Background: Because of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures. Methods: We demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump. Results & Discussion: Implemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption.
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