Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.
Journal
The Analyst
ISSN: 1364-5528
Titre abrégé: Analyst
Pays: England
ID NLM: 0372652
Informations de publication
Date de publication:
07 Jul 2021
07 Jul 2021
Historique:
pubmed:
10
6
2021
medline:
2
7
2021
entrez:
9
6
2021
Statut:
ppublish
Résumé
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 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% CO 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.
Sections du résumé
BACKGROUND
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
METHODS
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% CO
RESULTS & DISCUSSION
CONCLUSIONS
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.
Identifiants
pubmed: 34106111
doi: 10.1039/d1an00041a
pmc: PMC8239994
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
4326-4339Références
Redox Biol. 2019 Apr;22:101165
pubmed: 30877854
PLoS Biol. 2010 Oct 19;8(10):e1000514
pubmed: 21049082
Nat Commun. 2020 Jan 3;11(1):102
pubmed: 31900386
BMC Biol. 2014 May 27;12:34
pubmed: 24884669
Drug Discov Today. 2008 Mar;13(5-6):268-74
pubmed: 18342804
Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18131-6
pubmed: 19826085
Magn Reson Med. 1991 Oct;21(2):233-41
pubmed: 1745122
Annu Rev Physiol. 2001;63:15-48
pubmed: 11181947
NMR Biomed. 2017 Jun;30(6):
pubmed: 28301071
Mol Cell Biochem. 1992 Jul 6;113(1):1-15
pubmed: 1640933
J Biol Chem. 2016 Mar 4;291(10):5157-71
pubmed: 26703469
Anal Chem. 2015 Jul 21;87(14):7402-10
pubmed: 26121119
Oncogene. 2010 Jan 21;29(3):313-24
pubmed: 19881548
Philos Trans R Soc Lond B Biol Sci. 1980 Jun 25;289(1037):425-36
pubmed: 6106217
Biochim Biophys Acta. 2005 May 1;1734(1):1-12
pubmed: 15866478
Anal Chem. 2018 Jul 3;90(13):7912-7921
pubmed: 29863330
Biorheology. 2010;47(3-4):165-78
pubmed: 21084742
Lab Chip. 2016 May 24;16(11):2079-85
pubmed: 27149932
Sci Adv. 2017 Jun 16;3(6):e1700341
pubmed: 28630930
J Pharmacol Toxicol Methods. 2019 Jan - Feb;95:70-78
pubmed: 30502390
Curr Protoc Mol Biol. 2005 Aug;Chapter 28:Unit 28.3
pubmed: 18265368
Biomolecules. 2015 Jun 29;5(3):1319-38
pubmed: 26131977
NMR Biomed. 2002 Oct;15(6):375-84
pubmed: 12357551
Magn Reson Chem. 2016 Jun;54(6):451-6
pubmed: 26248898
Physiology (Bethesda). 2017 Jul;32(4):266-277
pubmed: 28615311
J Neurosci Res. 2007 Nov 15;85(15):3386-97
pubmed: 17628504
Nat Protoc. 2007;2(11):2692-703
pubmed: 18007604
J Inherit Metab Dis. 2006 Apr-Jun;29(2-3):327-31
pubmed: 16763895
NMR Biomed. 2015 Sep;28(9):1141-9
pubmed: 26202449
Cell. 2006 Jun 30;125(7):1241-52
pubmed: 16814712
Magn Reson Med. 2001 May;45(5):749-55
pubmed: 11323800
Biophys J. 1988 Feb;53(2):227-33
pubmed: 3345331
Nucleic Acids Res. 2018 Jan 4;46(D1):D608-D617
pubmed: 29140435
J Proteome Res. 2014 Dec 5;13(12):6033-45
pubmed: 25382592
J Vis Exp. 2015 Dec 12;(106):e53464
pubmed: 26709455
Curr Opin Struct Biol. 2015 Jun;32:39-47
pubmed: 25727665
Mol Cell Biochem. 2004 Jul;262(1-2):1-16
pubmed: 15532704
Int J Oncol. 2010 Feb;36(2):301-6
pubmed: 20043062
Proc Natl Acad Sci U S A. 1979 Mar;76(3):1274-8
pubmed: 286310
Chem Commun (Camb). 2012 Jan 21;48(6):811-3
pubmed: 22143456
Drug Discov Today. 2010 Aug;15(15-16):610-21
pubmed: 20601091
J Magn Reson. 2019 Sep;306:202-212
pubmed: 31358370
Sci Rep. 2019 Dec 10;9(1):18699
pubmed: 31822748
PLoS One. 2015 May 29;10(5):e0128478
pubmed: 26024484
Cancer Res. 2000 Sep 15;60(18):5179-86
pubmed: 11016646
Methods Mol Biol. 2013;979:65-70
pubmed: 23397389
Int J Biochem Cell Biol. 2011 Jul;43(7):990-1001
pubmed: 20633697
Magn Reson Med. 2002 Nov;48(5):819-25
pubmed: 12417996
Biophys J. 2019 Jan 22;116(2):239-247
pubmed: 30580921
Biotechnology (N Y). 1990 Dec;8(12):1282-5
pubmed: 1366962
Metabolites. 2018 May 24;8(2):
pubmed: 29795000
Am J Respir Cell Mol Biol. 2019 Nov;61(5):554-555
pubmed: 31100006
Biomaterials. 2010 Nov;31(32):8454-64
pubmed: 20684983
Interface Focus. 2017 Apr 6;7(2):20160100
pubmed: 28382199
Cell. 2012 Mar 16;148(6):1145-59
pubmed: 22424226
Cell Rep. 2015 Nov 3;13(5):981-9
pubmed: 26565911
J Proteome Res. 2010 Nov 5;9(11):5877-86
pubmed: 20815413
Toxicol Sci. 2018 Mar 1;162(1):15-23
pubmed: 29340618
Analyst. 2017 Jan 26;142(3):465-471
pubmed: 28074201
Mol Genet Metab. 2008 May;94(1):16-37
pubmed: 18243024
Skin Pharmacol Physiol. 2016;29(4):204-9
pubmed: 27441370
Biotechnol Bioeng. 2004 Sep 30;87(7):835-48
pubmed: 15334410
J Comput Assist Tomogr. 1985 Nov-Dec;9(6):1012-9
pubmed: 4056130
Metabolomics. 2019 Feb 28;15(3):32
pubmed: 30830487
Appl Microbiol Biotechnol. 2014 Oct;98(19):8367-75
pubmed: 24946863
Nat Protoc. 2014 Feb;9(2):421-38
pubmed: 24457333
Magn Reson Med. 2010 Feb;63(2):322-9
pubmed: 20099325
Expert Opin Drug Discov. 2009 Jul;4(7):785-97
pubmed: 23489169
Methods Mol Biol. 2009;520:273-95
pubmed: 19381962
Cytotechnology. 2018 Feb;70(1):375-386
pubmed: 29032507
NMR Biomed. 2010 Jun;23(5):473-9
pubmed: 20225233
Magn Reson Med. 1993 Feb;29(2):196-204
pubmed: 8429783