HepaRG culture in tethered spheroids as an in vitro three‐dimensional model for drug safety screening

Abstract

Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver-specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg-Gly-Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening.

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, ). Conventional differentiation of HepaRG involves plating the progenitor cells on 2D substrates, allowing them to grow to confluence for 14 days before switching to a differentiation medium for another 14 days (Hoekstra et al, ; Leite et al, ; Wang et al, ) (Fig.…”

“…Given that a microfluidic 3D environment has been helpful in the maintenance of primary hepatocyte morphology and functions (Banaeiyan et al, ; Goral et al, ; Lee et al, ; van Duinen et al, ), we postulated that the differentiation of HepaRG‐HLCs can be similarly enhanced in such a microenvironment. Since this microfluidic system allowed cells to be packed three‐dimensionally at high density with extensive cell–cell contact (Wang et al, ), we could by‐pass the 14‐day expansion phase required in the 2D differentiation protocol and directly initiate perfusion culture with the differentiation medium (Fig. Aiii).…”

“…In all, our results indicated that the microfluidic 3D perfusion culture system could significantly improve the generation of functional HepaRG‐HLCs over standard 2D static differentiation by reducing the differentiation period to 14 days; improving the HLC derivation efficiency from 50% to 92%; and increasing liver‐specific functions. Various research groups have explored the use of different culture configurations to improve the functionality of HepaRG‐HLCs (Leite et al, ; Maschmeyer et al, ; Wang et al, ), although the majority attempted to augment the functionality of HepaRG‐HLCs by putting them into 3D cultures or microfluidic devices only after they have been fully differentiated using the standard 28‐days 2D protocol (Maschmeyer et al, ; Schimek et al, ; Wang et al, ). This strategy cannot address the long duration of the standard 2D differentiation protocol, which takes up to 28 days, nor the inherent limits of hepatic differentiation efficiency in 2D cultures (Fig.…”

“…Human stem cells derived‐hepatocytes offers an attractive alternative as a sustainable human cell source for microfluidic liver models (Okita et al, ; Schwartz et al, ; Scott 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, ).…”

Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction

“…CYP activity was assayed at Day 5, after two days of culture and three days of compound exposure, a protocol which has been previously used in literature [52]. Different lengths of culture time and compound exposure could affect levels of CYP activity as shown in literature [24,53]. Moreover, assaying at a single point in time may have missed more acute changes in CYP activity.…”