Three-Dimensional HepaRG Model As An Attractive Tool for Toxicity Testing

Leite, Sofia B.; Wilk-Zasadna, Iwona; Zaldı́var, J.M.; Airola, Elodie; Reis-Fernandes, Marcos A.; Mennecozzi, Milena; Guguen‐Guillouzo, Christiane; Chesne, C.; Guillou, Claude; Alves, Paula M.; Coecke, Sandra

doi:10.1093/toxsci/kfs232

Cited by 88 publications

(88 citation statements)

References 40 publications

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“…Three-dimensional culture systems have been shown to promote enhanced cellular structure and function in many cell and tissue types, including mammary epithelial cells [25], mesenchymal stem cells [26], neural cells [27], and hepatic cells [19,[28][29][30][31][32][33][34]. In those studies employing mouse, rat or pig primary hepatocytes, cells were allowed to adhere to moderate to low attachment surface to enhance spheroid formation [33,35].…”

Section: Discussionmentioning

confidence: 99%

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells

Subramanian

Owens

Raju

et al. 2014

Stem Cells and Development

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Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

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Section: Discussionmentioning

confidence: 99%

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells

Subramanian

Owens

Raju

et al. 2014

Stem Cells and Development

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“…Leite et al developed a 3D culture system that utilised human hepatocytes cultured in multicellular spheroids [97]. It is reported that such a culture system requires fewer cells, are technically easier to grow and can be used for high-throughput studies relative to bioreactor systems [91].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

“…It is reported that such a culture system requires fewer cells, are technically easier to grow and can be used for high-throughput studies relative to bioreactor systems [91]. Using this system, the authors showed that HepaRG cells maintained their liver-specific functionality for several weeks, a vast improvement over conventional 2D culture techniques, and also showed that cells demonstrated a dosedependent effect in response to acetaminophen exposure [97]. Gunness et al then developed this model further to compare liver-specific functions to 2D cell culture as well as analysing acute toxicity in response to acetaminophen, troglitazone and rosiglitazone [91].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Life in 3D is never flat: 3D models to optimise drug delivery

Fitzgerald

Malhotra

Curtin

et al. 2015

Journal of Controlled Release

191

126

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“…Cells readily adhere to the walls of the MOC channel, create a confluent monolayer, and elongate along the shear stress ( Figure 2B). Furthermore, cells cover the entire circumference of the channels, as reported previously In a further experiment, consistent disk-shaped liver cell spheroids are formed from HepaRG and HHSteC during two days of hanging drop culture, as this model system was previously reported as being suitable for drug metabolism studies [11][12][13] . For demonstration purposes, one tissue culture compartment of each MOC circuit was seeded with 20 spheroids in 96-well cell culture inserts and tissues were cultivated over 14 days under dynamic conditions using non-endothelialized MOCs.…”

Section: Representative Resultsmentioning

confidence: 60%

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Materne

Maschmeyer

Lorenz

et al. 2015

JoVE

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The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for longterm multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells.For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

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Three-Dimensional HepaRG Model As An Attractive Tool for Toxicity Testing

Cited by 88 publications

References 40 publications

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells

Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells

Life in 3D is never flat: 3D models to optimise drug delivery

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

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