HepG2 liver cells show remarkable liver functions in Mimetix® scaffolds

HepG2 human liver carcinoma cells are widely-used as a research tool for drug metabolism and hepatotoxicity studies. They are easy to culture and readily available in contrast to primary hepatocytes but only express the major metabolising CYP450 enzymes to a very limited extent in 2D culture (Hewitt and Hewitt, 2004). HepG2 cells cultured in the 3D Mimetix tissue culture plate retain their basic functionality (urea and albumin production) and re-acquire metabolic CYP450 activity and phase II enzyme activity over 28 days.

Hewitt, N. J., Hewitt, P. (2004). Phase I and II enzyme characterization of two sources of HepG2 cell lines. Xenobiotica 34(3):243–256.

Experimental details: Albumin and urea production in HepG2 liver cells were followed for 28 days in Mimetix scaffolds and compared to a standard 2D 96-well plate. Cells were seeded at 10,000 cells/well. Albumin was quantified using the Abcam kit #108788. Urea was quantified using the Abcam kit #83362.

CYP activity

HepG2 cells demonstrate substantial activity in six selected CYP enzymes (data for the two major ones shown below) for up to 28 days when cultured in 3D in the Mimetix  scaffold. The corresponding CYP gene expression levels match the activity profiles. CYP activities after 21 days of culture in the Mimetix scaffold and 48 h substrate exposure were significantly higher than those of primary hepatocytes at classic assay conditions.

Experimental details: CYP activities in HepG2 liver cells were followed for 28 days in Mimetix scaffolds and compared to a standard 2D 96-well plate. Cells were seeded at 10,000 cells/well. Substrates were chosen according to FDA and EMA guidelines, activity was measured using LC-MS (Pharmacelsus, Germany).

Metabolic activity (measured as ATP)

HepG2 cells cultured in the 3D Mimetix scaffold remain viable and metabolically active for 28 days. The signal (proportional to the number of viable cells) remained stable over the entire time period, indicating that the cells did not proliferate. Instead, they re-acquired hepatocyte functions, as shown in the following.

Experimental details: Metabolic activity was measured over 28 days in the Mimetix scaffold by quantifying the ATP content using the CellTiter-Glo® Luminescent Cell Viability from Promega. Cells were seeded at 10,000 cells/well.

Basic functionality

HepG2 actively detoxify and produce albumin in Mimetix scaffolds as shown by a regular increase in urea and albumin production for 28 days.

Albumin is the most abundant protein in human blood plasma and is produced in the liver. Its secretion level is a good indicator for the basic functionality of hepatocytes.

HepG2 cells actively produce albumin when cultured in 3D Mimetix scaffolds, as shown by a regular increase in albumin synthesis over 28 days.

Urea is a metabolite of amino acids and excessive ammonium ions, which are highly toxic when they accumulate. Basal urea production of HepG2 cells was measured over 28 days.

HepG2 cells actively detoxify when cultured in 3D Mimetix scaffolds, as shown by a regular increase in urea over the entire time period.

Phase II enzyme activity

HepG2 actively detoxify and produce albumin in Mimetix scaffolds as shown by a regular increase in urea and albumin production for 28 days.

 The expression levels of genes coding for the 4 main phase II enzymes, (2 UGT and 2 SULT) are either maintained (UGT1A1) or increased (UGT2B7, SULT1A1 and SULT2A1) over 28 days in cells grown in the Mimetix  plate.

Bile acid production

HepG2 cells cultured in Mimetix plates produce bile acids (Pharmacelsus, Germany).

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