Recent Advancements in Fish-on-Chip: A Comprehensive Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this review article the authors review the current state of “fish-on-chip” literature. The current state of the art for microfluidics in embryo culture, zebrafish imaging, drug and disease modeling, stimulation and transport, microinjection and sperm retention, and zebrafish farming. Overall, the manuscript provides a comprehensive and timely review of the current literature. However, there are several aspects that need to be addressed prior to publication. Please see the provided document for more in depth comments.

Comments for author File: Comments.pdf

Author Response

Rebuttal for reviewer #1

We would like to thank the reviewer for conducting a thorough study of our work and providing constructive feedback. Our response to his/her various comments/suggestions is as follows:

Major Comments:

• For all the figures in the manuscript only information regarding the microfluidic devices is provided. The results of the study are not included. It would be extremely helpful to the readers of the review if the “take-home” results of the studies are provided in the figures in addition to the text.

 Response: We have included visual representations of the results wherever possible. However, in some instances, the inclusion of additional figures was limited due to potential copyright costs associated with exceeding the allowance for free-use images.

• The majority of Section 5 (Refs 85 – 89) appears to be focused on the iterations of a single device / one long study. This seems to provide too much focus on a single group’s work instead of providing an overview of all the studies being done in this portion of the field.

 Response: We have added a few more recent studies in the field of Stimulation and transportation methods in section 5. Regarding Rheotaxis and electrotaxis we did not get enough of the very recent studies except that group.

• It would be extremely useful to the reader if a table was included in the manuscript summarizing the advancements in the field and what references focus on each advancement. This would make it much easier for the reader to utilize the resources that the manuscript provides.

Response:  A table summarizing the advancements in the field along with the relevant references has been included in the Introduction section to help readers easily access the key resources.

 

Minor Comments:

• There are several statements within the manuscript that warrant a citation, but nothing is referenced. Please provide references for the following sentences below:

o Introduction

• Sentence beginning on line 37

Response: The earlier lines contained the references, merged both lines for better clarity. Line 39 of the revised manuscript.

• Sentence beginning on line 43

Response: added at line 47-51 of the revised manuscript

• Sentence beginning on line 51

Response:  added at line 55 of the revised manuscript

• Sentence beginning on line 53

Response: added at line 57 of the revised manuscript

                   o Automated capture and culturing of zebrafish embryos

   Sentence beginning on line 71

Response: added at line 110 of the revised manuscript

Sentence beginning on line 73

Response: added at line 111 of the revised manuscript  

             o High-resolution and advanced imaging techniques

Sentence beginning on line 285

Response: added at line 300 of the revised manuscript

 o Applications in drug and disease states

Sentence beginning on line 539

Response: added at line 600 of the revised manuscript

Sentence beginning on line 633  

Response: added at line 738 of the revised manuscript

Sentence beginning on line 635

Response: added at line 740 of the revised manuscript

o Novel stimulation and transportation methods

Sentence beginning on line 819

Response: added at line 999 of the revised manuscript  

Sentence beginning on line 950

Response: added at line 1196 of the revised manuscript

o Development of a smart microfluidics-based fish farm for zebrafish screening

Sentence beginning on line 1291

Response: added at line 1743 of the revised manuscript  

• Please confirm that the resolution of all the figures is high enough that all text can be read (ex. Fig 21a)

Response: We have checked and confirmed that the resolution of all figures is high enough for the text to be legible. Regarding Figure 21a, it uses the same resolution as in the original publication by the author.

• Figures 1 and 2 have quite a bit of overlap in the information they are showing. It could be helpful for the flow of the manuscript to reduce these into a single figure.

Response: We have combined Figures 1 and 2 into a single figure (Figure 2) to improve the flow of the manuscript.

• If possible, could you please provide a figure to help with understanding Ref 50 that is discussed in detail in Section 2 of the manuscript?

Response: Due to the high cost of obtaining copyright licenses for the figures related to Ref. 50, we were unable to include them. However, we have provided detailed descriptions in the text to aid in understanding the relevant concepts.

• For Figure 3, please add in a panel showing the device with embryos in it.

Response: We have added it. (New figure 3)

• Figures 4 and 5 have quite a bit of overlap in information as well. Please consider combining them into a single figure
• Response: We have combined into single figure and added the result of the study as well. (new figure 4)
• Several of the sections have a “takeaway” paragraph summarizing the section at the end, while some do not (ex. Section 2). I believe that it would be extremely helpful to the reader if a summarization paragraph was provided at the end of each section.

Response: We have added 'takeaway' paragraphs at the end of each section, including Section 2, to provide a summary and enhance the reader's understanding

• In Figure 6, several variations of the device are shown. It would be extremely helpful if these different variants were labeled withing the figure.
• Response: We have labeled it (New figure 5)
• Many of the panels in the figures are not referenced in the text (ex. Figure 6A – 6I). Please reference all panels in a figure, or remove them if they are not critical enough to be discussed in the text.

Response:  We have thoroughly reviewed the manuscript and addressed the issue by referencing all panels in the figures where applicable. Panels that were not critical to the discussion have been removed.

• Please provide a more descriptive caption for Figure 8. What is the rotating dish for? How do the rotating dish and rotating capillary fit together? Etc.
• Response: A more descriptive caption for Figure 8 has been provided, including details about the purpose of the rotating dish and how it interacts with the rotating capillary.
• For Ref 62, the setup is described as high throughput. Please provide more details on how high throughput it is and how this compares to current methods.

Response: While the control algorithm for decelerating the larvae (to improve viability) reduces the overall throughput compared to traditional methods, it improves the orienting and focusing time (3.2 sec per larva versus 5.6 sec for the traditional approach). This comparison has been added and described in lines 460–465.

• If possible, please provide a figure to visually show the work from Refs 65 and 66 that are described in detail in the text.

Response: Due to the high cost of obtaining copyright licenses for the figures related to Refs. 65 and 66, we were unable to include them. However, we have provided detailed descriptions in the text to offer readers a clear understanding of the work.

• Please provide a more descriptive caption describing the device in Figure 10a and 10b.

Response: A more descriptive caption for the device in Figures 10a and 10b has been provided, and the details can be found in lines 700–704.

• In the caption for Figure 12, there are (i), (ii), (iii), and (iv) mentioned in 12a, but they are not in the figure itself. Please include them.

Response: The (i), (ii), (iii), and (iv) references have been removed from the caption. Instead, the description is now provided in sequence from top to bottom in the figure.

• The “H” in Figure 12 appears to be inconsistent with the rest of the figure (much larger and capitalized). Please correct this.

Response: We have corrected it.

• Please provide a figure caption for panel f in Figure 14.

Response:  We have given it.

• Section 5 utilizes the abbreviation “µfluidic” while the rest of the manuscript does not. Please either change this in Section 5 or use the abbreviation throughout.

Response:  We have changed it in section 5.

• There are 2 “b”s and “c”s in Figure 15. This makes it quite confusing to understand which one is being discussed. Please change the sub labeling in 15b to (i), (ii), etc.

Response: We have removed those . Just kept the discussion as b and c only. (New figure 17 & 18)

• It seems a little strange to combine microinjection techniques and sperm retention into a single section (Section 6). I would advise the authors to consider splitting this section up.

Response: Given the relatively limited amount of work in these areas, and the absence of figures due to high copyright costs in some discussions, we found it more practical to combine the topics of microinjection techniques and sperm retention into a single section, with appropriate introductory discussion. 

• If possible, please provide figures to visually show the work from Refs 96 and 97 that are described in detail in the text.

 Response: Due to the high cost of obtaining copyright licenses for the figures related to Refs. 96 and 97, we were unable to include them. However, we have provided detailed descriptions in the text to give readers a clear understanding of the work.

• The manufacturers are included in Section 7.1.2, but this is not done for the rest of the manuscript.

 Response: We have standardized the manuscript by removing the manufacturers and generalizing the content throughout, as done in Section 7.1.2.

• For Ref 100, could you please provide how many zebrafish can be cultured at once in the device shown in Figure 20?

Response: 30 at a time mentioned at line 1791 of the revised script.

• Section 7 being split into subsections is inconsistent with the rest of the manuscript. Was there any specific reason that this was done?

Response: The subsections in Section 7 were initially added to help readers easily identify different components of a single study. However, in response to the suggestion, we have removed the subsections and aligned the format with the rest of the manuscript for consistency.

• In Section 7.3, what drugs were tested?

Response: We have mentioned this in line 1834-1835

• The future perspective sections could be expanded. In addition to mentioning what advancements will be made, discuss the current challenges that still need to be addressed, what new challenges may come from these advancements, etc.

Response:  We have updated this section with more detailed analysis and study. 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Major revision

This manuscript reviews the application of Fish-on-Chip (FOC) technology in zebrafish research. With advancements in microfluidics, FOC has enhanced the precision and efficiency of zebrafish experiments, particularly in high-throughput screening and disease studies. The manuscript covers six key applications of FOC in zebrafish research and briefly discusses future directions, including its integration with other biomedical technologies. Overall, it offers valuable insights into the progress and future optimization of FOC technology in zebrafish research.

However, the manuscript still contains several issues that require careful revision and proofreading by the authors:

1、The article is divided into seven sections, each discussing the use of microfluidics and MEMS technology in various aspects of zebrafish research. While the content is rich, the logical independence between the sections makes the structure somewhat loose. It is recommended that the authors add Figure 1 to visually present the writing logic of the article, which would enhance logical coherence and improve readability.

2、There is a lack of clear logical progression between each sections. It is suggested that the authors revise the section titles and introductory paragraphs of each section during the writing process to form a more rigorous logical structure.

3、In the "Applications in drug and disease studies" section (Section 4), the authors review the application of microfluidics technology in drug and disease research, but the content does not directly relate to “microbiome research”. Therefore, the sentence mentioning "microbiome" in Lines 543-546 is confusing. The authors are advised to clarify, supply or correct this point.

4、The review of the studies cited as [50], [65], [96], and [97] in the manuscript lacks corresponding figures to support the discussion.

5、Zebrafish, as an important tool for drug screening and disease modeling, has gained considerable attention in the academic community. In this context, the authors should provide more detailed examples of zebrafish applications in drug screening and disease modeling in the "Applications in drug and disease studies" section. The focus should not only be on the use of zebrafish in microfluidic chips, but also on how microfluidic technology helps advance zebrafish research in drug and disease studies.

6、The author’s some opinions differ from the original references and concepts. It is recommended that the author re-examine the opinions of each paragraph to ensure that the statements are faithful to the original text. For example, as cited in reference [85], Peimani et al. developed a microfluidic device to quantitatively investigate rheotaxis. The opinion in this manuscript is stated in lines 957-959: “This microfluidic platform offers a precise and efficient tool for studying rheotaxis and flow sensing mechanisms in zebrafish larvae.” However, the original paper not only claims that the device is a precise and efficient tool, but more importantly, it allows for individual experiments on larval fish, eliminating the possibility of altered group behavior; it is also amenable to high-throughput investigations simply by parallelizing the screening microchannel into tens of side-by-side units. Please carefully check throughout the manuscript to see if there are similar issues.

7、Why was the subsection format used in Section 7? It is recommended that the authors maintain consistency with the format used earlier in the article.

8、Section 7 should emphasize the advantages of "a smart microfluidics-based fish farm" over the traditional Petri Dish, particularly in terms of automation and high-throughput. The current discussion is rather brief, and it is suggested that the author provide a more detailed explanation.

9、The conclusion and future outlook sections at the end of the article do not fully reflect the author's personal views and evaluations of existing research, nor do they provide an in-depth discussion on the future development and application of the technology. Please expand these sections carefully.

10、The limitations of FOC technology in practical applications should be discussed further to help readers understand its shortcomings and potential improvements.

11、Many of the images used in this manuscript are excerpts and compilations from references. The author should be aware of potential copyright issues with the papers and images. Please ensure that all images used comply with copyright regulations.

 

Author Response

We would like to thank the reviewer for conducting a thorough study of our work and providing constructive feedback. Our response to his/her various comments/suggestions is as follows:

1、The article is divided into seven sections, each discussing the use of microfluidics and MEMS technology in various aspects of zebrafish research. While the content is rich, the logical independence between the sections makes the structure somewhat loose. It is recommended that the authors add Figure 1 to visually present the writing logic of the article, which would enhance logical coherence and improve readability.

Response: We have added Figure 1 in the Introduction section to visually illustrate the logical relationships between the different sections of the manuscript, enhancing coherence and readability.

2、There is a lack of clear logical progression between each section. It is suggested that the authors revise the section titles and introductory paragraphs of each section during the writing process to form a more rigorous logical structure.

Response: We have enhanced the Introduction section to clearly describe the interconnections between different sections. Additionally, we have revised the introductory paragraphs of each section to improve logical flow and coherence.

3、In the "Applications in drug and disease studies" section (Section 4), the authors review the application of microfluidics technology in drug and disease research, but the content does not directly relate to “microbiome research”. Therefore, the sentence mentioning "microbiome" in Lines 543-546 is confusing. The authors are advised to clarify, supply or correct this point.

Response: The mention of 'microbiome' in Lines 543-546 has been removed to maintain the consistency and focus of the section.

4、The review of the studies cited as [50], [65], [96], and [97] in the manuscript lacks corresponding figures to support the discussion

Response: We have provided as much detail as possible to give readers a clear understanding. Unfortunately, obtaining the necessary copyright licenses for these figures is quite costly, which limits our ability to include them

5、Zebrafish, as an important tool for drug screening and disease modeling, has gained considerable attention in the academic community. In this context, the authors should provide more detailed examples of zebrafish applications in drug screening and disease modeling in the "Applications in drug and disease studies" section. The focus should not only be on the use of zebrafish in microfluidic chips, but also on how microfluidic technology helps advance zebrafish research in drug and disease studies.

 Response: Our review primarily focuses on the latest advancements in interconnected fields, particularly the use of novel FOC devices in zebrafish research, which has not been extensively covered in previous reviews. However, in response to the recommendation, we have incorporated additional insights into drug screening and disease modeling by highlighting key findings from relevant studies to further illuminate this area.

6、The author’s some opinions differ from the original references and concepts. It is recommended that the author re-examine the opinions of each paragraph to ensure that the statements are faithful to the original text. For example, as cited in reference [85], Peimani et al. developed a microfluidic device to quantitatively investigate rheotaxis. The opinion in this manuscript is stated in lines 957-959: “This microfluidic platform offers a precise and efficient tool for studying rheotaxis and flow sensing mechanisms in zebrafish larvae.” However, the original paper not only claims that the device is a precise and efficient tool, but more importantly, it allows for individual experiments on larval fish, eliminating the possibility of altered group behavior; it is also amenable to high-throughput investigations simply by parallelizing the screening microchannel into tens of side-by-side units. Please carefully check throughout the manuscript to see if there are similar issues. –

Response: The capability of the device for high-throughput investigations was addressed in our original manuscript in lines 954–957: 'The study highlights the need to extend the length of the channel for further investigations into rheotactic behavior at higher velocities. Additionally, the throughput of the device could be enhanced by incorporating multiple parallel channels, enabling simultaneous analysis of larger populations.' In response to the reviewer’s suggestion, we have also explicitly mentioned the ability to conduct individual investigations on larval fish (lines 1203–1204) and have carefully reviewed the manuscript to ensure all statements accurately reflect the original references.

7、Why was the subsection format used in Section 7? It is recommended that the authors maintain consistency with the format used earlier in the article.

Response: Since this section focused on a single study, we initially structured it with subsection titles to help readers easily identify different components. However, in response to the suggestion, we have removed the subsections and aligned the formatting with the rest of the manuscript for consistency.

8、Section 7 should emphasize the advantages of "a smart microfluidics-based fish farm" over the traditional Petri Dish, particularly in terms of automation and high-throughput. The current discussion is rather brief, and it is suggested that the author provide a more detailed explanation.

Response: We have expanded our discussion and provided a more detailed explanation of the advantages of 'a smart microfluidics-based fish farm' over the traditional Petri dish, particularly in terms of automation and high throughput. These additions can be found in lines 1824–1840.

9、The conclusion and future outlook sections at the end of the article do not fully reflect the author's personal views and evaluations of existing research, nor do they provide an in-depth discussion on the future development and application of the technology. Please expand these sections carefully.

Response: We have revised and expanded the conclusion and future outlook sections to provide a more comprehensive evaluation of the current research landscape. Additionally, we have included a more in-depth discussion on the future development and applications of technology.

10、The limitations of FOC technology in practical applications should be discussed further to help readers understand its shortcomings and potential improvements.

Response: We have expanded the discussion to further address the limitations of FOC technology in practical applications, highlighting its shortcomings and potential improvements.

11、Many of the images used in this manuscript are excerpts and compilations from references. The author should be aware of potential copyright issues with the papers and images. Please ensure that all images used comply with copyright regulations

Response: The copyright licenses for all figures used in the manuscript have been uploaded along with submission of the manuscript. We have also carefully addressed copyright considerations for any excerpts or compilations, ensuring that proper permissions and sources are acknowledged for each figure

Reviewer 3 Report

Comments and Suggestions for Authors

In this review, a comprehensive analysis of 18 very recent advancements in Fish-on-Chip (FOC) technologies with a focus on their applications in 19 zebrafish research, including trapping, imaging, transportation, and studies involving drug 20 screening and disease modeling, was provided. Furthermore, recent efforts in retaining progressively 21 motile zebrafish sperm, which is increasingly critical to meeting the rising demand for diverse 22 zebrafish lines, were discussed. This work provides a comprehensive review of recent advancements in Fish-on-Chip, which contains some worthwhile information for the researchers. This work can be published in the journal without revision.

Author Response

We would like to thank the reviewer for conducting a thorough study of our work and providing constructive feedback. We have made various revisions to improve the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, the authors have done a great job addressing my concerns from the first review. I only have one very minor comment (please see below). Otherwise, I believe that the manuscript is appropriate for publication.

-For Figure 4, panel f is the first panel on the second row (if reading from left to right), while panels b - e are to its right. It may be helpful to reorganize this figure to have panel f be on the far right.