An Open-Source Automated Pipeline for Quantitative Morphological Analysis of 3D-Bioprinted Cancer Cell Spheroids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript does not meet the standards of a scientific paper; its level is comparable to that of a lab report.

References are mentioned only in the introduction section.

The Methods section constitutes about 75% of the manuscript and is written in the style of a lab report, lacking consistency and scientific structure.

The first paragraph of the Results section actually describes methods rather than results.

The results are presented in only half a page and one plot.

There is no Discussion section and no comparison to previously published research.

Overall, the manuscript is of very low quality, with no significant results presented and discussed.

Author Response

We appreciate the insightful comments regarding the format and structure of the manuscript. The manuscript is formatted according to the publisher’s template to provide the reader with a straightforward protocol for performing the techniques described therein. We acknowledge that this format leads to heavy emphasis on the Methods section and a condensed Results and Conclusions section. In this manuscript, we have outlined the utility, limitations, and plans for future improvements.

 

• Critique: The manuscript does not meet the standards of a scientific paper; its level is comparable to that of a lab report.
• Response/Action: The manuscript was written to fit the template for a protocol submission.
• Critique: References are mentioned only in the introduction section.
• Response/Action: References are mostly location in the introduction section, but additional works have been cited elsewhere in the manuscript where appropriate.
• Critique: The Methods section constitutes about 75% of the manuscript and is written in the style of a lab report, lacking consistency and scientific structure.
• Response/Action: The reason the methods take up a significant portion of the manuscript is because it is the most important aspect of the protocol submission. We have written it in similar style and structure to other submissions to the journal.
• Critique: The first paragraph of the Results section actually describes methods rather than results.
• Response/Action: The first paragraph of results section demonstrates the methods in action for the reader.  The first part of the Results section also introduces the quantitative methodology (Form Factor, Solidity, Mesenchymal Morphology Score (MS)) necessary to interpret the experimental results, which is key to the pipeline's utility. The formulas for these quantitative metrics, including the MS formula (1/(Form Factor * Solidity)), were added to the results section.
• Critique: The results are presented in only half a page and one plot.
• Response/Action: The results section demonstrates that the customized image analysis pipeline and 3D culture model successfully measured a biphasic morphological response in spheroids treated with oleic acid over 10 days, serving as a proof-of-concept for the protocol’s utility.
• Critique: There is no Discussion section and no comparison to previously published research.
• Response/Action: The Conclusions section outlines the unique integration of precise bioprinting and open-source analysis for accessible and reproducible tumor models. It also includes a detailed discussion of the limitations of the current analysis workflow and specifies extensive plans for future work, such as integrating additional features, utilizing fluorescent imaging, and quantifying proteins associated with EMT to provide stronger biological interpretations.
• Critique: Overall, the manuscript is of very low quality, with no significant results presented and discussed.
• Response/Action: The manuscript now highlights the utility of the approach in high-throughput drug screening, investigation of tumor-stroma interactions, and personalized therapeutic modeling.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors provide an excellent protocol for their novel, open-sourced pipeline to enable efficient analysis of spheroid morphological characteristics. While the authors are only submitting a protocol, they also use this opportunity to address a hypothesis that is interesting to them. This allows for a “real-world” deployment of the developed pipeline, which is the primary objective of the protocol. For the most part, the authors do an excellent job writing, organizing, and producing this protocol. There are some minor concerns that I draw attention to that require addressing.

 

Minor Revisions:

1. Is there supposed to be a gap at the end of the introduction and the start of experimental design?
2. Experimental design, in particular materials, some of the materials were lacking cat #s or vendor information. For example, lines 78 and 95. There was also no information in the materials section on cell culture materials, such as flasks or other materials used for passage.
3. Unless I miss it, I don’t think ABS was defined, line 117.
4. The harvesting spheroids section seemed a bit vague, especially the “pipette tip with expanded orifice”.
5. Bioprinting section could have used more information, especially for the “user-defined printing protocol”. Also, the settings used where they standard or optimized previously?
6. The spheroid-GelMA mixing/printing section was very sparse and again, not sure what size pipette tip was used. Even a guesstimate in diameter would help. For step 8, was UV crosslinking performed after each droplet or at the end when all droplets were dispensed? After step 8, what happened to the droplets? Media was added, or placed in incubator?
7. In section 3.2 and 4, are those supposed to be sub-sub headings? Microscopy, image analysis, etc.
8. Scale bars for Figures 1 and 2 would help give readers and understanding of scale.
9. Formula for MS would also be nice, even though you define in text.
10. Figure 3 layout is odd, and I believe the legend for B is missing. It might be simpler to combine into 1 graph to compare 50, 75, and 150 as I believe the BSA for each is the same. Also, would it be possible that ANOVA (1 or 2-way depending on what comparisons you want to make) might be better for stats?
11. Sometimes protocols have a notes section with common troubleshooting issues really help the reader work through the setup and use of the pipeline.
12. A cool graphical abstract outlining the big picture process for the protocol could also be useful, but not necessary.

 

In conclusion, this protocol is very interesting, and I am personally excited to access and use it in my own laboratory. It would have also been nice to see the authors potentially mention the applicability outside of mono-cellular culture (co- or tri-cultured spheroids with CAFs, stem cells, or immune cells) or non-cancerous spheroids. However, they did outline specific limitations and their thoughts on future updates to address them. Many of the revisions are simple and are suggestions. Overall, very nice manuscript and only minor revisions are required!

Author Response

We acknowledge the constructive feedback, especially regarding the clarity and completeness of the materials and procedure sections. We have addressed these comments by improving the manuscript’s structure, adding a graphical abstract, providing greater contextual detail for the bioprinting steps, and providing greater clarity for the protocol, which is essential given the growing need for straightforward analysis of 3D cultures in laboratories.

• Critique: Is there supposed to be a gap at the end of the introduction and the start of experimental design?
• Response/Action: The gap is due to the missing graphical abstract; this will be fixed with the addition of the missing graphical abstract.
• Critique: A cool graphical abstract outlining the big picture process for the protocol could also be useful, but not necessary.
• Response/Action: The graphical abstract will be added.
• Critique: Experimental design, in particular materials, some of the materials were lacking cat #s or vendor information. There was also no information in the materials section on cell culture materials, such as flasks or other materials used for passage.
• Response/Action: Materials were edited to include cell culture passaging materials.
• Critique: Unless I miss it, I don’t think ABS (adult bovine serum) was defined.
• Response/Action: "(ABS)" was added at the very first location where adult bovine serum was mentioned.
• Critique: The harvesting spheroids section seemed a bit vague, especially the “pipette tip with expanded orifice”.
• Response/Action: Writing was adjusted to include an estimate of the diameter of the tip (approximately 2 – 3 mm in diameter).
• Critique: Bioprinting section could have used more information, especially for the “user-defined printing protocol”. Also, the settings used—were they standard or optimized previously?
• Response/Action: Clarification is needed on how the bioprinting protocol was developed, and a reference to Cellink should be added. The settings used were optimized from previous methods.
• Critique: The spheroid-GelMA mixing/printing section was very sparse and again, not sure what size pipette tip was used.
• Response/Action: Writing was adjusted to include an estimate of the diameter of the wide bore pipette tip (2 – 3 mm in diameter) used to load the mixtures into cartridges.
• Critique: For step 8, was UV crosslinking performed after each droplet or at the end when all droplets were dispensed? After step 8, what happened to the droplets? Media was added, or placed in incubator?
• Response/Action: Crosslinking is performed for each droplet that has been dispensed, using a 405 nm LED light source for 5 seconds. A new sub-section (3.1.8) was added to address media addition and incubation (This is Day 0).
• Critique: In section 3.2 and 4, are those supposed to be sub-sub headings? Microscopy, image analysis, etc.
• Response/Action: Yes, Microscopy and Image analysis should be sub headings as they fall under Post-printing Analysis. Labels "3.2.1." for Microscopy and "3.2.2" for Image analysis were added.
• Critique: Formula for MS would also be nice, even though you define in text.
• Response/Action: The Mesenchymal Morphology Score (MS) formula: $\text{Mesenchymal Morphology Score} = 1/(\text{Form Factor} \times \text{Solidity})$, was added to the Results section.
• Critique: Also, would it be possible that ANOVA (1 or 2-way depending on what comparisons you want to make) might be better for stats?
• Response/Action: While ANOVA is usually better for comparing the means of groups, the results from the ANOVA tests would be no different from the t-test results since the authors only compare two groups at a time.
• Critique: Sometimes protocols have a notes section with common troubleshooting issues that really help the reader work through the setup and use of the pipeline.
• Response/Action: Table 2, containing a list of common problems encountered during the bioprinting process and how to address them, was added.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript, “An Open-Source Automated Pipeline for Quantitative Morpho-2 logical Analysis of 3D-Bioprinted Cancer Cell Spheroids”, presents a workflow integrating 3D bioprinting with automated image analysis for tumor spheroid characterization. The approach is promising, but additional experiments and clearer presentation of key advantages are needed to enhance scientific rigor and practical relevance.

Major Comments

1. The current method appears effective mainly for spheroids with clear boundaries and regular morphology. Its performance on irregular, partially degraded, or hydrogel-embedded cell structures is unclear. Expanding the pipeline to handle heterogeneous morphologies and incorporating functional metrics (e.g., migration, invasion) would improve its robustness and utility.
2. Although the manuscript emphasizes 3D bioprinting for high-throughput spheroid production, the results do not clearly illustrate its practical advantages. Comparative data on uniformity, reproducibility, or throughput would strengthen the claims. Demonstrating synergy between bioprinting and automated analysis would further support the workflow’s relevance.

Minor Comments

1. A schematic of the image-analysis workflow would help the reader understand.
2. The Discussion could better highlight applications in drug screening and personalized medicine.

 

Author Response

We thank the reviewer for their comments, which highlighted areas where we could strengthen the claims regarding the high-throughput nature of the bioprinting process and the utility of the analysis pipeline. We have addressed these critiques by adding a graphical abstract and better framing our limitations and long-term goals to provide context for the growing need for straightforward analysis of 3D cultures in research laboratories.

• Critique: (Major Comment) The current method appears effective mainly for spheroids with clear boundaries and regular morphology. Its performance on irregular, partially degraded, or hydrogel-embedded cell structures is unclear. Expanding the pipeline to handle heterogeneous morphologies and incorporating functional metrics (e.g., migration, invasion) would improve its robustness and utility.
• Response/Action: Future work plans include integrating additional features such as texture or intensity profiles, utilizing fluorescent imaging to analyze irregularly shaped spheroids, and adding a protocol to harvest spheroids to quantify proteins associated with EMT. This expansion aims to provide more biological insight.
• Critique: (Major Comment) Although the manuscript emphasizes 3D bioprinting for high-throughput spheroid production, the results do not clearly illustrate its practical advantages. Comparative data on uniformity, reproducibility, or throughput would strengthen the claims.
• Response/Action: The manuscript noted the limitation that summarizing the output from the pipeline is currently only minimally automated (requiring manual data analysis and plotting). The long-term objective is to use image-derived morphology scores combined with robust transcriptomic or proteomic datasets to provide a medium-to-high-throughput method to evaluate treatment effects on 3D tumor models.
• Critique: (Minor Comment) A schematic of the image-analysis workflow would help the reader understand.
• Response/Action: The graphical abstract will help address this, and the authors could also refer back to the two examples provided in Figure 4 (Figure 3 in the original manuscript).
• Critique: (Minor Comment) The Discussion could better highlight applications in drug screening and personalized medicine.
• Response/Action: The manuscript highlights applications in drug screening, investigation of tumor-stroma interactions, and personalized therapeutic modeling.

Reviewer 4 Report

Comments and Suggestions for Authors

In the manuscript titled “An Open-Source Automated Pipeline for Quantitative Morphological Analysis of 3D-Bioprinted Cancer Cell Spheroids”, the authors describe a detailed and reproducible protocol that combines GelMA-based bioprinting with CellProfiler-based image analysis for assessing the morphology of 3D-cultured tumor spheroids. The procedure is clearly structured, and the use of open-source software to achieve automated quantification adds accessibility and reproducibility to the workflow. Overall, this protocol is valuable and practical, with clear relevance to researchers in cancer biology, bio-fabrication, and image-based analytics. The manuscript is well prepared and scientifically sound, only several minor revisions are required to further enhance clarity and completeness.

1. Introduction The introduction clearly explains the motivation for transitioning from 2D to 3D culture models; however, the referenced literature is somewhat dated and limited. To improve contextual depth and align with current advances in bio-fabrication and hydrogel-based 3D culture, please consider incorporating the following key references:

• S. R. Caliari; J. A. Burdick et al., Nature Methods 2016, 13, 405 – 414: “A practical guide to hydrogels for cell culture,” which comprehensively discusses 2D – 3D matrix transitions.
• T. Qiao et al., Nature Protocols 2025, 20, 1221: “Bioink design for organ-scale projection-based 3D bioprinting,” which highlights the latest GelMA-based bioink modifications.

These additions would modernize the introduction and situate the protocol within the broader landscape of contemporary 3D bioprinting research.

2. Line 137 Please define the exact range (e.g., 20 – 25 °C) to ensure reproducibility, especially since photoinitiator solubility and GelMA viscosity are temperature-sensitive.
3. Line 148 Please remove the redundant space in “5% GelMA” to maintain uniform formatting.
4. Section 3.1.7 The description of temperature adjustments (4 °C ↔ 37 °C) during GelMA handling is appropriate, but please discuss whether such fluctuations may affect cell viability, especially for non-cancerous or temperature-sensitive primary cells. If the authors intend this protocol to be broadly applicable, a brief comment on potential thermal effects and mitigation (e.g., minimizing exposure time) would enhance completeness.
5. Line 210 Please insert a space between “500” and “µm.”
6. Results and Conclusion The manuscript effectively uses spheroid circularity as a morphological metric for EMT, and the conclusion appropriately notes the limitation of relying solely on boundary-defined spheroid geometry. Since fluorescence imaging is already mentioned as a complementary approach, the authors may consider briefly discussing or referencing fluorescent staining of canonical EMT markers, such as E-cadherin, vimentin, or F-actin organization. This would help validate circularity-based EMT assessments and address the stated limitation more explicitly.

Author Response

We thank the reviewer for the detailed technical comments, particularly concerning temperature control, formatting, and contextual literature. We have updated the manuscript by incorporating the suggested references, clarifying technical parameters for reproducibility, and strengthening the discussion around cell viability and morphological validation, thereby improving the structural integrity and providing context for the growing need for straightforward analysis of 3D cultures in laboratories.

 

• Critique: Introduction The referenced literature is somewhat dated and limited. Consider incorporating Nature Methods 2016 ("A practical guide to hydrogels for cell culture") and Nature Protocols 2025 ("Bioink design for organ-scale projection-based 3D bioprinting").
• Response/Action: Both references have been included in the appropriate places in the introduction.
• Critique: Line 137 Please define the exact range (e.g., 20 – 25 °C) to ensure reproducibility, especially since photoinitiator solubility and GelMA viscosity are temperature-sensitive.
• Response/Action: The optimized settings specify the printhead temperature control to $27 \text{ °C}$ and the printbed to $20 \text{ °C}$.
• Critique: Line 148 Please remove the redundant space in “5% GelMA” to maintain uniform formatting.
• Response/Action: (Implied formatting correction).
• Critique: Section 3.1.7 Please discuss whether temperature fluctuations ($4 \text{ °C} \leftrightarrow 37 \text{ °C}$) during GelMA handling may affect cell viability, especially for non-cancerous or temperature-sensitive primary cells. A brief comment on potential thermal effects and mitigation... would enhance completeness.
• Response/Action: A comment on potential thermal effects and mitigation (e.g., minimizing exposure time) will be included in the manuscript.
• Critique: Line 210 Please insert a space between “500” and 'µm'.
• Response/Action: (Implied formatting correction). The text now reads "500 µm".
• Critique: Results and Conclusion Since fluorescence imaging is already mentioned as a complementary approach, the authors may consider briefly discussing or referencing fluorescent staining of canonical EMT markers, such as E-cadherin, vimentin, or F-actin organization.
• Response/Action: Future work was updated to state that the authors will confirm mesenchymal characteristics using immunostaining for specific mesenchymal markers (N-Cadherin, Vimentin, and Zeb1) as previously described. Additionally, the authors plan to harvest spheroids to quantify proteins associated with EMT to validate data generated from the analysis pipeline.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have responded appropriately to all comments, and the manuscript could be accepted after a minor language revision.

Author Response

Comment1: The authors have responded appropriately to all comments, and the manuscript could be accepted after a minor language revision.

Response 1: Thank you to the reviewer for taking the time to review our manuscript. The authors have reorganized the manuscript and attempted to clarify language and correct grammatical issues to improve readability of the text. We believe our corrections have accomplished the intent of the the reviewers comments.