An image-guided microfluidic system for single-cell lineage tracking

Mahmut Aslan Kamil; Camille Fourneaux; Alperen Yilmaz; Stavrakis Stavros; Romuald Parmentier; Andras Paldi; Sandrine Gonin-Giraud; Andrew J. deMello; Olivier Gandrillon

doi:10.1371/journal.pone.0288655

Peer Review History

Original SubmissionApril 4, 2023
18 May 2023 Decision Letter - Hon Fai Chan, Editor PONE-D-23-09681An Image-Guided Microfluidic System for Single-Cell Lineage TrackingPLOS ONE Dear Dr. Gandrillon, 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. Please submit your revised manuscript by Jul 02 2023 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. 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Thank you for stating the following in your Competing Interests section: "NO" Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 5. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. Additional Editor Comments: Changes required for acceptance: Please address the reviewers' comments by providing detailed description of the processes of cell separation and cell extraction for sequencing. In addition, please correct the typos found in the manuscript. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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 ****** 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: In this paper, the authors have developed a multilayer microfluidic device and an experimental workflow for tracking non-adherent cell divisions at the single-cell level. The microfluidic platform could trap single-cells in eight independently controlled proliferation chambers, isolate sister cells after division and extract them for downstream analysis. They demonstrated that the platform could track cells over at least two generations. In comparison with related reports, the design is original and novel and the single-cell extraction volume is down to 500 nL. However, there are typos in the context need to be corrected like on page 3, line 42 a period is missing and so on. Reviewer #2: This paper present a setup of microfluidic system for monitoring cell division and isolation of sister cells for post process such as single cell sequencing. The technique looks fine in general. Some revisions or clarifications are needed before publication for better reproducibility. 1. After division, the sister cells are close to each other even after dissociation. The author mentioned the sister cells were to separate into two different chambers. Please give detailed operation process on how to move one cell into another chamber while keep the other cell stay, as demonstrated in Fig. 3. Wouldn’t both of the cells move with the flow to new places? 2. Single cells were extracted with capillary glass for sequencing. How was the capillary glass inserted into the well? Through PDMS slab or through any hole punched in PDMS? I don’t find description on this part. Directly plugging a capillary glass through PDMS sounds quite difficult, especially into a micron sized well precisely. However, if a hole was pre-punched, wouldn’t the fluid flow out due to the driven pressure? Please describe in details for clarification. ****** 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. 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/. 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Revision 1
27 Jun 2023 Author Response Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Done 2. Thank you for stating the following financial disclosure: "This work was supported by funding from the French agency ANR (SinCity; ANR-17-CE12-0031)." Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Done 3. Thank you for stating the following in your Competing Interests section: "NO" Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. Done 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Done 5. Please include your full ethics statement in the 'Methods' section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. Not applicable (no human subjects involved). Review Comments to the Author Reviewer #1: In this paper, the authors have developed a multilayer microfluidic device and an experimental workflow for tracking non-adherent cell divisions at the single-cell level. The microfluidic platform could trap single-cells in eight independently controlled proliferation chambers, isolate sister cells after division and extract them for downstream analysis. They demonstrated that the platform could track cells over at least two generations. In comparison with related reports, the design is original and novel and the single-cell extraction volume is down to 500 nL. However, there are typos in the context need to be corrected like on page 3, line 42 a period is missing and so on. This typo has been corrected. Reviewer #2: This paper present a setup of microfluidic system for monitoring cell division and isolation of sister cells for post process such as single cell sequencing. The technique looks fine in general. Some revisions or clarifications are needed before publication for better reproducibility. 1. After division, the sister cells are close to each other even after dissociation. The author mentioned the sister cells were to separate into two different chambers. Please give detailed operation process on how to move one cell into another chamber while keep the other cell stay, as demonstrated in Fig. 3. Wouldn't both of the cells move with the flow to new places? To answer this comment the following modifications were made in the manuscript: The following statement was added line 261-274: The sister cells, resulting from the first division of the mother cell, were separated using Accutase as described above and were moved towards the separation zone which incorporates two control valves (Fig 1B). The fluid flow was precisely controlled with the pressure pump by applying a pressure of approximately 10 mbars. Accordingly, sister cells moved into a long serpentine channel that connects cell chambers to the separation area in a controlled manner. When the cells were flowing through the serpentine channel to the separation area a sufficient cell-to-cell distance or spacing (a few hundreds of microns) always occurred. In the separation area, actuation of one of the control valves ensured that one of the cells was driven towards relocation area (or extraction area), while the other cell remained trapped (S1 video). The feedback channel allowed relocation of sister cells after division from the separation zone into the cell trapping chambers. Sister cells separated after division circulated through the feedback channel upon actuation of the control valves and fine control of flow pressure, and were subsequently placed in individual trapping chambers (Fig 3). We also added supplementary video 1 showing sister separation in the separation area for relocation or extraction. The following statement was added line 570: S1 : Video of sister cells exiting the chambers area and moving toward the separation area. 2. Single cells were extracted with capillary glass for sequencing. How was the capillary glass inserted into the well? Through PDMS slab or through any hole punched in PDMS? I don't find description on this part. Directly plugging a capillary glass through PDMS sounds quite difficult, especially into a micron sized well precisely. However, if a hole was pre-punched, wouldn't the fluid flow out due to the driven pressure? Please describe in details for clarification. To answer this comment the following modifications were made in the manuscript: The selection paragraph was rewritten as follows (line 290-306): Each selected cell was delivered to the pre-punched extraction well by applying 10 mbar of pressure from the medium inlet, resulting in a cellular velocity of 10 µm/s, with fluorescence imaging being used to track single-cells after their delivery into the extraction well. Next, single-cells were extracted from the device using a thin graduated capillary tube (Fig 4A). The glass capillary tube was inserted into the well to extract the cell via capillarity. The extraction area includes two independently addressable, 1 mm diameter and 3 mm depth open wells for the collection of sister cells (Fig 1C). Cells are led to the pre-punched extraction wells, then, the capillary glass is plugged in the well. Since the tip diameter of the capillary glass is smaller than the well diameter, the capillary glass can be easily introduced into the well. As aforementioned, the driven pressure is very low (~10 mbars) which results in a cell velocity of 10 µm/s allowing an easy tracking of the cell inside the extraction well. After a single cell was driven inside the well, fluid flow was stopped during the extraction process and the single cell was collected via the capillary flow. Therefore, the fluid, included the single cell, did not flow out of the well. Additionally, the well has a reservoir volume of 2.5 µL significantly larger than the extraction volume and thus the fluid containing a single cell would not flow out during the extraction process. Attachments Attachment Submitted filename: response_to_reviewers_3.docx https://doi.org/10.1371/journal.pone.0288655.r002
3 Jul 2023 Decision Letter - Hon Fai Chan, Editor An Image-Guided Microfluidic System for Single-Cell Lineage Tracking PONE-D-23-09681R1 Dear Dr. Gandrillon, 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. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Hon Fai Chan, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): The manuscript is now acceptable for publication. 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 ******** 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 ****** 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 ****** 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: (No Response) Reviewer #2: In the revised version, the authors have addressed all my concerns. I think it is good for publication. ****** 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 ******** https://doi.org/10.1371/journal.pone.0288655.r003
Formally Accepted
21 Jul 2023 Acceptance Letter - Hon Fai Chan, Editor PONE-D-23-09681R1 An Image-Guided Microfluidic System for Single-Cell Lineage Tracking Dear Dr. Gandrillon: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Hon Fai Chan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0288655.r004

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