Comparative analysis of 3D culture methods on human HepG2 cells (original) (raw)

Abstract

Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

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Abbreviations

2D:

Two-dimensional

3D:

Three-dimensional

ACTB:

β-Actin

AhR:

Aryl hydrocarbon receptor

Alv:

Alvetex

CAR:

Constitutive androstane receptor

Col:

Collagen

CTB:

CellTiter-Blue

CYP:

Cytochrome-P450 monooxygenase(s)

DPPIV:

Dipeptidyl-peptidase IV

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

Mg:

Matrigel

OAZ1:

Ornithine decarboxylase antizyme 1

PXR:

Pregnane-X receptor

qPCR:

Real-time quantitative PCR

RFU:

Relative fluorescence units

SD:

Standard deviation

UGT:

UDP-glucuronosyltransferase(s)

ZO-1:

Zona occludens-1

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Acknowledgments

This work was financed by the Federal Institute for Risk Assessment (grant number 1322-591) and by the Robert Bosch Foundation, Stuttgart. We thank Beatrice Rosskopp, Markus König (Stuttgart) and Igor Liebermann (Stuttgart) for their excellent technical assistance.

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Authors and Affiliations

1. Department of Food Safety, Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589, Berlin, Germany
   Claudia Luckert, Christina Schulz, Nadja Lehmann, Albert Braeuning, Alfonso Lampen & Stefanie Hessel
2. Department of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558, Nuthetal, Germany
   Claudia Luckert
3. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstraße 112, 70376, Stuttgart, Germany
   Maria Thomas & Ute Hofmann
4. University of Tuebingen, Tuebingen, Germany
   Maria Thomas & Ute Hofmann
5. Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU Dortmund University, Ardeystrasse 67, 44139, Dortmund, Germany
   Seddik Hammad & Jan G. Hengstler
6. Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, 83523, Qena, Egypt
   Seddik Hammad

Authors

1. Claudia Luckert
2. Christina Schulz
3. Nadja Lehmann
4. Maria Thomas
5. Ute Hofmann
6. Seddik Hammad
7. Jan G. Hengstler
8. Albert Braeuning
9. Alfonso Lampen
10. Stefanie Hessel

Corresponding author

Correspondence toStefanie Hessel.

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The authors declare no conflict of interest.

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Luckert, C., Schulz, C., Lehmann, N. et al. Comparative analysis of 3D culture methods on human HepG2 cells.Arch Toxicol 91, 393–406 (2017). https://doi.org/10.1007/s00204-016-1677-z

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• Received: 09 November 2015
• Accepted: 28 January 2016
• Published: 12 February 2016
• Issue date: January 2017
• DOI: https://doi.org/10.1007/s00204-016-1677-z

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