Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.

Hertig, Damian; Maddah, Sally; Memedovski, Roman; Kurth, Sandra; Moreno, Aitor; Pennestri, Matteo; Felser, Andrea; Nuoffer, Jean-Marc; Vermathen, Peter (2021). Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy. Analyst, 146(13), pp. 4326-4339. Royal Society of Chemistry 10.1039/d1an00041a

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Radiology, Neuroradiology and Nuclear Medicine (DRNN) > Institute of Diagnostic, Interventional and Paediatric Radiology > DCR Magnetic Resonance Spectroscopy and Methodology (AMSM)
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Pavillon 52 > Abt. Magnetresonanz-Spektroskopie und Methodologie, AMSM
04 Faculty of Medicine > Department of Radiology, Neuroradiology and Nuclear Medicine (DRNN) > Institute of Diagnostic and Interventional Neuroradiology
04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Institute of Clinical Chemistry
04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Hertig, Damian; Kurth, Sandra; Felser, Andrea Debora; Nuoffer, Jean-Marc and Vermathen, Peter

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1364-5528

Publisher:

Royal Society of Chemistry

Language:

English

Submitter:

Maria de Fatima Henriques Bernardo

Date Deposited:

29 Jun 2021 10:56

Last Modified:

01 Jul 2021 01:33

Publisher DOI:

10.1039/d1an00041a

PubMed ID:

34106111

BORIS DOI:

10.48350/156826

URI:

https://boris.unibe.ch/id/eprint/156826

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