Zdrazilova 2022 PLOS ONE

Zdrazilova L, Hansikova H, Gnaiger E (2022) Comparable respiratory activity in attached and suspended human fibroblasts. PLoS ONE 17:e0264496. https://doi.org/10.1371/journal.pone.0264496

» Open Access

Zdrazilova Lucie, Hansikova Hana, Gnaiger Erich (2022) PLOS ONE

Abstract: Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are cultured in monolayers, but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached versus suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell. No differences were observed between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated respirometric database.

• Keywords: attached cells; ace, suspended cells; sce, fibroblasts, high-resolution respirometry; HRR, Oroboros O2k, Seahorse Bioanalyzer XF24, ROUTINE respiration; R, LEAK respiration; L, electron transfer capacity; E, residual oxygen consumption; Rox, bioenergetic cluster analysis; BCA • Bioblast editor: Gnaiger E • O2k-Network Lab: AT Innsbruck Oroboros, CZ Prague Zeman J

ORCID: Zdrazilova Lucie, Hanskova Hana, Gnaiger Erich

Data availability

Original files are available Open Access at Zenodo repository: 10.5281/zenodo.5518059

Support

This work was partially funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770, NextGen-O2k project, and by Institutional project GAUK110119. Contribution to COST Action CA15203 MitoEAGLE with financial support of Short-Term Scientific missions (LD).

MitoFit Preprints

Comparable respiratory activity in attached and suspended human fibroblasts

Cited by

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. https://doi.org/10.26124/mitofit:2021-0008

Labels: MiParea: Respiration, Instruments;methods 

Organism: Human  Tissue;cell: Fibroblast  Preparation: Intact cells 

Regulation: Coupling efficiency;uncoupling  Coupling state: LEAK, ROUTINE, ET  Pathway: ROX  HRR: Oxygraph-2k 

SUIT-003, MitoEAGLEPublication, MitoFit 2021 BCA