Chemical‐based primary human hepatocyte monolayer culture for the study of drug metabolism and hepatotoxicity: Comparison with the spheroid model

Zhong, Yixiang; Yu, Jun; Wang, Xiaoqiong; Binas, Bert; Yoo, Hye Hyun

doi:10.1096/fj.202001629rr

Cited by 6 publications

(11 citation statements)

References 44 publications

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“…Monolayer 2D culture systems using immortalized cell lines and primary hepatocytes have been widely used for liver toxicity testing [ 8 ]. However, 2D culture systems cannot recapitulate the liver microenvironments that are strongly correlated with hepatic cellular functions; hepatocytes in monolayer 2D culture systems lose viability rapidly and decrease liver-specific functionality within a few days [ 11 , 12 , 13 ]. A sandwich-culture system of culturing primary hepatocytes between two layers of collagen improved cell viability, polarized architecture, and liver-specific functions [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

Niu

Lin

Lee

2022

Gels

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Drug-induced liver injury (DILI) is a leading cause of attrition in drug development or withdrawal; current animal experiments and traditional 2D cell culture systems fail to precisely predict the liver toxicity of drug candidates. Hence, there is an urgent need for an alternative in vitro model that can mimic the liver microenvironments and accurately detect human-specific drug hepatotoxicity. Here, for the first time we propose the fabrication of an albumin methacryloyl cryogel platform inspired by the liver’s microarchitecture via emulating the mechanical properties and extracellular matrix (ECM) cues of liver. Engineered crosslinkable albumin methacryloyl is used as a protein-based building block for fabrication of albumin cryogel in vitro models that can have potential applications in 3D cell culture and drug screening. In this work, protein modification, cryogelation, and liver ECM coating were employed to engineer highly porous three-dimensional cryogels with high interconnectivity, liver-like stiffness, and liver ECM as artificial liver constructs. The resulting albumin-based cryogel in vitro model provided improved cell–cell and cell–material interactions and consequently displayed excellent liver functional gene expression, being conducive to detection of fialuridine (FIAU) hepatotoxicity.

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Section: Introductionmentioning

confidence: 99%

An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

Niu

Lin

Lee

2022

Gels

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“…3D spheroid PHHs retain the in vivo hepatic phenotype of human liver in culture for several weeks and stably express CYPs and transporters, which is a prerequisite for in vitro to in vivo extrapolation. 15,16,18,19,24 Previously, it was shown that high baseline expression of CYP3A4 mRNA in 3D spheroid PHHs resulted in clinically more relevant magnitude of induction than in 2D monolayer PHHs. 23 Moreover, 3D spheroid PHHs were able to identify inducers that failed to induce CYP3A4 in 2D monolayer PHHs.…”

Section: Discussionmentioning

confidence: 99%

“…16,24 3D spheroid PHHs have outperformed 2D monolayer and sandwich cultured PHHs in hepatotoxicity assays 15,20 and they retain drug metabolism activity of important CYPs for several weeks allowing long-term experiments. 15,16,18,19,24,25 Assessment of drug clearance and identification of low-abundant metabolites is also feasible in 3D spheroid PHHs. 22,25,26 In contrast to 2D monolayer PHHs, 3D spheroid PHHs express CYP3A4 mRNA at almost 200-fold higher levels.…”

Section: Introductionmentioning

confidence: 99%

“…6,13 However, PHHs lose their phenotype within days in most culture formats. [14][15][16][17][18][19] To improve in vitro modelling of human liver, a 3D spheroid culture of primary human hepatocytes (3D spheroid PHHs), without addition of extracellular matrices or scaffolds, has been characterized and investigated for several pharmacological applications. 16,[18][19][20][21][22][23][24][25][26] 3D spheroid PHHs retain the hepatic phenotype of native tissue at the protein and RNA levels, and are viable for several weeks.…”

Section: Introductionmentioning

confidence: 99%

“…[14][15][16][17][18][19] To improve in vitro modelling of human liver, a 3D spheroid culture of primary human hepatocytes (3D spheroid PHHs), without addition of extracellular matrices or scaffolds, has been characterized and investigated for several pharmacological applications. 16,[18][19][20][21][22][23][24][25][26] 3D spheroid PHHs retain the hepatic phenotype of native tissue at the protein and RNA levels, and are viable for several weeks. 16,24 3D spheroid PHHs have outperformed 2D monolayer and sandwich cultured PHHs in hepatotoxicity assays 15,20 and they retain drug metabolism activity of important CYPs for several weeks allowing long-term experiments.…”

Section: Introductionmentioning

confidence: 99%

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3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction

Järvinen

Hammer²,

Pötz³

et al. 2022

Preprint

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Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. 3D spheroid PHHs from three different donors were treated for four days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole or β-naphthoflavone. Induction of CYPs 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, P-gp/ABCB1, MRP2/ABCC2, ABCG2, OCT1/SLC22A1, SLC22A7, SLCO1B1 and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19 and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of 5- to 6-fold for rifampicin, which closely correlates to induction observed in clinical studies. Similar estimates were found for dicloxacillin and flucloxacillin, which also correlates to findings from clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, while the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, while the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2 and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

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3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction

Järvinen

Hammer²,

Pötz³

et al. 2023

Clin Pharma and Therapeutics

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Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug–drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or β‐naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P‐glycoprotein (P‐gp)/ABCB1, multidrug resistance‐associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five‐ to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9‐ and 12‐fold, whereas the protein levels of these CYPs reached 2‐ and 3‐fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4‐fold, whereas the induction of CYP2C9 mRNA was over 2‐fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2‐fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug‐metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

show abstract

Chemical‐based primary human hepatocyte monolayer culture for the study of drug metabolism and hepatotoxicity: Comparison with the spheroid model

Cited by 6 publications

References 44 publications

An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

An Engineered Protein-Based Building Block (Albumin Methacryloyl) for Fabrication of a 3D In Vitro Cryogel Model

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction

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