Comparison of 2D, 3D In Vitro, and Ex Vivo Platforms for Modeling the Rat Small Intestine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript takes the rat small intestine as the research model to validate a 3D bioelectronic transmembrane platform, and conducts a multi-dimensional comparative analysis of this platform with the traditional 2D insert-based culture model and the ex vivo rat small intestinal tissue model. The platform integrates a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) scaffold, which can support the co-culture of rat intestinal epithelial cells (IEC-6) and rat fibroblasts (208F), and realize real-time monitoring of the electrical signals of the intestinal barrier via electrochemical impedance spectroscopy (EIS). The study tracked the barrier formation process through a 21-day culture experiment and found that the barrier resistance of the model increased in a time-dependent manner. By means of an Ethylene Glycol Tetraacetic Acid (EGTA)-induced calcium switch assay, it was verified that the 3D model and the ex vivo tissue model exhibited reversible barrier disruption after EGTA treatment, whereas the 2D culture model had limited barrier recovery capacity. The results demonstrated that this 3D platform can more faithfully recapitulate the structure and function of native small intestinal tissue with response characteristics highly similar to those of ex vivo tissue, and it can serve as a robust tool for drug screening, intestinal barrier studies and preclinical gastrointestinal research. This manuscript is rich in data and possesses high academic research value and practical application reference significance, yet appropriate revisions and improvements are still required before its publication.

1. It is suggested that the authors supplement the core differences or advantages of this 3D platform compared with existing similar 3D models in the Introduction section.
2. Please add relevant functional markers of intestinal epithelial cells to the immunofluorescence experiments.
3. It is recommended that the authors supplement the potential limitations of this 3D platform and propose future optimization directions in the Conclusion section.
4. The section numbering is inconsistent: the section labeled "3.4. Imaging" should be corrected to "2.4. Imaging".
5. It is recommended to unify the labeling positions of subfigure identifiers in Figure 3 to ensure clear and consistent placement of the labels.
6. The title of Figure 4 uses a colon and contains an underline; both issues should be corrected in accordance with standard formatting.
7. There are vertical lines on both sides of the Figure 2 image, which need to be removed and corrected.
8. The formatting of the reference section is disorganized: some references lack DOI links, the citation formats of Chinese and English literatures are inconsistent, and the proportion of recent relevant literatures in the field is relatively low. All these aspects require standardization and supplementation.
9. Some details still need attention, and the language of the manuscript needs professional English polishing.
10. It is recommended to cite more recent relevant literatures in the reference section to better reflect the latest research progress in this field. The Introduction should also be supplemented with relevant literature on bone organoids and scaffold-based culture systems, such as the studies with DOI: 10.36922/OR8294, DOI: 10.12336/biomatertransl and DOI: 10.1016/j.bioactmat.2025.02.033.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to the Authors

The manuscript presents a novel platform for in vitro modelling of the rat small intestine, based on a PEDOT: PSS scaffold that supports the co-culture of epithelial cells and fibroblasts within a previously designed electronic transmembrane device. Interestingly, this system enables the establishment of a functional barrier after 21 days in culture and is further validated using a calcium-switch assay to assess barrier disruption and recovery following EGTA exposure. Compared to a 2D model, the 3D platform exhibits barrier behaviour comparable to ex vivo conditions, thus demonstrating its ability to mimic physiological responses. Overall, the study addresses a highly relevant topic as the increasing demand for non-animal–based approaches and the development of reliable in vitro platforms for drug screening and preclinical research. The methodology is described in detail, and the results are clearly presented with appropriate figures and graphs. Only minor revisions are suggested to improve clarity and the translational relevance of the work. Please find the specific comments below.

1. Please define all acronyms at first occurrence, including iPS at line 52, and DBSA and PEGDE at lines 91 and 92.
2. Please correct typographical errors, including the missing bracket at line 174.
3. The manuscript does not describe the sterilisation procedures for either the scaffold or the device. Please include this information, as it is critical for reproducibility and biological validation of the platform.
4. Please compare the reported TEER values obtained from the 2D and 3D models with values reported in the literature and discuss whether these are physiologically relevant for intestinal barrier function to strengthen the validation of the model.
5. To enhance the readability of the Results section, I recommend subdividing it into subsections dedicated to the different characterisation approaches (e.g., imaging, EIS).
6. The schematic of the equivalent circuit model currently reported in the Supplementary Material (Figure S2) could be moved next to Figure 3 to facilitate understanding of how the barrier resistance was estimated.
7. In Section 3, I suggest adding a brief paragraph at the end discussing the limitations of the current study and identifying what aspects still require optimisation before the platform could be considered robust enough for its usability in preclinical research and possible commercialisation.
8. The Conclusions section is too brief. Please expand it to provide a more detailed summary of the study objectives, rationale, and key findings, and to outline potential future developments and applications of the platform.
9. Several references are older than 15 years (e.g., reference 6 from 2008 and reference 13 from 2009). Please update these citations with more recent and relevant literature where available.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The research article focusing on the Comparison of 2D, 3D In vitro, and Ex vivo Platforms for Modeling the Rat Small Intestine is well designed and scientifically well written. The manuscript is well suitable for the journal standards. However, few sections are the article needs to be improved for detailed information and better understanding. The authors are suggested to address the below comments:

1. The abstract needs to be improved. The current content flow is not satisfactory and is not providing with an overview of background, objective, methods, results and conclusion.
2. In the introduction, authors are suggested to provide a detailed background of any such models which were currently being used or previously researched and in what way the current research add a value.
3. Authors are suggested to include section 2.1 as materials and list all the materials, chemicals utilized in the current work along with the information for the source
4. In the methods wherever incubation is mentioned, please provide any specific temperature conditions if applicable
5. Can authors please clarify, following incubation how were the cells separated from the media?
6. Can authors please explain how TEER, Rb and resistance are comparable between 2D, 3D and ex vivo studies?
7. Can authors please strengthen the results with proper justification and supporting literature?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf