Dataset Information

A closer look at Aspergillus: Online monitoring via scattered light enables reproducible phenotyping

ABSTRACT: Development of microtiter plate based microbioreactor cultivation for Aspergillus giganteus with quasi-continuous online measurements. Different parameters such as well geometry, shaking frequency and morphology controlling agents were investigated in order to optimize the microtiter plate cultivation and scattered light signal towards reproducibility and homogeneity. An optimized medium was developed and scalability into stirred tank bioreactor cultivation was analyzed. As a transferability indicator the supernatant of both cultivation systems was analyzed for secreted protein patterns with a focus on an antifungal protein (AFP) and alpha-sarcin. These proteins were identified via LC-MS/MS.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Aspergillus Giganteus

SUBMITTER: Bianca Klein

LAB HEAD: Marco Oldiges

PROVIDER: PXD013751 | Pride | 2019-08-12

REPOSITORIES: Pride

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A closer look at <i>Aspergillus</i>: online monitoring via scattered light enables reproducible phenotyping.

Jansen Roman P RP Beuck Carina C Moch Matthias M Klein Bianca B Küsters Kira K Morschett Holger H Wiechert Wolfgang W Oldiges Marco M

Fungal biology and biotechnology 20190805

<h4>Background</h4>Filamentously growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. However, limited experimental throughput in bioprocess development still hampers taking advantage of their full potential. Miniaturization and automation are powerful tools to accelerate bioprocess development, but so far the application of such technologies has mainly been ...[more]

PMID: 31396392

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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.

Dataset Information

A closer look at Aspergillus: Online monitoring via scattered light enables reproducible phenotyping

Publications

A closer look at <i>Aspergillus</i>: online monitoring via scattered light enables reproducible phenotyping.

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