A Blood Vessel Organoid Model Recapitulating Aspects of Vasculogenesis, Angiogenesis and Vessel Wall Maturation

Round 1

Reviewer 1 Report

1. The title is too ambitious and misleading. Please remove "An easy and cost effective"..... from the title.
2. This is another vascular organoid paper, with no new mechanism. For example, Peninger has already shown this. Question is what is new here. Please describe something new to the audience. 
3. Figures and data have no statistics or no quantification. 
4. It is unclear if the blood vessels are perfused.
5. Can these organoids be frozen in liquid Nitrogen for a month and regrow in culture? 
6. What is the efficiency of this technology?
7. How do we know that iPSC used (Sendai virus mediated iPSCs) are not actually tumors? 
8. Methodology, page 3, lines 99 and 110: Why use 20% CO2? Please describe.
9. Please provide some statistical information. Do we really know if this technology is reproducible? Without statistics, we should not say reproducible. This is a major limitation of this report.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, the authors describe a design study of a new blood vessel organoid model focused on vasculogenesis, angiogenesis and vessel wall maturation. Blood vessel organoid models have already been described; this model focuses on the self-organization of blood vessels, which makes it different from already published protocols. Also, the model used a reduced amount of culture supplements, making it more cost-effective and easy to use than already existing models. All these findings are novel and very interesting.

Specific comments:

1. The introduction lacks information in terms of scientific background, novelty, and societal impact, please expand.
2. The figures are displayed well; however, information such as colour descriptions and time-points are lacking in some figures. 
3. The authors focused on the analysis of the organoid by use of imaging with a very limited form of quantitative data. Especially for vessel integrity - the paper would benefit from quantitative data from mechanical testing.   
4. The authors mentioned that this organoid model does not use mice or rats for the maturation of the vessel. However, it uses chicken without mentioning why this would be better. Also, the author mentioned that use of microfluidics would be explored to eliminate the use of animals completely. This paper would benefit from data suggesting microfluidics are a promising replacement. 
5. Please provide a future perspective for this organoid model. A general perspective on human blood vessel organoids is insufficient since they already exist.
6. Please include how this system will work across different tissues and organ-specific vascular beds in the discussion section, including the recent evidence of tissue-specific vascular changes in ageing and relevant references - PMID: 33536212, PMID: 33215738 and other...

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Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Overall, the revised manuscript addressed my concerns.  

Title: "Robust" is equally an ambitious word, please remove. Please keep it simple and straightforward. 

There are minor grammatical issues, such as double verbs in a single sentence. While sentences make sense, but it is distracting.

Please make sure the references are aligned with the texts.

Author Response

Comment 1:

We agree with the reviewer and remove the word "robust“ from the title.

Comments 2:

We will carefully read the manuscript and address these issues.

Comments 3:

Thanks for your comments. The references are aligned with the texts in the revised manuscript.

Reviewer 2 Report

The authors have responded to my comments.