PONE-D-20-05469

Isolation of intact extracellular vesicles from cryopreserved samples.

PLOS ONE

Dear Professor Stott,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration y two reviewers, we feel that your study 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.

Specifically, concerns were raised regarding the experimental design and whether the results support the conclusions of the manuscript. Addressing the issue of whether the decrease in particle concentration is attributed to the loss of EVs rather than to the loss of other extracellular particles is of particular concern. In addition, one reviewer had difficulties in interpreting the figures and provided suggestions for improving them. Both reviewers also requested more information on how the experiments were conducted.

If you believe you can address all of the reviewer concerns, we would appreciate receiving your revised manuscript by May 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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We look forward to receiving your revised manuscript.

Kind regards,

Colin Johnson, Ph.D.

Academic Editor

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: Partly

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

Reviewer #1: No

Reviewer #2: 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

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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

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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: Isolation of intact extracellular vesicles from cryopreserved samples

by Shannon N. Tessier , Lauren D. Bookstaver, Cindy Angpraseuth, Cleo J. Stannard, Beatriz Marques, Berent Aldikacti, Eduardo Reátegui, Mehmet Toner, and Shannon L. Stott.

This manuscript addresses the negative effects that cryopreservation may have on extracellular vesicles. The effect of two different cooling regimes, thawing regimes, storage periods, and RNA isolation kits, in addition of addition of the cryopreservation agent DMSO, on EV number, RNA yield and RNA quality was observed.

Overall the subject of the study is most interesting and the results should be useful for researchers in the EV field. I do have some concerns, however, that I think warrant a major revision of the manuscript. These comments apply in particular to the presentation of the results and the thoroughness of the Materials and Methods section.

Major concerns:

1. Figures

The figures are not all easy to interpret. For example, in Figure 2A, what is really Stored:Fresh? And what comprises one data point? Samples from the same donor? I think the presentation you use in Figure 2B should be included for all your figures, i.e., separate columns for fresh and stored samples. And it is quite unclear what the n value corresponds to. This should be described more clearly in the Materials and Methods section.

And in the manuscript text you write that the plasma-derived EV concentration of stored samples was only 38% of fresh, non-frozen samples. When I look at Figure 2C, it appears as though the particle concentration at all particle diameters is much lower than this. If the figure cannot be interpreted as such, please explain.

Additionally, you keep referring to D0 and D10 in the figure legends while these annotations are nowhere to be found (only in the supplementary figures).

You never explain to the reader what an electropherogram depicts (you mention it very briefly in the Discussion, but that is not sufficient).

In Figure 4, please chose a color other than blue. It is not that easy to distinguish it from the black line.

2. Materials and Methods

This section is generally lacking information. Please be more thorough, as suggested below:

-line 157: Add a comment that the method is described below

-line 158: Please explain the rationale for choosing 10-12 days. This amount of days seems in many respects meaningless low. In my own personal experience we normally have to store the EVs for much longer than that. I would think that holds true in many laboratories. While it certainly would have been impractical to wait for years (although I hope you stored some of your samples for a future study), you could have chosen the middle road and selected a few months.

-line 161: For how long were the samples kept in the freezing container?

-line 164: How many hours?

-line 167: How much 10% DMSO did you add?

-line 175, starting with “For ultracentrifugation,…”: Please revise the sentence; there is a grammar issue here.

-line 179: You mix verb tenses several places, including here. Stick to one tense.

-line 192: What is the rationale for choosing these two kits? Please explain here or in the Introduction.

-line 204: Again, I am having trouble deciphering the exact number of samples in your different experiments. With n=4 it is troublesome to perform a t-test. Additionally, did you check for normality?

Additional comments:

1. Check singular vs. plural verb forms, f.ex. in line 32.

2. Shouldn´t there be a space before listing the reference number in the text?

3. Line 66-73: Cumbersome language; please revise.

4. Line 109: Which are “all experimental conditions”?

5. Line 111 starting with “Further, …”: It is unclear what you mean.

6. Line 115: Specify that you mean drawing of blood.

7. Line 115: Please move “including liposomes”. As it stands, it reads that lipid bilayers are sensitive to liposomes.

8. Line 121: What do you mean by “biologics”?

9. Line 217: Please explain what particle rate signifies.

10. Line 236: This belongs in the Materials and Methods section.

11. Line 238, starting with “This is…”: This belongs in the Materials and Methods section.

12. There is no sense in reporting p-values to four decimals.

13. Line 247: Please explain the RIN value in the Material and Methods section. Not all readers will understand what it signifies.

14. Line 268: This belongs in the Materials and Methods section.

15. Line 271, starting with “Secondly, …”: A bit unclear. What do you mean by traverse in this respect?

16. Line 286: “bucket”?

17. Line 329: Please explain the issue of EV count vs. RNA yield a bit more closely.

18. Line 337: Please discuss if your results could have been affected by the rather harsh ultracentrifugation process. Ideally, you should have used an additional EV isolation method.

19. Line 357: This explanation should have been included in the Materials and Methods section. Same for line 363 (slow vs. fast).

20. Line 374: Sentence unclear. Please revise.

21. Line 387: Please explain glassy state/glass transition some more.

22. Line 397: What do you mean with “While longer storage durations are required”?

23. Line 457: The names of authors in reference 14 are all in capital letters.

Reviewer #2: Tessier et al. study the impact of a freeze thaw cycle of blood plasma on the integrity of EVs and their associated RNA cargo. Blood plasma was collected from healthy donors and stored at -80°C for 10-12 days in the presence or absence of cryoprotectant (DMSO) versus freshly processed. Stored samples were frozen directly or through means of freezing containers; and thawed at room temperature versus 37°C. EVs were separated from fresh or stored blood plasma by differential ultracentrifugation. Particle distributions were obtained by qNano measurements on EV preparations. In addition, RNA was isolated and yield was quantified. This study aims to contribute towards setting standards for blood processing for downstream EV analysis. Although of broad interest for the EV research field, there are several concerns.

1) Since the authors separate EVs from blood plasma by differential ultracentrifugation the EV preparations are contaminated with other extracellular particles including RNA containing ribonucleoprotein complexes and lipoprotein particles. qNano cannot distinguish different types of extracellular particles. Thus, from the qNano experiments it is impossible to conclude that the decrease in particle concentration is attributed to the loss of EVs rather than to the loss of other extracellular particles (or a combined effect).

2) The same holds true for the observed decrease in RNA yield. Is the decreased yield due to disruption of EVs or other extracellular particles? Did the authors measure RNA yield on total blood plasma, prior to EV separation? Would the authors observe the same losses if EV preparations from both fresh blood plasma and cryopreserved blood plasma were treated with a combination or not of RNase, protease and detergent?

3) Comment 1 and 2 also account for testing the use of freezing containers, thawing at 37°C and adding DMSO to blood plasma prior to storage.

4) From the experiments presented in the manuscript it cannot be concluded that the observed decrease in number of particles and RNA yield can be attributed to EVs. Additional experiments should be performed including:

-assessing RNA yield in total blood plasma (prior to EV separation)

-assessing RNA and particle yield after RNase and/or protease and/or detergent treatment

-performing further characterization of EV preparations by electron microscopy and protein analysis (EV and non-EV associated proteins) to assess the composition of EV preparations (cfr MISEV2018 guidelines)

5) An alternative strategy that can be implemented is by the spiking an EV reference material, as is recently increasingly reported in literature, in blood plasma prior to testing different storage and thawing conditions. If not possible to include in the current experimental design of the study, it can be discussed by the authors.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-05469R1

Isolation of intact extracellular vesicles from cryopreserved samples.

PLOS ONE

Dear Dr. Stott,

I am pleased to state that both reviewers found the revised manuscript significantly improved. One reviewer listed a few minor changes to the text which would remove the ambiguity to lines 43, 81, and 157.

Please submit your revised manuscript by Apr 23 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Colin Johnson, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (No Response)

Reviewer #2: 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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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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

**********

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: Please see attached document for my specific comments for this manuscript, I recomennd minor revision at this point

Reviewer #2: (No Response)

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: reviewer comments.docx

Isolation of intact extracellular vesicles from cryopreserved samples.

PONE-D-20-05469R2

Dear Dr. Stott,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Colin Johnson, Ph.D.

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

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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: (No Response)

**********

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

Reviewer #1: (No Response)

**********

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: (No Response)

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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: (No Response)

**********

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)

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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