, the mechanisms underlying remodeling of the cytoplasm of cultured hepatocytes have not been well characterized. In liver during fasting and cultured hepatocyes after nutrient withdrawal or hormone induction, mitochondria and other organelles such as peroxisomes are eliminated through a dynamic mechanism called autophagy, or macroautophagy.…”

Section: Introductionmentioning

confidence: 99%

Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes

Rodrı́guez-Enrı́quez

Kai²,

Maldonado³

et al. 2009

Autophagy

106

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In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serum-containing Waymouth’s medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1α mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.

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“…In contrast, when hepatocytes were cultured on an extracellular matrix derived from the EngelbrethHolm-Swarm mouse sarcoma (EHS gel) [3,4,9,10], on a liver-derived biomatrix [11,12], or, on a heparin-containing collagen gel [13], liver-specific functions are maintained for relatively long periods of time. A sandwich culture of hepatocytes between two layers of type I collagen (TIC) gel [6,14] or between TIC and an EHS gel [5,15,16], or culture the cells in an EHS gel [17] also have been reported to preserve liver functions. In addition to these observations, we recently reported that a fibronectin-containing TIC gel overlay lead to the induction of liver-specific genes in primary hepatocytes [18].…”

Section: Introductionmentioning

confidence: 99%

Differential Regulation of Liver-Specific and Ubiquitously-Expressed Genes in Primary Rat Hepatocytes by the Extracellular Matrix

Otsu

Ito²,

Kuzumaki³

et al. 2001

Cell Physiol Biochem

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Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase and β-actin) increased. When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (an EHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression in primary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole or actinomycin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of primary hepatocytes by inducing the transcription of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.

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Ultrastructural and functional differentiation of hepatocytes under long‐term culture conditions

Cited by 57 publications

References 37 publications

Dorsal and Ventral Stimuli in Cell–Material Interactions: Effect on Cell Morphology

Dorsal and Ventral Stimuli in Cell–Material Interactions: Effect on Cell Morphology

Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes

Differential Regulation of Liver-Specific and Ubiquitously-Expressed Genes in Primary Rat Hepatocytes by the Extracellular Matrix

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