Development of microfluidic chip for entrapping tobacco BY-2 cells

Kazunori Shimizu; Yaichi Kawakatsu; Ken-ichi Kurotani; Masahiro Kikkawa; Ryo Tabata; Daisuke Kurihara; Hiroyuki Honda; Michitaka Notaguchi

doi:10.1371/journal.pone.0266982

Peer Review History

Original SubmissionFebruary 14, 2022
23 Mar 2022 Decision Letter - Hiroshi Yabu, Editor PONE-D-22-04583Development of Microfluidic Chip for Entrapping Tobacco BY-2 CellsPLOS ONE Dear Dr. Notaguchi, 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 May 07 2022 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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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by grants from the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (18K05373 and 20H05501 to RT, 18KT0040, 18H03950, and 21H00368 to MN and 20H03273 to KK and MN), Grant-in-Aid for Scientific Research on Innovative Areas (20H05358 to DK), the Japan Science and Technology Agency PRESTO program (JPMJPR18K4 to DK), and the Canon Foundation (R17-0070 to MN)." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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 Reviewer #1: The authors prepared microfluidic device to trap BY-2 cells and to evaluate physiological activity. Basically, the work shown in the manuscript is technically sounds and give a new device to evaluate cells especially those having filament-like morphology. But some points listed below should be revised before publication. 1) The authors designed microfluidic channels as shown in Figure 2, but what is the principle of the design? In order to trap cells the channel sizes and shapes should be well-considered and it should has specific design. What is the key parameters to determine the design of microfluidic channels? 2) Basically, the end of channel is open, therefore most of cells can be flow away and did not entrapped in the channels. Why did the authors design this type of open channels? 3) Regarding photobleaching, the recovery of fluorescent was not obvious. Is it sufficient intensity to say "recover"? Furthermore, please explain how the authors detect those slight difference. 4) Figure resolution is not high enough. Please upload more clear images. Reviewer #2: This paper describes a study of trapping plant cells with a microchannel device for in-situ culture and observation. The cell filament trapped in the microchannel were shown to retain cell proliferation ability in it. Furthermore, the authors have succeeded in obtaining important information on cell-to-cell protein transport from fluorescence observations such as FRAP. Therefore, this manuscript should be published after some minor revisions, (1) Page12, Line207, the authors claim that the entrapment frequency is 22.3%. On the other hand, in Ref.28, although it is a microparticle, it is trapped more efficiently. It is necessary to describe the difference from the case of cell filaments. (2) In fig.3C More than half of the trapped filaments consists of two cells. Why is the number 2?, Is there a correlation with the length of the minor axis of the approximate minimum ellipse? (3) In fig.4D the mitotic indexes of the trapped BY-2 cell is slightly larger than that of the sorted filament. Why? (4) In the FRAP experiment, please describe the wavelength and power of the irradiation laser light. (5) Line 285, the remained rate of the filament during cultivation in microdevice is discussed. The authors claim that it is likely due to medium flow. However, the number of filament cells after 4 days increased 13.6 times (Fig. 4C), so is the effect possible? (6) In Fig. 5A, is the photobleached area part of a “single cell”? ****** 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: Yes:** Yasutaka Matsuo [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. https://doi.org/10.1371/journal.pone.0266982.r001
Revision 1
29 Mar 2022 Author Response For Reviewer #1’s comments, 1) The authors designed microfluidic channels as shown in Figure 2, but what is the principle of the design? In order to trap cells the channel sizes and shapes should be well-considered and it should has specific design. What is the key parameters to determine the design of microfluidic channels? 2) Basically, the end of channel is open, therefore most of cells can be flow away and did not entrapped in the channels. Why did the authors design this type of open channels? Response: We appreciate the reviewer’s careful reading and providing thoughtful comments to our study. For the design of the microfluidic channels, the determination of parameters and the layout of the channels were based on the design of a previous study, Kim et al (Lab Chip, 2014). The key factor in this device design was the ratio of the width of each channel. According to their fluidic simulation, a trap zone was placed between the main channel and the side channel, and the pressure difference between the two attracted cells to the trap zone. We also modified the width according to the size of the cells. The size parameters of the microfluidic channel were based on their result that the ratio of the width of the inlet and outlet of trap zone, main channel, and side channel were critical for cell trapping. 3) Regarding photobleaching, the recovery of fluorescent was not obvious. Is it sufficient intensity to say "recover"? Furthermore, please explain how the authors detect those slight difference. Response: We have replaced the paper cited in the FRAP experiment with a more appropriate one. We consider the values in previous report, Martens et al. (Plant Physiol., 2010) on fluorescence recovery using FRAP to be in a similar range. (When photobleaching reduced fluorescence intensity in intercellular FRAP to about 15% of that before bleaching, it recovered to about 25% after 20 minutes.) The fluorescence values were quantified in ImageJ from images taken with confocal laser microscopy with the same observation parameters. 4) Figure resolution is not high enough. Please upload more clear images. Response: Image resolution was obtained at the optimal value for each observation in our experimental systems. The blurred focus was due to the thickness of the PDMS resin. For Reviewer #2’s comments, (1) Page12, Line207, the authors claim that the entrapment frequency is 22.3%. On the other hand, in Ref.28, although it is a microparticle, it is trapped more efficiently. It is necessary to describe the difference from the case of cell filaments. Response: We appreciate the reviewer’s careful reading and providing some suggestions. We added sentences “The width of the trap zone was determined from the standard short axis size of BY2 cells in the 1-cell state. The efficiency to trap BY-2 cell filaments in the trap zones was smaller than previously reported for Giardia cysts, probably because of the geometry complexity of filament-forming BY-2 cells [28]” (2) In fig.3C More than half of the trapped filaments consists of two cells. Why is the number 2?, Is there a correlation with the length of the minor axis of the approximate minimum ellipse? Response: We apologize for the missing figure citation in Fig 3A, which was difficult to understand, and correct it. And the distribution of the lengths of the minor and major axes of the approximate minimum ellipse of the cell filaments extracted in Fig 3A was added as S2 Fig. As you said, there is some correlation between the length of the short axis and the number of cells in the filament. As shown in Fig. 3A and new S2 Fig, by passing the cell strainer through the cell strainer, we succeeded in collecting cell filaments with approximate minimum ellipse lengths of less than 60 µm and normally distributed with a peak at 35 µm. Filaments composed of two cells may explain much of this distribution. (3) In fig.4D the mitotic indexes of the trapped BY-2 cell is slightly larger than that of the sorted filament. Why? Response: This difference may not be significant, although the possibility cannot be ruled out that the supply of nutrients, oxygen, etc. was better than under normal culture conditions. (4) In the FRAP experiment, please describe the wavelength and power of the irradiation laser light. Response: Thank you for pointing this out. More details are added in Materials and methods and main text. (5) Line 285, the remained rate of the filament during cultivation in microdevice is discussed. The authors claim that it is likely due to medium flow. However, the number of filament cells after 4 days increased 13.6 times (Fig. 4C), so is the effect possible? Response: There was no large medium flow when fluorescence observation was being performed but it was possible during daily exchange of the liquid medium by pipetting. We haven’t observed the events during pipetting, it was not clear how the trapped filaments were actually lost. Therefore, we added sentences “Although it was not determined how the trapped cell filaments were actually lost, a large and fast flow of liquid medium during pipetting may cause removal of the trapped BY-2 cell filaments from the entrapped zones.” (6) In Fig. 5A, is the photobleached area part of a “single cell”? Response: The area enclosed by a square targeting almost one entire cell was photobleached, except for the boundary with the adjacent cell. Attachments Attachment Submitted filename: Shimizu_et_al_Rebuttal_letter.docx https://doi.org/10.1371/journal.pone.0266982.r002
31 Mar 2022 Decision Letter - Hiroshi Yabu, Editor Development of Microfluidic Chip for Entrapping Tobacco BY-2 Cells PONE-D-22-04583R1 Dear Dr. Notaguchi, 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, Hiroshi Yabu, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): The authors responded to whole queries from reviewers and I recommend to accept the manuscript to be accepted in PLoS One. https://doi.org/10.1371/journal.pone.0266982.r003
Formally Accepted
5 Apr 2022 Acceptance Letter - Hiroshi Yabu, Editor PONE-D-22-04583R1 Development of Microfluidic Chip for Entrapping Tobacco BY-2 Cells Dear Dr. Notaguchi: 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 Dr. Hiroshi Yabu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0266982.r004

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