PONE-D-19-18862

Effects of amotosalen treatment on human platelet lysate bioactivity

PLOS ONE

Dear Dr Sigurjonsson,

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 comprehensively addresses the points raised by both referees during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Andre van Wijnen

Academic Editor

PLOS ONE

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We note that one or more of the authors are employed by a commercial company:

Platome Biotechnology, Hafnarfjörður, Iceland

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

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: Christensen and colleagues report results of an ex-vivo study of bone-marrow derived MSCs expansion and differentiation with human platelet lysates (hPL) with two different pathogen inactivation processes. Specifically, this study addresses the issue that clinical application of MSCs usually required and in vitro expansion step for clinically relevant numbers, but this is done with supplementation of basal mammalian cell culture medium with growth factors. The concept of avoiding using supplements with animal substances, hPL produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However globally, most transfusion units are not pathogen inactivation. They aimed to assess the quality of hPL produced from expired platelet concentrates with pathogen inactivation applied after platelet lysis (compared to conventional after collection), as well as its ability to support MSC proliferation and tri-lineage differentiation.

They compared results of expanding and differentiation bone marrow MSCs using hPL derived from pathogen inactivated platelet lystes (with pathogen inactivation after lysis of expired platelets) versus using hPL produced from conventional expired pathogen inactivated human platelet concentrates (with pathogen inactivation applied soon applied soon after blood donation). THEY FOUND THAT PATHOGEN INACTIVATION AFTER LYSIS HAD LOWER CONCENTRATIONS OF SOLUBLE GROWTH FACTOR AND CYTOKINES THAN THOSE WITH CONVENTIONAL PATHOGEN INACTIVATION, AND THAT IN CELL CULTURE THE MSCS PROLIFERATED AT A REDUCED BUT MORE CONSISTENT RATE THAN THE CONVENTIONAL INACTVATION. THEY FOUND THE ABILITY TO SUPPORT TRILINEAGE DIFFERENTIATION WAS COMPARABLE BETWEEN THE LYSATES.

They interpreted their results as supportive of hPL being produced from expired and untreated platelet lysates by post lysis pathogen inactivation for global hPL production methods to increase the pool of starting material and meet future demand for animal-free supplements in human cell culturing.

The writing is clean, the methods address the aims, the results clear, the table and figures well done and interpretable, but the interpretation of the results is overreaching and the lack of comparison to the standard of fetal bovine serum disappointing.

Major issues:

1. The very large ranges in the % differences in the growth factors between the two batches of PIPC and PIPL makes it hard to know what a representative ‘baths’ of these hPL really are. I believe to really make any comment on the concentrations of growth factors in this setting, and to really interpret the results, more than 2 batches must be compared. Not only are there differences between the two hPL pathogen inactivation techniques, but also between the batches of the same technique…. This is a problem for interpreting the results.

2. It is disappointing that there is not a control with the standard fetal bovine serum. Given the literature suggests that conventional pathogen inactivated hPL is as good or better than standard fetal bovine serum, it would be important to compare this method to the conventional AND post-lysate pathogen inactivation methods. This is especially important given the concentrations of selected soluble growth factors were lower in the post-lysate pathogen inactivation group, because it is important to know if these concentrations are of equivalence to the standard bovine fetal serum (as there may be a ceiling effect in the amount of growth factor needed for proliferation and differentiation, further supporting less importance on the growth factor differences and more importance on the biologic effects)

3. Along these lines, the study is missing a limitations section that should be added, and should include discussion of the important issues with changing from a non-human source to a human blood-product source need to be mentioned as the pathogen reduction techniques may not completely mitigate the risk of viral transmissions.

4. The interpretation of the results should be tempered. Given there were lower concentrations of key growth factors, lower cumulative population doublings, no comparison to standard fetal bovine serum, and no study of retention of genomic stability of avoidance of tumorigenicity, it is unclear to me whether this successfully supports long term cell proliferation.

5. Please discuss the decision to store for 3 weeks of -80 degrees. Why was this chosen? The LOS at -80 may effect results and needs to be studied going forward.

Minor issues:

1. Please spell out the growth factors in the manuscript and in the table, they cannot be assumed to be known.

Reviewer #2: Overall the study titled “effects of amotosalen treatment on human platelet lysate bioactivity” by Christensen et al. is sound and reveals interesting results. However, we believe the authors should address the following concerns:

1. All the experiments were done using fresh platelets, but authors let the platelets expire. We think that experiments using the banked expired platelets should be performed to complement current findings.

2. In every section of the results, author should provide the rationale for each of the experiments, what they did, explain the results and their inferences, as much as possible and a brief manner.

3. In equations 1 and 3, authors should “write” multiplication symbol instead of using a “dot”.

3. In every place authors should correct CO2/CO2 (should be CO2).

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6. 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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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-19-18862R1

Effects of amotosalen treatment on human platelet lysate bioactivity

PLOS ONE

Dear Dr Sigurjonsson,

Thank you for submitting your revised manuscript to PLOS ONE. Your paper was re-reviewed by the original reviewers and one of them feels you have adequately addressed their comments, but Reviewer#1 has residual concerns and recommends against acceptance of your work. Because this decision is split, we are providing you with a second opportunity to revise this study with the guidance of a second set of comments from the reviewers. 

We would appreciate receiving your revised manuscript by Mar 22 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Andre van Wijnen

Academic Editor

PLOS ONE

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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: No

Reviewer #2: Yes

**********

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: (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: 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: Christensen and colleagues have submitted a revision of their reported results of an ex-vivo study of bone-marrow derived MSCs expansion and differentiation with human platelet lysates (hPL) with two different pathogen inactivation processes. Specifically, this study addresses the issue that clinical application of MSCs usually require an in vitro expansion step for clinically relevant numbers, but this is done with supplementation of basal mammalian cell culture medium with growth factors. Addressing the concept of avoiding using supplements with animal substances, hPL produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally most transfusion units are not pathogen inactivated, so they aimed to assess the quality of hPL produced from expired platelet concentrates with pathogen inactivation applied AFTER platelet lysis (compared to conventional after collection), as well as its ability to support MSC proliferation and tri-lineage differentiation.

They compared results of expanding and differentiation of bone marrow MSCs using hPL derived from pathogen inactivated platelet lystes (with pathogen inactivation after lysis of expired platelets) versus using hPL derived from conventional expired pathogen inactivated human platelet concentrates (with pathogen inactivation applied soon applied soon after blood donation). THEY FOUND THAT PATHOGEN INACTIVATION AFTER LYSIS HAD LOWER CONCENTRATIONS OF SOLUBLE GROWTH FACTOR AND CYTOKINES THAN THOSE WITH CONVENTIONAL PATHOGEN INACTIVATION, AND THAT IN CELL CULTURE THE MSCS PROLIFERATED AT A REDUCED BUT MORE CONSISTENT RATE THAN THE CONVENTIONAL INACTVATION. THEY FOUND THE ABILITY TO SUPPORT TRILINEAGE DIFFERENTIATION WAS COMPARABLE BETWEEN THE LYSATES. They interpreted their results as supportive of hPL being produced from expired and untreated platelet lysates (by post lysis pathogen inactivation) for global hPL production methods to increase the pool of starting material and meet future demand for animal-free supplements in human cell culturing.

Unfortunately, based on my prior review, very few changes have been made in the manuscript (only a few short sentences added and a couple of words changed), and their responses do not adequately address my concerns. They have now called this study a ‘proof-of-concept’ study in the reviewer responses, (but this is mentioned only once in their discussion). I would suggest, that if this is indeed a ‘proof-of-concept’ study, this be clarified throughout the ENTIRE manuscript including adding those words to the title so this is not misleading to readers. As previously mentioned, the findings are not interpretable without more than two batches due to the very large ranges in the % differences in the growth factors between the two batches of PIPC and PIPL (not only are there differences between the two hPL pathogen inactivation techniques, but also between the batches of the same technique), and the missing bovine fetal serum comparison group (given the citations of direct comparisons performed between hPL and bovine fetal serum by this author group, there should be no reason why this cannot be performed for this study). The couple short sentences added to the manuscript without a focus on a full limitations section (as previously suggested), will not suffice given the significant limitations in current form. Given the many limitations, these should be addressed together in a true limitations section and furthermore need to include the issue raised of storage for 3 weeks at -80 especially given it was decided based on practical reasons.

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 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.

Effects of amotosalen treatment on human platelet lysate bioactivity: a proof-of-concept study

PONE-D-19-18862R2

Dear Dr. Sigurjonsson,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Andre van Wijnen

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

**********

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

**********

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

Reviewer #1: 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

**********

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

**********

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: Christensen and colleagues have submitted a revision of their reported results of an ex-vivo study of bone-marrow derived MSCs expansion and differentiation with human platelet lysates (hPL) with two different pathogen inactivation processes.

On their last revision, very few of my comments had been addressed. I appreciate that they have addressed that and have reframed the study throughout as a proof of concept study. By reframing, the issue of not being able to generalize these results beyond the two batches, and the missing bovine fetal serum comparison group are much improved. The expanded limitations section is much appreciated.

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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