Publication:
Neue Ansätze zur systembiologischen Untersuchung der Mitochondrien und des Metabolismus in tierischen Zellkulturen

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Date

2014

Authors

Bahnemann, Janina Stephanie

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Research Projects

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Abstract

Metabolomic analysis constitutes a crucial step for understanding cellular functions at a genomic level. In particular for systems biology studies, e.g. to describe the interplay of metabolic reactions and their dynamics, a reliable metabolomic analysis under physiological or in vivo conditions is required. While genomic, proteomic, and transcriptomic data can be obtained with high accuracy nowadays, the determination of metabolic profiles and fluxes still proves to be complicated. On the one hand, this is due to the fast turnover rates, the interaction between various metabolic pathways, and the chemical diversity of metabolites. On the other hand, the compartmentalization of metabolism in mammalian cells poses a huge challenge because of the dynamic range of metabolite concentration within the cytosol and the mitochondria. To overcome these limitations of metabolomic analysis, attempts have been made in this work to achieve a fast and efficient separation of the cytosolic and mitochondrial compartments by using new technological approaches in both macro and microscales. Due to its significant value, the well-known industrial cell line CHO-K1 was used as a model organism within this research project. To investigate the isolation of mitochondria in macroscale, different cell disruption methods were modified and tested. The efficiency and sensitivity of each were determined by the measure of mitochondrial yield and the integrity of isolated mitochondria. Here, the highest yields of intact mitochondria were obtained using ultrasound for cell disruption and subsequent differential centrifugation. In addition to the large-scale procedures, a microfluidic device for metabolic analysis, providing a complete sample preparation (including cell disruption) within seconds, was developed in project cooperation. In this work, a prototype of the Lab-on-a-Chip (LoaC) was successfully integrated into a bioreactor and operated for a period of 18 h. Continuous sampling was carried out by applying overpressure onto the bioreactor. Besides dynamic pulse experiments, this microchip-bioreactor system implemented an efficient separation of cells and extracellular media. Moreover, a selective cell lysis using digitonin and a subsequent separation of released cytosol from the remaining cellular and mitochondrial compartments was realised on-chip. Thus, this integrated microfluidic system provides high potential to overcome the limitations of analyses concerning compartmentalized metabolism. In the last part of this work, CHO-K1 cells were cultivated in a controlled small-scale bioreactor in order to perform systems biology studies. Therefore, detailed analyses of extracellular metabolites using LC-MS were performed under batch and continuous cultivation conditions. In addition, the mitochondrial structure and distribution of mitochondrial proteins were studied using confocal and high resolution STED microscopy. Furthermore, the relative level of enzymes involved in metabolic reactions was measured intracellularly by using the in-cell ELISA technology. To investigate the dependence of metabolic reactions on different cell cycle phases, the cultivation of synchronized cells was performed. For this purpose, cells from heterogeneous populations were enriched in the G1- and S-phase, respectively, using the centrifugal elutriation method. The synchrony during batch cultivation was determined flow cytometrically by analysing the DNA content and the cell size distribution. In addition, the cell count was observed continuously due to the effective integration of an at-line microscope. Metabolomic and microscopic studies of synchronized cells were performed during cultivation and the resultant data were compared to that obtained during cultivation of heterogeneous cell populations. The cell cycle dependency of metabolism was shown by the increased consumption rates of glucose and glutamine within the S-phase and the shifted increased lactate production during G1-phase, amongst others.

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Zellkompartimentierung, Mitochondrien, kompartimentierter Stoffwechsel, CHO-K1-Zellen, Zellaufschluss, Mitochondrienisolation, Mikrofluidik, cell compartmentalization, mitochondria, compartmentalized metabolism, CHO-K1 cells, cell disruption, mitochondria isolation, microfluidics

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