Functional polymer‐dependent 3D culture accelerates the differentiation of HepaRG cells into mature hepatocytes

Abstract

Aim:The hepatoma-derived cell line HepaRG is regarded as an in vitro model of drug metabolism because fully differentiated HepaRG cells demonstrate functional metabolic responses comparable to those of primary human hepatocytes. Recently, it was demonstrated that the 3D culture of HepaRG cells enhanced their metabolic functions and toxicological responses. We approached the mechanisms underlying these enhancement effects.Methods: We compared 2D-cultured HepaRG cells with 3D-cultured HepaRG spheroids in the gene expression patterns and the metabolic functions. In the present study, we performed 3D culture of HepaRG cells using functional polymers (FP). To reveal the in vivo differentiation ability, we transplanted the 3D-cultured HepaRG spheroids into TK-NOG mice.Results: A comparison between 2D and 3D cultures revealed that 3D-cultured HepaRG spheroids demonstrated reductions in bile duct marker expression, accelerated expression of cytochrome P450 3A4, and increases in the ratio of albuminexpressing hepatocytes. Furthermore, catalytic activities of cytochrome P450 3A4 were modified by omeprazole and rifampicin in the 3D-cultured HepaRG spheroids. Transplantation analysis revealed that 3D-cultured HepaRG spheroids formed hepatocyte-like colonies rather than cholangiocytes in vivo. Conclusion:Our results indicated that the enhancement of hepatic functions in 3D-cultured HepaRG cells was induced by selective hepatocyte differentiation and accelerated hepatocyte maturation. HepaRG spheroids reproduced the metabolic responses of human hepatocytes. Therefore, FP-dependent 3D-cultured HepaRG cells may serve as an excellent in vitro model for evaluating the hepatic metabolism and toxicity.

A pump‐free microfluidic 3D perfusion platform for the efficient differentiation of human hepatocyte‐like cells

“…HepaRG is a bipotent human liver progenitor cell line that is capable of differentiating into hepatocyte‐like cells (HLCs) (Higuchi et al, ) and biliary epithelial‐like cells (BECs) (Cerec et al, ; Leclerc et al, ; Leite et al, ). HepaRG‐derived hepatocyte‐like cells (HepaRG‐HLCs) have been shown to exhibit similar metabolic functions as primary human hepatocytes (Hoekstra et al, ; Leite et al, ), and have been extensively explored as an alternative human hepatocyte cell source in drug metabolism and toxicity testing (Leite et al, ; Wang et al, ) as well as disease modeling (Marion et al, ).…”

“…While human stem cell‐derived hepatocytes differentiated on conventional 2D cultures are being extensively investigated for applications in disease modeling as well as drug metabolism and toxicity testing (Leite et al, ; Wang et al, ), their differentiation and maturation in a microfluidic 3D hepatocyte culture platform have not yet been demonstrated. Similar to primary hepatocytes, stem cell‐derived hepatocyte‐like cells (HLCs) cultured in a 3D configuration as spheroids in micro‐wells and scaffolds showed a functionally more mature phenotype with higher liver‐specific functions (Gieseck et al, ; Higuchi et al, ; Tasnim et al, ; Wang et al, ). Therefore, we postulate that a liver‐sinusoid‐inspired microfluidic 3D microenvironment can enhance the differentiation and maturation of human liver progenitor cells into HLCs.…”

Fabrication and Characterization of Heparin/Collagen Sponge for in Vitro Differentiation of Wharton’s Jelly-Derived Mesenchymal Stem Cells into Hepatocytes

“…The current study showed that culturing MSCs in heparinized 3D scaffold at the presence of hepatogenic media led to an increase in the frequency of albumin-and cytokeratin 19-positive cells and also a rise in the intensity of the reaction for all tested liverspecific markers. Heparinized collagen sponges have been detected to improve hepatocyte functions (20). Indocyanine green clearance test showed MSC-derived hepatocyte functions also are promoted in heparin/collagen sponges, as well.…”

“…Also, an improvement in hepatocyte function and bio-mimicking of toxicity response has been reported through 3D culture (19). The 3D culture condition can lead to the acceleration of hepatocyte maturation (20). GAGcontaining collagen scaffolds have been previously used in tissue engineering context for both in vivo and in vitro studies to improve cellular attachment, metabolism, viability, as well as cell-matrix interactions (18).…”

Combining Chimeric Mice with Humanized Liver, Mass Spectrometry, and Physiologically-Based Pharmacokinetic Modeling in Toxicology

“…Recently, transplantation of three-dimensional-cultured hepatoma-derived cell line HepaRG cells has been reported to yield hepatocyte-like colonies in in vivo mouse bodies, like primary human hepatocytes, suggesting a possible human cell source for steady generation of humanized liver TK-NOG mice. 54,55 Humanized mice reconstituted with human immune systems are also essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. A novel transgenic NOG strain bearing human interleukin-3 and granulocyte macrophage colony-stimulating factor genes has been developed.…”