Comparable respiratory activity in attached and suspended human fibroblasts
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 whet...
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| Veröffentlicht in: | PloS one 2022-03, Vol.17 (3), p.e0264496-e0264496 |
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| creator | Zdrazilova, Lucie Hansikova, Hana Gnaiger, Erich |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0264496 |
| format | Article |
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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. 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Repeatability and reproducibility are required for building a validated respirometric database.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0264496</identifier><identifier>PMID: 35239701</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Antibiotics ; ATP synthase ; Biology and Life Sciences ; Biotechnology ; Calibration ; Cell culture ; Cell Respiration - physiology ; Electron transfer ; Fibroblasts ; Glucose ; Health aspects ; Hospitals ; Humans ; Medical research ; Medicine and Health Sciences ; Medicine, Experimental ; Metabolic disorders ; Mitochondria ; Morphology ; Oligomycin ; Oxidative Phosphorylation ; Oxygen ; Oxygen consumption ; Oxygen Consumption - physiology ; Pediatrics ; Physical Sciences ; Physiological aspects ; Physiology ; Platforms ; Reproducibility ; Reproducibility of Results ; Residual oxygen ; Respiration ; Respirometry ; Sensors ; Titration ; Toxicology</subject><ispartof>PloS one, 2022-03, Vol.17 (3), p.e0264496-e0264496</ispartof><rights>COPYRIGHT 2022 Public Library of Science</rights><rights>2022 Zdrazilova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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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. 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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. 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| subjects | Antibiotics ATP synthase Biology and Life Sciences Biotechnology Calibration Cell culture Cell Respiration - physiology Electron transfer Fibroblasts Glucose Health aspects Hospitals Humans Medical research Medicine and Health Sciences Medicine, Experimental Metabolic disorders Mitochondria Morphology Oligomycin Oxidative Phosphorylation Oxygen Oxygen consumption Oxygen Consumption - physiology Pediatrics Physical Sciences Physiological aspects Physiology Platforms Reproducibility Reproducibility of Results Residual oxygen Respiration Respirometry Sensors Titration Toxicology |
| title | Comparable respiratory activity in attached and suspended human fibroblasts |
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