PONE-D-21-34792Comparable respiratory activity in attached and suspended human fibroblastsPLOS ONE

Dear Dr. Zdrazilova,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

While the reviewers have underlined the technical interest of your paper in research on mitochondria, it is however clear that the paper would benefit from a more precise writing, alond the lines mentioned by reviewer 3. In particular, the time range during which suspended and attached fibroblats keep similar respiration is a key issue that should be at the very least thoroughly discussed, if not experimentally verified. If you authors have any experiments in store along this line it would be a great idea to include them in a revision.

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Zdrazilova et al. contains a valuable bioenergetic/respirometric comparison of either attached or suspended fibroblasts. This comparison was done in two setups, the attached cells were analyzed in the Seahorse analyzer and the suspended cells in the Oroboros O2k. No differences were observed in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. Thus, both approaches may be comparatively or alternatively used.

The authors further find that respiratory values have greater standard deviations in the Seahorse instrumentation. Further, these measurements may also be limited in the number of possible additions, thereby resulting in a potentially underestimated maximal respiratory capacity due to a potential oligomycin overdose or due to a too low dose of uncoupler. On the other hand, the O2k has a limited sample number that can be analyzed in parallel and also requires around ten times more cells per measurement.

For “mitochondriologists”, i.e. bio-medical researchers interested in bioenergetic analyses, this is a valuable paper, as to my knowledge, such direct methodological comparisons hardly exist. Furthermore, the idea to further develop a unifying data handling to allow comparisons of the different approaches is most welcome.

Specific point of criticism/improvement are:

1. With respect to the methodological comparison, a table summarizing the most important differences (e.g. possible sample replicates, required cell amount, number of possible additions, detection mode, sample volume, etc…) between the approaches would be very helpful for the reader to decide which approach to use.

2. I doubt that the unifying respiratory capacity per single cell unit is a good one. First, the resulting attomol values are not really of practical use. Second, cell research typically occurs on “millions”. Thus, such a more practical number could be better/alternatively introduced. Third, when looking at the standard deviations of 30% or more (see the mentioning of R, L and E that are 37 ± 12; 6 ± 3; and 78 ± 26 amol∙s1∙x1 in reference 18), one may think of alternative/more precise parameters?

3. The authors state: “Our results suggest that ROUTINE respiration was not different in attached cells studied after overnight seeding and freshly suspended cells measured one hour after harvesting.” This comes with an important question: how long can detached solubilized cells be “safely” measured until clear respiratory deficits with respect to attached cells can be encountered? It would be wonderful if the authors could do such measurements at different time points to provide data for this. Clearly, such an information would further increase the value of this manuscript for the interested readers.

Minors:

1. At different passages in the text the authors speak about “physiological or near-physiological conditions”. In which sense? For example, the oxygen concentration in the measurement chambers is assumed to be much higher than in tissue? Thus, please specify.

2. One/two sentence/s explaining why fibroblasts were chosen for this study would be appreciated.

3. The authors find that ROX and LEAK measurements were higher in Seahorse than in the O2k and with a higher scatter. Could this be due to the detection via fluorescence that may be non-linear in this low range of change? Please comment why this could be in the discussion.

Reviewer #2: The study by Zdrazilova and co-workers addresses an important point regarding the assessment of the mitochondria-driven cell respiration in intact cells. The recent introduction of methodologies for measuring O2 consumptions, alternative to the traditional Clark-electrode-based polarography, have raised the possibilities of differences in determining the absolute oxygen consumption rates. In addition to a complete different instrumental design, the Seahorse approach carries measurements in attached cells while the electrodic measurements are conducted in suspended cells. This raises the reservation of a different bioenergetics response of the cell samples depending if they are anchored (better mimicking a physiological situation) or are freely floating in suspension. Zdrazilova and co-workers uncover this gap of knowledge comparing the respirometric parameters of human fibroblasts attained by real-time multiwall and high resolution oximetry. The results reported do not unveil significant differences between adherent and short-term suspended cells in the so called Routine and Leak respirations (after correction for mitochondria-independent respiration) but a slower oxygen consumption rate (i.e. the maximal capacity) in suspended cells that the authors explain as due to an inhibitory effect of oligomycin used at higher concentrations in the Seahorse standard protocol.

The study is well-conducted, the results are clearly presented in the Figures and table and supported by a robust statistical analysis. The recommendations presented in the supplements are also relevant and helpful in providing a standardized protocol to set reproducibility in this specific assay to extend respirometric database.

Only the following minor points are asked to replay:

1. Can the authors anticipate, if they have preliminary evidence, that the same conclusions attained in this study using primary cell cultures might be extended to cancer cell lines that are the samples more widely used in cellular biochemistry investigations?

2. The oxygen consumption rate under uncoupled condition is taken as a measure of the maximal capacity of the respiratory chain that is untied by the controlling protonmotive force. However, anionic respiratory substrates (such as pyruvate or glutamate) enter into the mitochondria by transporters utilizing the mt-DpH gradient. Therefore, under conditions dissipating the delta-mu-H+ the respiratory capacity is, in any case, limited by the respiratory substrate availability. It is also possible that under this condition the cell switches to substrates whose entry into mitochondria is not delta-mu-H+-driven (such as fatty acids). Can the authors comment on this and suggest an alternative term in place of the somewhat confusing “maximal capacity”?

Reviewer #3: In this study, Lucie Zdrazilova and coworkers have compared the oxygen consumption of human dermal fibroblast cell lines cultured in monolayers, either directly in their adherent form, or in suspension after trypsinization. To address this question, they used two instruments, the Seahorse XF Analyser (Agilent, US) which is designed to measure respiration on adherent cells and the Oroboros O2k (Oroboros Instruments, Austria) which is designed for suspended cells. Their main conclusion is that short-term suspension of fibroblasts does not affect respiratory activity and coupling control.

This article needs some proofreading to improve the English. As an example, the last sentence of the abstract is particularly muddled: “Consistent results obtained with different platforms provide a test for reproducibility and allow for building an extended respirometric database” which could be rewritten as “Obtaining consistent results across different platforms is a good measure of reproducibility and could help build a comprehensive respirometric database.”

The introduction and the discussion sections are insufficiently developed. The introduction is particularly short. In contrast, the materials and methods and results sections are better developed and provide sufficient information. This paper is methodological and the study is centered on the use of the new Seahorse apparatus. However the experiments are in my opinion not very original and do not present an important breakthrough. While the results do not show significant differences between measurements in adherent or in suspended conditions, the authors suggest that it could probably be different after longer incubation times in suspension, but show no experiments to support this hypothesis.

The introduction lacks a clear explanation of the biological interest and the real goal of this study. The bibliographic references of the introduction are particularly old and are not cited with sufficient relevance. For example, at the beginning of the introduction “After blebbing, cells undergo membrane reorganization and attain a spherical shape to prevent membrane loss [3].” This phrase alone, taken out of context, is very hard to fully understand and in my opinion does not accurately convey the message of reference 3.

The works cited in the discussion are also not clearly exposed.

At the beginning of the discussion, for example: “Suspending fibroblasts causes an immediate drop of mRNA synthesis to about 20 % of controls within minutes [6]. The remaining mRNA is inactivated, such that the total cell mRNA content remains unchanged.” As I did not quite grasp the meaning of these sentences, I read the cited article. My impression is that the authors have mixed up two separate concepts. Indeed, the cited article states that the mRNAs were in fact stabilized so that the total amount of mRNA remained constant, and that they were also probably transiently inactivated to explain the drop in protein synthesis and the rapid increase of protein synthesis which occured upon reattachment to a solid substrate (prior to mRNA neosynthesis). But it is incorrect to say that the inactivation of the remaining mRNA explains “that the total cell mRNA content remains unchanged”.

The discussion does not give a strong argument to justify the technical and or biological interest of this study. However, this study could probably be helpful to address technical questions about the Seahorse instrument. For example, does this study prove that trypsinization induces no bias when measuring respiration on suspended cells?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Comparable respiratory activity in attached and suspended human fibroblasts

PONE-D-21-34792R1

Dear Dr. Zdrazilova,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Thierry Rabilloud

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have sufficiently satisfied the requests of this reviewer although the point relating to the different utilization of respiratory substrates by mitochondria in the presence of a decoupler has been evaded.

Reviewer #3: I appreciate the efforts of the authors to answer to my questions and to improve their article. The new version is good for publication. The authors are acknowledged for their reply to the issues raised.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Nazzareno Capitanio

Reviewer #3: No