Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression in a three-dimensionally cultivated human hepatocellular carcinoma (HCC) model

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 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Uta Fernekorn

PROVIDER: E-GEOD-41962 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: The main focus of the work was the evaluation of gene expression differences between our established NSCLC 3D cell culture model and the 2D cell culture in regard to the use of our model for drug screening applications. Methods: The non-small cell lung cancer (NSCLC) cell lines Colo699 and A549 were cultivated as monolayer (2D) on cell culture plates for five days or as microtissues (3D) in a hanging-drop system for five and ten days, respectively. Cells and microtissues were harvested and Affymetrix chip analyses were performed with the prior isolated RNA. This was repeated in three independent experiments. Subsequent biostatistical data analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. Results: The analyses revealed a high interassay correlation within the distinct culture systems, thus proving a high validity of our data. The comparison of 3D versus 2D cell cultures revealed significant differences in RNA expression (979 genes for A549; 1106 genes for Colo699), but the overlap of changes in RNA profiles between the cell lines at the individual gene level was small (149 genes), potentially reflecting overall heterogeneity and their origin, i.e. primary vs pleural effusion. Nevertheless, these RNA expression changes affected most relevant cancer-associated pathways as DNA methylation, cell cycle, rRNA expression and meiosis pathways. Furthermore, the expression differences between 2D and 3D were more evident after longer cultivation time, which supports the hypothesis of cultivation related mechanisms and the usage of long-time cultivation systems. Conclusion: In summary, our data support the need of innovative 3D drug testing systems to close the gap between in-vitro drug screening and in-vivo data. Thus, our 3D NSCLC model might provide a model to address the challenge of microenviroment associated resistance mechanisms, as well as cell-cell interaction related effects.

Project description:Development of systems allowing the maintenance of native properties of mesenchymal stromal cells (MSC) is a critical challenge for studying physiological functions of skeletal progenitors, as well as towards cellular therapy and regenerative medicine applications. Conventional stem cell culture in monolayer on plastic dishes (2D) is associated with progressive loss of functionality, likely due to the absence of a biomimetic microenvironment and the selection of adherent populations. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow cells within the pores of 3D scaffolds in a perfusion-based bioreactor system, followed by enzymatic digestion for cell retrieval. The 3D-perfusion system supported MSC growth while maintaining cells of the hematopoietic lineage, and thus generated a cellular environment mimicking some features of the bone marrow stroma. As compared to 2D-expansion, sorted CD45- cells derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) maintained a 4.3-fold higher clonogenicity and exhibited a superior differentiation capacity towards all typical mesenchymal lineages, with similar immunomodulatory function in vitro. Transcriptomic analysis performed on MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability as well as a significant upregulation of multipotency-related gene clusters following 3D-perfusion as compared to 2D expansion. The described system offers a model to study how factors of a 3D engineered niche may regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems. Nucleated cells were isolated from 5 fresh human bone marrow aspirates by means of red blood cells lyses buffer and then were seeded into a 3D perfusion bioreactor system using a pure hydroxyapatite 3D scaffold and in conventional Petri dishes (2D). After culture for 19 days, cells from both systems were enzymatically retrieved and sorted using anti-CD45-coated magnetic beads. Total RNA was extracted from CD45- cells, QCed and hybridized to Affymetrix microarrays.

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Gene expression in a three-dimensionally cultivated human hepatocellular carcinoma (HCC) model

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