Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

Shulman, Maria; Nahmias, Yaakov

doi:10.1007/978-1-62703-125-7_17

Cited by 98 publications

(77 citation statements)

References 49 publications

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“…[1] This limits or prevents their use for clinical, engineering and research purposes. The prolonged maintenance of hepatocyte phenotype could e.g.…”

Section: Introductionmentioning

confidence: 99%

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

et al. 2014

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The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

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“…[1] This limits or prevents their use for clinical, engineering and research purposes. The prolonged maintenance of hepatocyte phenotype could e.g.…”

Section: Introductionmentioning

confidence: 99%

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

et al. 2014

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“…When cultured in vitro , hepatocytes are also known to accumulate fat when exposed to high levels of glucose[34]. Therefore the media conditions used in this model were selected to have lower, more physiologically relevant, glucose and insulin concentrations when compared to standard hepatocyte culture media[35]. This media composition allowed us to study the specific effect of triglyceride accumulation on hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease

Kostrzewski

Cornforth

Snow

et al. 2017

WJG

118

110

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AIMTo develop a human in vitro model of non-alcoholic fatty liver disease (NAFLD), utilising primary hepatocytes cultured in a three-dimensional (3D) perfused platform.METHODSFat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids (FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds.RESULTSHepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factor-binding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantly reduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known anti-steatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium.CONCLUSIONThe 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.

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“…Primary rat hepatocytes were purchased from Tebu-Bio (Barcelona, Spain) and cultured in a collagen sandwich configuration68. Rat hepatocytes were seeded into 6-well plates (3 × 10 5 cells/well) and grown in Complete Growth Medium [DMEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium (Invitrogen, Paisley, UK), 40 ng/mL dexamethasone (Sigma), 20 ng/mL epidermal growth factor (Sigma) and antibiotic-antimycotic] for 24 h. Then, cells were serum-starved for 24 h and stimulated with increasing concentrations (10, 100, 1000 pmol/L) of acylated and desacyl ghrelin, (Tocris, Ellisville, MO, USA) for 24 h. These physiological and supraphysiological concentrations of ghrelin isoforms to carry out the experiments were chosen on the basis of previous studies performed in our laboratory2038.…”

Section: Methodsmentioning

confidence: 99%

Acylated and desacyl ghrelin are associated with hepatic lipogenesis, β-oxidation and autophagy: role in NAFLD amelioration after sleeve gastrectomy in obese rats

Ezquerro

Méndez‐Giménez

Becerril

et al. 2016

Sci Rep

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Bariatric surgery improves non-alcoholic fatty liver disease (NAFLD). Our aim was to investigate the potential role of ghrelin isoforms in the resolution of hepatic steatosis after sleeve gastrectomy, a restrictive bariatric surgery procedure, in diet-induced obese rats. Male Wistar rats (n = 161) were subjected to surgical (sham operation and sleeve gastrectomy) or dietary interventions [fed ad libitum a normal (ND) or a high-fat (HFD) diet or pair-fed]. Obese rats developed hepatosteatosis and showed decreased circulating desacyl ghrelin without changes in acylated ghrelin. Sleeve gastrectomy induced a dramatic decrease of desacyl ghrelin, but increased the acylated/desacyl ghrelin ratio. Moreover, sleeve gastrectomy reduced hepatic triglyceride content and lipogenic enzymes Mogat2 and Dgat1, increased mitochondrial DNA amount and induced AMPK-activated mitochondrial FFA β-oxidation and autophagy to a higher extent than caloric restriction. In primary rat hepatocytes, the incubation with both acylated and desacyl ghrelin (10, 100 and 1,000 pmol/L) significantly increased TG content, triggered AMPK-activated mitochondrial FFA β-oxidation and autophagy. Our data suggest that the decrease in the most abundant isoform, desacyl ghrelin, after sleeve gastrectomy contributes to the reduction of lipogenesis, whereas the increased relative acylated ghrelin levels activate factors involved in mitochondrial FFA β-oxidation and autophagy in obese rats, thereby ameliorating NAFLD.

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Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

Cited by 98 publications

References 49 publications

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease

Acylated and desacyl ghrelin are associated with hepatic lipogenesis, β-oxidation and autophagy: role in NAFLD amelioration after sleeve gastrectomy in obese rats

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