Immunocompetent High-Throughput Gut-on-Chip Model for Intestinal Microbes—Host Interaction Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Topic of Article: Immunocompetent High-Throughput Gut-on-Chip Model for Intestinal Microbes–Host Interaction Studies

 

Dear Author,

 

Thank you for submitting your manuscript on this innovative and timely topic. The study explores the development of an immunocompetent high-throughput Gut-on-Chip model, which has significant implications for understanding host-microbe interactions in intestinal health and disease. While the manuscript has potential, there are several areas where improvements can be made to enhance clarity, structure, and scientific rigor. Below are my detailed comments and suggestions:

   

1. Abstract Structure
   The abstract is not well-structured, making it challenging for readers to follow the key elements of the study. I recommend dividing it into distinct sections (e.g., Introduction, Methods, Results, and Conclusion):

   • Introduction: Briefly introduce the importance of studying host-microbe interactions and the need for advanced models like Gut-on-Chip.
   • Methods: Summarize the experimental design, including the use of the Gut-on-Chip model and microbiota-host interaction studies.
   • Results: Highlight the key findings, such as the validation of the model using specific parameters (e.g., TEER, PBMC responses).
   • Conclusion: Summarize the implications of your findings for future research or applications in intestinal health and disease.
     This structured approach will create a strong first impression and encourage readers to explore the full article.
    

2. Keyword Optimization
   The keywords section omits important terms such as "microbiota," "PBMC (peripheral blood mononuclear cells)," and "TEER (Trans Epithelial Electrical Resistance)." These terms are central to the study and would significantly increase the manuscript's discoverability and alignment with its content. Including these keywords will also enhance the visibility of your research in academic databases.

3. In-Text Citation Format
   The in-text citation format is inconsistent, with improper numbering and formatting issues in line no. 55. For example, the reference to (Ciorba 2024) appears to be incorrectly formatted for [Ciorba, M. A., (2024). Inflammatory bowel diseases 2024. Current opinion in gastroenterology, 40(4), 233-234.] or improperly numbered. If this is a typographical error, please provide clarification or justification. Proper formatting and accurate numbering of in-text citations are essential for maintaining the manuscript's professionalism and credibility.

 

4. Methodology Clarification
   In the The methodology section specially Bacteria co-culture in Organo Plate, line no. 102-107. the author mentions using a single pure colony of each strain after 48 hours of culture but also describes filtration and washing of cells three times. If the purpose of washing is to remove liquid broth remains, why is it necessary to repeat the washing process three times when a single pure colony is used? Please clarify whether there is additional information missing or provide a proper justification for this step. This clarification will ensure the reproducibility and transparency of the methodology.

 

5. Conclusion Section
   The manuscript lacks a separate conclusion section, which is a critical component of any scientific article. A dedicated conclusion section should summarize the key findings and their implications, linking them back to the research objectives. This will help readers understand how the results justify the research title and objectives.

6. Acknowledgments and Funding Information
   The manuscript does not include an acknowledgments or funding information section. It is essential to acknowledge individuals, institutions, or funding agencies that contributed to the research. Additionally, if the study was funded by a grant or organization, this information must be disclosed to ensure transparency and compliance with ethical guidelines.

Author Response

Summary

We would like to thank you for the time you invested in the review of our manuscript. Following your suggestions we have improved the overall structure for better clarity and added a Conclusion section to summarize the main findings and implications.
Please find below the detailed response to your comments.

Detailed reply

Comment 1:

The abstract is not well-structured, making it challenging for readers to follow the key elements of the study. I recommend dividing it into distinct sections (e.g., Introduction, Methods, Results, and Conclusion):

• Introduction: Briefly introduce the importance of studying host-microbe interactions and the need for advanced models like Gut-on-Chip.
• Methods: Summarize the experimental design, including the use of the Gut-on-Chip model and microbiota-host interaction studies.
• Results: Highlight the key findings, such as the validation of the model using specific parameters (e.g., TEER, PBMC responses).
• Conclusion: Summarize the implications of your findings for future research or applications in intestinal health and disease.
  This structured approach will create a strong first impression and encourage readers to explore the full article.

Response:  We revised the entire abstract following these general guidelines although we did not specifically keep the proposed sub-sectioning since this study relies on our recently published model making it difficult to distinguish between Introduction and Methods while also introducing the Objectives sentence.

Comment 2:

The keywords section omits important terms such as "microbiota," "PBMC (peripheral blood mononuclear cells)," and "TEER (Trans Epithelial Electrical Resistance)." These terms are central to the study and would significantly increase the manuscript's discoverability and alignment with its content. Including these keywords will also enhance the visibility of your research in academic databases.

Response:   we added “Microbiota” and “peripheral blood mononuclear cells” to the keywords. We consider that the proposed "TEER (Trans Epithelial Electrical Resistance)" keyword is redundant with the “Intestinal epithelial barrier” we used.

Comment 3:

The in-text citation format is inconsistent, with improper numbering and formatting issues in line no. 55. For example, the reference to (Ciorba 2024) appears to be incorrectly formatted for [Ciorba, M. A., (2024). Inflammatory bowel diseases 2024. Current opinion in gastroenterology, 40(4), 233-234.] or improperly numbered. If this is a typographical error, please provide clarification or justification. Proper formatting and accurate numbering of in-text citations are essential for maintaining the manuscript's professionalism and credibility.

Response: the reference was properly formatted (Line 58)

Comment 4:

In the the methodology section specially Bacteria co-culture in Organo Plate, line no. 102-107. the author mentions using a single pure colony of each strain after 48 hours of culture but also describes filtration and washing of cells three times. If the purpose of washing is to remove liquid broth remains, why is it necessary to repeat the washing process three times when a single pure colony is used? Please clarify whether there is additional information missing or provide a proper justification for this step. This clarification will ensure the reproducibility and transparency of the methodology.

Response: In fact, a single colony was transferred to a 5 mL liquid growth medium and then cultured overnight. We then removed (and filtered) the supernatants while the pelleted bacteria were washed twice more to ensure that no residuals of the over-night supernatants are present. This way we could distinguish the effect of the secreted molecules alone (in the supernatants) from the effect of the live bacteria co-culture.

The paragraph was modified for better clarity: “To eliminate the residuals of the supernatant, bacteria pellets were washed twice in PBS by repeating resuspension and centrifugation steps and then set to an optical density (600nm) of 1 in a MEM medium supplemented with… » (Lines 123- 125)

Comment 5:

The manuscript lacks a separate conclusion section, which is a critical component of any scientific article. A dedicated conclusion section should summarize the key findings and their implications, linking them back to the research objectives. This will help readers understand how the results justify the research title and objectives.

Response the conclusions are now improved and moved to a separate section. (Lines 356-371)

Comment 6:

The manuscript does not include an acknowledgments or funding information section. It is essential to acknowledge individuals, institutions, or funding agencies that contributed to the research. Additionally, if the study was funded by a grant or organization, this information must be disclosed to ensure transparency and compliance with ethical guidelines.

Response: the research was funded solely by Bioaster institute itself and performed by the employees of the institute that are mentioned in the authors list

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This is a relevant and promising study demonstrating microphysiological co-culture of intestinal epithelium, PBMCs, and anaerobes under reduced oxygen. The platform has potential for screening immunomodulatory effects. To enhance clarity, rigor, and utility, please address the points below.

Abstract and Keywords

• Please add a single, explicit objective sentence so the reader immediately understands what the Gut-on-Chip experiment is designed to test.
• Please make the abstract explicitly quantitative (state readouts, effect sizes/direction, timepoints, n, and significance) and indicate whether effects come from live bacteria or supernatant.
• Calibrate the closing sentence to a proof-of-concept framing (e.g., has potential for screening) rather than implying full validation for broad use.
• The abstract refers to the platform as “Gut-on-Chip,” while the keywords include “organ-on-chip.” Please standardize to one form across the abstract, keywords, and main text, preferably “gut-on-chip” unless you are naming a brand (e.g., OrganoPlate).
• The keywords are generally relevant, but they need refinement. Please replace “epithelial barrier” with “intestinal epithelial barrier,” and “anaerobe” with “anaerobic bacteria.” Align “organ-on-chip” with your use of “gut-on-chip” in the abstract. Write “CCL2 (MCP-1)” to capture common search terms.

Introduction

• Please note that the word “underline” is not correct in this context; the right term is “underlie” (or “underpin”). Also, the sentence is overly long and would read better if divided into two; we suggest shortening and restructuring for clarity.
• Ensure cytokines are written in standard form (e.g., IL-1β, TNF-α), and acronyms like live biotherapeutic products (LBPs) are consistent (LBPs, not LPBs).
• The citation “Ciorba 2024” is not consistent with the journal’s numbered citation style and does not appear in the reference list; please revise this by either adding the complete reference and assigning it the correct number or by replacing it with another relevant source already formatted according to the journal’s requirements.
• It would also strengthen the section to briefly state what is new in your model compared to previously published Gut-on-Chip systems.

Methods

• Please separate or clearly label the Caco-2/TC7 and PBMC subsections to avoid mixing conditions, and keep PBMC ethics/source details within the PBMC subsection.
• Standardize terminology: use Caco-2/TC7 consistently; write Minimum Essential Medium (MEM) when mentioned for the first time; keep supplement names in sentence case; and write 2 × 10⁴ (not 2*10⁴).
• State exactly when penicillin–streptomycin is removed before live-bacteria co-culture (include any wash steps), and clarify whether antibiotics are absent from both epithelial and PBMC compartments during co-culture. Indicate whether FBS is heat-inactivated/adjusted and justify serum-free MEM with a brief viability check.
• For PBMCs, report donor number (n), anticoagulant, and time from draw to isolation.
• Use a consistent supplier format for all reagents (supplier, city, country).
• Use standard density-gradient wording: blood layered over Ficoll-Paque Plus, then centrifuged at RT for 30 min at 400 × g, with 'no break' not 'without break'.

Results

• Use numbers rather than vague terms: report effect sizes, 95% CIs, and exact p-values.
• Show absolute TEER (with baseline) alongside % change.
• Report absolute concentrations of cytokines plus fold-change.
• Figures: overlay individual data points, label units/time points, annotate n, and standardize notation such as 2 × 10⁴).
• Clarify that the TEER decrease reflects PBMC-mediated injury rather than direct epithelial LPS exposure.

Discussion

• Ground key statements in numbers (effect sizes, exact p-values) and use “associated with” unless you show causality.
• Discuss the drop of C. scindens at 4% O₂ as a limitation that may explain weaker protection.
• Standardize names such as CXCL8 (IL-8)
• Quantify morphology (e.g., ZO-1 junction continuity; F-actin metric) rather than relying only on images.
• Clarify your novelty/throughput versus prior gut-on-chip work.
• Acknowledge model limits and close with “captures selected features”.

References

• Keep a single “References” heading; remove the duplicate.
• Add missing article numbers or page ranges where the journal uses e-IDs or pages: references 1, 3, 4, 14, 20, and 27.
• Verify/correct implausible page/article details: reference 18 shows “17(3):9966,” which appears incorrect.
• Complete incomplete citations by adding volume/issue/pages (or article number): references 29 (year only) and 34 (“49” only; needs issue and pages/article number).
• Align preprint handling: reference 19 is labeled [Preprint]. Replace it with a peer-reviewed source.
• Standardize page-range formatting with en dashes and ascending order: reference 6 should read “R67–R82”; reference 26 has been presented as 120–111 and needs ascending pages.
• Unify journal abbreviations/capitalization to NLM form across the list: references 1, 21, 23, and 36 need normalization (e.g., Cold Spring Harb Perspect Biol, SLAS Technol…. etc.).
• Add the missing source cited in text: “Ciorba 2024” appears in the manuscript but is absent from the list; include it with a proper numbered entry or replace the in-text mention to maintain consistency.

Comments on the Quality of English Language

The English is generally clear but would benefit from light editing for clarity and consistency. Please shorten long sentences, correct word choice (e.g., use “underlie,” not “underline”), and standardize terminology and notation (consistent “gut-on-chip,” cytokines as IL-1β/TNF-α).

Author Response

Summary

We would like to thank you for your thorough review of our manuscript. Besides rewriting it for a better readability and coherence, we incorporated quantitative statements throughout the main text, added a supplementary figure with absolute cytokine values and reformatted the references according to the journal’s guidelines.
Please find below the detailed response to your comments.

Detailed reply

Comment 1:

• Please add a single, explicit objective sentence so the reader immediately understands what the Gut-on-Chip experiment is designed to test.
• Please make the abstract explicitly quantitative (state readouts, effect sizes/direction, timepoints, n, and significance) and indicate whether effects come from live bacteria or supernatant.
• Calibrate the closing sentence to a proof-of-concept framing (e.g., has potential for screening) rather than implying full validation for broad use.

Response:  The explicit objective sentence was added: “The present study aimed to evaluate the feasibility of co-culturing anaerobic members of the human intestinal microbiota within this model and to assess their effects on inflammation-induced epithelial damage.” (Lines 16 -18).
We found that making the abstract explicitly quantitative in a concise way (as only 200 words are allowed in the abstract!) is difficult in light of variable results for different read-outs and for different bacteria and their supernatants. We preferred rather to focus on the main global message of the abstract while the quantitative data can be found in the main text and figures.
The closing sentence was revised as suggested: “By recapitulating some of the key aspects of intestinal inflammation, we suggest that the current Gut-on-Chip model has potential as an easy-to-use platform for screening next-generation probiotics and live biotherapeutics with homeostatic and immunomodulatory properties.” (Lines 25 – 28)

Comment 2:

The abstract refers to the platform as “Gut-on-Chip,” while the keywords include “organ-on-chip.” Please standardize to one form across the abstract, keywords, and main text, preferably “gut-on-chip” unless you are naming a brand (e.g., OrganoPlate).

Response:  Keywords provides an opportunity to use a synonym that potential readers might look for. Therefore, as we use “gut-on-chip” throughout the manuscript, we’d like to keep the “intestine-on-chip” term in the keywords. (Lines 29 - 32)

Comment 3:

The keywords are generally relevant, but they need refinement. Please replace “epithelial barrier” with “intestinal epithelial barrier,” and “anaerobe” with “anaerobic bacteria.” Align “organ-on-chip” with your use of “gut-on-chip” in the abstract. Write “CCL2 (MCP-1)” to capture common search terms.

Response: we updated the keywords to include “intestinal epithelial barrier,” “anaerobic bacteria.” CCL2 was excluded from the keywords. We’d like to keep the “Intestine-on-chip” term (a synonym of Gut-on-chip) so potential readers could find the article even in case they search for this term instead of Gut-on-chip. (Lines 29 - 32)

Comment 4:

Please note that the word “underline” is not correct in this context; the right term is “underlie” (or “underpin”). Also, the sentence is overly long and would read better if divided into two; we suggest shortening and restructuring for clarity.

Response: The sentence was revised. “Disruption of this barrier, referred to as the “leaky gut,” and the resulting inflammation contribute to the onset or progression of numerous human pathologies. These include gastrointestinal disorders such as inflammatory bowel disease, irritable bowel syndrome, and celiac disease, as well as extraintestinal conditions like rheumatoid arthritis, non-alcoholic fatty liver disease, diabetes, and Parkinson’s disease. (Lines 37 - 41)

Comment 5:

Ensure cytokines are written in standard form (e.g., IL-1β, TNF-α), and acronyms like live biotherapeutic products (LBPs) are consistent (LBPs, not LPBs).

Response: The cytokines names were harmonized and acronyms corrected along the manuscript

Comment 6:

It would also strengthen the section to briefly state what is new in your model compared to previously published Gut-on-Chip systems.

Response: the novelty of the proposed model is now better discussed in the Conclusion section: “Previously, we have set up the Gut-on-Chip inflammatory model using epithelial cell line and human PBMCs and demonstrated the homeostatic effect of probiotic supernatants. Besides enterocytes and circulating immune cells, the intestinal tissue comprises a variety of cell types, including secretory epithelial cells, intraepithelial and tissue-resident immune cells. Therefore, the current Gut-on-Chip model cannot fully replicate the entire complexity of inflammatory process in vivo. Nevertheless, it recapitulates selected key features of intestinal inflammation – epithelial barrier damage and pro-inflammatory cytokine secretion. Furthermore, in the present study we showed that some anaerobic bacteria from human intestine can be co-cultured in this model.” (lines 357-365)

Comment 7:

Please separate or clearly label the Caco-2/TC7 and PBMC subsections to avoid mixing conditions, and keep PBMC ethics/source details within the PBMC subsection.

Response The labeling was harmonized along the article and PBMC sourcing and isolation sections combined (lines 79 -94)

Comment 8:

Standardize terminology: use Caco-2/TC7 consistently; write Minimum Essential Medium (MEM) when mentioned for the first time; keep supplement names in sentence case; and write 2 × 10⁴ (not 2*10⁴).

Response: The article was revised to correct and harmonize these inconsistencies

Comment 9:

• State exactly when penicillin–streptomycin is removed before live-bacteria co-culture (include any wash steps), and clarify whether antibiotics are absent from both epithelial and PBMC compartments during co-culture. Indicate whether FBS is heat-inactivated/adjusted and justify serum-free MEM with a brief viability check.

Response:  “When indicated, 5 days after Caco-2 seeding, the medium in the opposite channel was exchanged with 10⁵ PBMCs in supplemented RPMI (without antibiotics),…” (Lines 112 – 113)
“…called here “MEM-co-culture medium”. Of note, this medium is free of antibiotics and serum to avoid interference with bacterial growth, while the addition of ITS maintains the mammalian cell viability in the absence of serum.” (Lines 127 – 130)

FBS heat-inactivation has been indicated (Lines 92-93)

• For PBMCs, report donor number (n), anticoagulant, and time from draw to isolation.

Use a consistent supplier format for all reagents (supplier, city, country).

Use standard density-gradient wording: blood layered over Ficoll-Paque Plus, then centrifuged at RT for 30 min at 400 × g, with 'no break' not 'without break'.

Response: The paragraph was revised:

“PBMCs from six human blood samples collected in lithium heparin tubes (Becton Dickinson and Co, NJ, USA) were isolated using standard procedure within 12 hours upon collection. Briefly, the whole blood was diluted 1:1 with phosphate-buffered saline (PBS), layered over Ficoll-Paque Plus (Cytiva, Marlborough, MA, USA) and centrifuged at room temperature (RT) for 30 min at 400 x g with no break. The interphase containing PBMCs was then recovered, and the cells were washed in PBS by centrifugation at 600 x g and then again at 450 x g, 10 minutes each time. PBMCs were kept in liquid nitrogen until use. On the day of experiment, PBMC were thawed, centrifuged at 450 x g for 10 minutes and set to 10⁶ cells/mL in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (HI FBS) and 1% penicillin/streptomycin (Sigma-Aldrich, Saint Louis, MO, USA)” (Lines 84 -94)

Comment 10:

Use numbers rather than vague terms: report effect sizes, 95% CIs, and exact p-values.

Response:  The relevant paragraphs were modified according to the remark:

“Intriguingly, while LPS stimulation of PBMCs provoked a 41% decrease in TEER of Caco-2 (from 144.0±25.89 to 84.83±4.22 Ω·cm²) at 24h post stimulation, co-culture with B. thetaiotaomicron or co-incubation with its supernatant completely protected the Caco-2 cells from the LPS-induced damage (Fig. 2C). On the other hand, the spent medium of C. scindens, but not bacteria themselves, demonstrated moderate mitigation of the epithelial barrier damage – 123.0±18.33 Ω·cm² compared to control medium (84.83±4.22 Ω·cm²).” (Lines 232 – 238)

“Specifically, B. thetaiotaomicron and its spent medium decreased the CCL2 concentration by 4.53 (p=7.63x10-6) and 5.76 (p=7.63x10-6) folds respectively, while only the bacteria, but not the supernatant, reduced the concentration of CCL20 by 5.85 (p=1.53x10-5) folds. Co-culture of C. scindens with the Caco-2 cells provoked even more profound decrease – 14.10 (p=7.63x10-6) folds – in the concentration of CCL20, however this tendency was reverted when only the spend medium of C. scindens was used provoking 2.3 (p=7.63x10-6) folds increase of this cytokine. Accordingly, the concentration of two pro-inflammatory cytokines IL-6 and IL-8 showed very moderate – 1.10 and 1.56 folds respectively, but still statistically significant (p=6.57x10-3 and p=8.03x10-3), increase when only the supernatant of C. scindens was added to the Lumen channel.” (Lines 260 -270)

Comment 11:

Show absolute TEER (with baseline) alongside % change.

The figures were revised, and the data is now presented as absolute TEER values (Ohm*cm2) (Fig. 1D, 2C)

Comment 12:

Report absolute concentrations of cytokines plus fold-change.

Response: Box plots showing absolute concentrations of cytokines are now presented in supplementary figure 1 (Fig. S1). We prefer to keep the original figures in the main text as they better demonstrate the effect of LPS-mediated inflammatory response (A and B) and the effect of bacteria and their supernatants (C and D) on cytokine profiles in each channel.

Comment 13:

Clarify that the TEER decrease reflects PBMC-mediated injury rather than direct epithelial LPS exposure.

Response: The introduction was modified to better clarify this point: “In this model E. coli LPS-stimulated PBMCs secrete a variety of pro-inflammatory cytokines that compromise the integrity of the epithelial barrier formed by Caco-2 cells (Lines 70-72)

Comment 14:

Figures: overlay individual data points, label units/time points, annotate n, and standardize notation such as 2 × 104).

Response: individual data points were overlayed as requested. The figures were harmonized to the best of our understanding of the remark.
Comment 15:

Ground key statements in numbers (effect sizes, exact p-values) and use “associated with” unless you show causality.

Response: The relevant paragraphs in the results section were modified to include precise data. For the Discussion section we prefer to focus on main biologically important trends rather than go into details of precise fold of change of a particular read-out. The manuscript was revised to exclude overstatement when no causality is proven.

Comment 16:

Discuss the drop of C. scindens at 4% O₂ as a limitation that may explain weaker protection.

Response: The suggested point was added to the discussion: “The rapid decline of cultivable bacteria number (~2 log CFU / day) might be one of the reasons for the ambiguous phenotype of C. scindens in the current model conditions.” (Lines 334 -336)

Comment 17:

• Standardize names such as CXCL8 (IL-8)

Response: The cytokines names were harmonized along the manuscript.

Comment 18:

Quantify morphology (e.g., ZO-1 junction continuity; F-actin metric) rather than relying only on images.

Response: we agree with this comment. In fact, we tried to quantitatively assess the morphological differences, but we realized that many more technical repeats should be done in order to obtain statistically meaningful results. Unfortunately, our institute (Bioaster) is closing at the end of this week and all lab activities were stopped 2 months ago so we have no possibility of pursuing any experiments. We still think that even only qualitative evaluation of the cell morphology that we present here is biologically meaningful and worth sharing with the scientific community.

Comment 19:

Clarify your novelty/throughput versus prior gut-on-chip work. Acknowledge model limits and close with “captures selected features”.

Response: The relevant paragraph was rewritten: “Previously, we have set up the Gut-on-Chip inflammatory model using epithelial cell line and human PBMCs and demonstrated the homeostatic effect of probiotic supernatants. Besides enterocytes and circulating immune cells, the intestinal tissue comprises a variety of cell types, including secretory epithelial cells, intraepithelial and tissue-resident immune cells. Therefore, the current Gut-on-Chip model cannot fully replicate the entire complexity of inflammatory process in vivo. Nevertheless, it recapitulates selected key features of intestinal inflammation – epithelial barrier damage and pro-inflammatory cytokine secretion. Furthermore, in the present study we showed that some anaerobic bacteria from human intestine can be co-cultured in this model” (Lines 355 -365)

 

Comment 20:

• Keep a single “References” heading; remove the duplicate.

Response: the duplication removed

• Add missing article numbers or page ranges where the journal uses e-IDs or pages: references 1, 3, 4, 14, 20, and 27.
• Verify/correct implausible page/article details: reference 18 shows “17(3):9966,” which appears incorrect.
• Complete incomplete citations by adding volume/issue/pages (or article number): references 29 (year only) and 34 (“49” only; needs issue and pages/article number).
• Align preprint handling: reference 19 is labeled [Preprint]. Replace it with a peer-reviewed source.
• Standardize page-range formatting with en dashes and ascending order: reference 6 should read “R67–R82”; reference 26 has been presented as 120–111 and needs ascending pages.
• Unify journal abbreviations/capitalization to NLM form across the list: references 1, 21, 23, and 36 need normalization (e.g., Cold Spring Harb Perspect Biol, SLAS Technol…. etc.).
• Add the missing source cited in text: “Ciorba 2024” appears in the manuscript but is absent from the list; include it with a proper numbered entry or replace the in-text mention to maintain consistency.

Response: the reference format was modified as requested

Comment 21:

The citation “Ciorba 2024” is not consistent with the journal’s numbered citation style and does not appear in the reference list; please revise this by either adding the complete reference and assigning it the correct number or by replacing it with another relevant source already formatted according to the journal’s requirements.

Response: the reference was modified to a proper format (Line 58)

Reviewer 3 Report

Comments and Suggestions for Authors

In this research paper, the authors presented an organ-on-a-chip system from the commercialized OrganoPlate to model intestinal microbes-host interactions by incorporating Caco-2 epithelial cells with PBMC and co-culturing them with several intestinal anaerobic bacteria to investigate their impact on epithelial barrier integrity. The group simulated inflammation in their system by subjecting the organ chip to LPS in stimulate inflammation-mediated epithelial damage. Please find my comments below.

 

• TEER values should be reported as Ohm*cm² instead of % TEER values since the standard practice for reporting TEER values is to report them in Ohm*cm². The reason why the TEER measurements need to be reported as Ohm*cm² is because the electrical resistance depends on both the intrinsic barrier properties AND the area of the membrane. Reporting these TEER values in Ohm*cm² allows for reproducibility and repeatability across research labs since it allows other researchers to compare across experiments, membrane sizes, and instruments. Without this normalization, the % TEER that’s been reported does not reflect true differences in barrier function.
• Caco-2 epithelial cells are well known to be lipopolysaccharide (LPS)-resistant since they express very little Toll-like receptor 4 (TLR4), which is the canonical receptor for LPS. Because of this, LPS alone rarely induces inflammatory cytokine release from Caco-2 cells. Therefore, how do we know whether the protective effect on epithelial barrier integrity from LPS stimulation is from the intestinal bacteria present in your system or from the inherent insensitivity of Caco-2 cells from LPS? The authors need to perform an experiment that decouples this. Therefore, other primary cell lines such as primary human intestinal organoids, which express higher TLR4 and are thus more sensitive to LPS, are needed to confirm whether the protective effect on barrier integrity from LPS, is in fact, from the intestinal gut microbes and not from the insensitivity of Caco-2 cells to LPS.

Author Response

Summary

We would like to thank you for the time and effort you spent reviewing our manuscript. Besides rewriting the manuscript, we have reanalyzed the TEER data and reported them as Ohm*cm² values to ensure proper normalization and reproducibility. However, some suggestions involving repeating the experiment are not possible, unfortunately, due to unavailability of the original samples and the actual closure of the lab, as well as the entire Bioaster institute in this month.
Please find below the detailed response to your comments.

Detailed reply

Comment 1: TEER values should be reported as Ohm*cm²instead of % TEER values since the standard practice for reporting TEER values is to report them in Ohm*cm². The reason why the TEER measurements need to be reported as Ohm*cm² is because the electrical resistance depends on both the intrinsic barrier properties AND the area of the membrane. Reporting these TEER values in Ohm*cm² allows for reproducibility and repeatability across research labs since it allows other researchers to compare across experiments, membrane sizes, and instruments. Without this normalization, the % TEER that’s been reported does not reflect true differences in barrier function.

Response: The figures were revised, and the data is now presented in Ohm*cm2 (Fig. 1D, 2C)

Comment 2: Caco-2 epithelial cells are well known to be lipopolysaccharide (LPS)-resistant since they express very little Toll-like receptor 4 (TLR4), which is the canonical receptor for LPS. Because of this, LPS alone rarely induces inflammatory cytokine release from Caco-2 cells. Therefore, how do we know whether the protective effect on epithelial barrier integrity from LPS stimulation is from the intestinal bacteria present in your system or from the inherent insensitivity of Caco-2 cells from LPS? The authors need to perform an experiment that decouples this. Therefore, other primary cell lines such as primary human intestinal organoids, which express higher TLR4 and are thus more sensitive to LPS, are needed to confirm whether the protective effect on barrier integrity from LPS, is in fact, from the intestinal gut microbes and not from the insensitivity of Caco-2 cells to LPS.

Response: In the present model, LPS does not directly target Caco-2 cells, but serves to stimulate PBMCs administrated in the Basal channel. No LPS is added to the Lumen channel with Caco-2 cells. PBMCs respond to LPS stimulation by producing various pro-inflammatory cytokines that eventually compromise the integrity of the epithelial barrier formed by Caco-2 cells. Thus, the effect observed on the epithelial barrier does not rely on the intrinsic sensitivity of Caco-2 cells to LPS, but rather on the communication between activated PBMCs and epithelial cells.

The introduction was modified to better explain this point: “In this model E. coli LPS-stimulated PBMCs secrete a variety of pro-inflammatory cytokines that compromise the integrity of the epithelial barrier formed by Caco-2 cells (Lines 70-72)

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the thorough and thoughtful revision. The manuscript now reads clearly, methods are reproducible, statistics are transparent, and the abstract/keywords align well with the study’s scope. Figures and legends are much clearer, the terminology is consistent, and the references conform to the journal’s style. I recommend acceptance in its current form.

Author Response

Dear reviewer, 
We'd like to thank you again for your contribution to the improvement of our manuscript.
Kind regards,

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for addressing my comments. In its present form, the revised manuscript is sufficient for publication. I have no further comments. 

Author Response

Dear reviewer, 
We'd like to thank you again for your  contribution to the improvement of our manuscript.
Kind regards,