Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression in a three dimensionally cultivated human hepatocytes

ABSTRACT: The development of more complex but reliable systems for compound testing in a pharmaceutical context is a challenging task to date. Three dimensional (3D), organ mimetic cell culture is aiming to become an alternative to common two dimensional (2D) cell culture or animal testing in that field. We developed a biocompatible 3D cell culture environment for a 3D hepatocyte cell culture that enables cellular maintenance in a polycarbonate scaffold structure. Our data indicate that an actively perfused three dimensional cell culture displays a more pronounced metabolic genotype than statically cultivated hepatocytes. Human hepatocytes of three donors were cultivated for five days under 2D and 3D statical and perfused conditions. Cultivation was started with 0.25 x106 in 2D and with 1x 106 vital cells for the 3D experiments. The day of seeding was defined as d0. The groups were classified as follows: 2D i.e.monolayer cultures, 3D i.e. statical 3D culture and BR denotes perfused 3D culture of hepatocytes. The perfusable bioreactor system was operated using a peristaltic pump. It houses the MatriGrid, a polycarbonate based microporous cellular support. For 3Dstatic cultivation, cell- inoculated MatriGrids were placed in wells of a 24 wells plate. Microarray experiments of three 2D (i.e. control), three 3D statically and three actively perfused 3D cultivations, respectively, were performed at SIRS-Lab GmbH (SIRS-Lab GmbH, Jena, Germany) according to the manufacturerM-bM-^@M-^Ys instructions (Illumina, San Diego, CA). Altogether, 8 RNA samples of hepatocyte cultures and an internal control RNA were hybridized on two HumanHT-12 v4 Expression BeadChips.

ORGANISM(S): Homo sapiens

SUBMITTER: Uta Fernekorn

PROVIDER: E-GEOD-44580 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The development of more complex but reliable systems for compound testing in a pharmaceutical context is a challenging task to date. Three-dimensional (3D), organ mimetic cell culture is aiming to become an alternative to common two-dimensional (2D) cell culture or animal testing in that field. We developed a biocompatible 3D cell culture environment for a hepatocellular carcinoma (HCC) model that enables cellular maintenance in a polycarbonate scaffold structure. Albumin, regarded as a differentiation marker, was elevated in statically 3D cultivated HepG2 cells. Expression of HCC tumor marker alpha-fetoprotein (AFP) was reduced compared to immunofluorescence stainings of 2D cultivated cells. Remarkably, expression of cytokeratin and pathophysiologically relevant beta-1 integrin (ITGB1) was found enhanced in nonperfused 3D cell culture. Changes in gene expression induced by the 3D cultivation environment were investigated using Ingenuity Pathway Analysis (IPA). Our findings revealed involvement of the insulin growth factor (IGF) signaling pathway in upregulation of matrix metalloproteinases (MMP) and ITGB1. The experimental data indicate a more differentiated state in 3D cultivated HepG2 cells than in the respective 2D experiments. Hence, scaffold-supported 3D cultivation of HepG2 cells may lead to a gain of information valuable for both drug testing and cancer research. HepG2 cells were cultivated for five days under 2D and 3D statical and perfused conditions. Cultivation was started with 0.25x10^6 cells in 2D and with 1x10^6 vital cells for the 3D experiments. The day of seeding was defined as d0. The groups were classified as follows: 2D, i.e., monolayer cultures, 3D, i.e., statical 3D cultures and BR, which denotes perfused 3D culture of HepG2 cells. The perfusable bioreactor system was operated using a peristaltic pump. It houses the MatriGrid, a polycarbonate-based microporous cellular support. For 3D static cultivation, cell-inoculated MatriGrids were placed in wells of a 24-well plate. Microarray experiments of three 2D (i.e., control), three 3D statically and three actively perfused 3D cultivations were performed at SIRS-Lab GmbH (SIRS-Lab GmbH, Jena, Germany) according to the manufacturer's instructions (Illumina, San Diego, CA). Altogether, 9 RNA samples of hepatocyte cultures and an internal control RNA were hybridized on two HumanHT-12 v4 Expression BeadChips.

Project description:The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional (2D) monolayers on plastic. However, many cellular features are impaired in these unnatural conditions and big alterations in gene expression in comparison to tumors have been reported. Three-dimensional (3D) cell culture models have become increasingly popular and are suggested to be better models than 2D monolayers due to improved cell-to-cell contacts and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput 3D drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in 2D, in polyHEMA coated anchorage independent 3D models and in Matrigel on-top 3D cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating or they were allowed to grow in 3D for four days prior to the drug treatment. Big variations in drug responses were observed between the models indicating that comparisons of culture model influenced drug sensitivities cannot be made based on effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in 2D cultures, while responses of cells grown in polyHEMA resembled those of 2D. Furthermore, comparison of gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study we also suggest that 3D cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode and be used as alternatives for traditional 2D screens towards better comparability to in vivo state. Gene expression analysis of JIMT1 breast cancer cells cultured as xenografts for 43 days, in two dimensional cultures for seven days (2D7d), in polyHEMA three dimensional cell culture models for four and seven days (PH7d and PH7d), and in Matrigel three dimensional cultures for four and seven days (MG4d and MG7d). Two biological replicates was included for each sample.

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Gene expression in a three dimensionally cultivated human hepatocytes

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