Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The review by Cheruku et al. provides a comprehensive overview of the development and necessity of vascular organoids and vessel-on-chip models. Following a clear and concise introduction, the authors explore two main themes: vascular organoids (VO) and vessel-on-chip (VOC) models. The current approaches for generating these models are described in a well-structured manner. Overall, this is an interesting and valuable review article; however, there are several major issues that need to be addressed before it can be considered for publication.

 

Major comments:

 

1) In the Abstract and Introduction, there are several mentions of how single-cell RNA sequencing and high-resolution imaging techniques have the potential to enhance the utility of organoids and vessels-on-chips (which is true). However, these topics are not discussed later in the manuscript.

 

2) Figure 1 is highly confusing. It should be noted that Wimmer et al. (Nature 2019) reported the first generation of vascular organoids. This protocol was also published in Wimmer et al. (Nature Protocols 2020). Werschler et al. (JoVE 2023) reported the same protocol, but with accompanying movies. Nikolova et al. (bioRxiv 2022) and Romeo et al. (Nature Communications 2023) also used the same protocol. Therefore, these four articles are based on the same protocol, and it is misleading to see four different branches in a diagram intended to show the different methods for generating vascular organoids. I suggest grouping these four approaches into one derived from Wimmer et al. (Nature 2019 and Nature Protocols 2020).

 

3) In the section "Transplantation studies" (l. 232), more examples can be provided. For vascular organoids, it should be noted that Nikolova et al. (bioRxiv 2022) showed, via scRNA-seq, enhanced maturation of organoids after transplantation into the kidney capsule of mice. Examples with other organoids include Takebe et al. (Nature 2013) using pre-vascularized liver organoids, Takahashi et al. (Cell Reports 2018) using pre-vascularized pancreatic islet organoids, and Cakir et al. (Nature Methods 2019) with cortical organoids.

 

4) In section “Challenges and limitations of vascular organoids” (l. 360), the sentence would be clearer with “inability to generate hierarchical vascular tree structures in vitro” instead of “inability to generate vascular structures in vitro”.

 

5) The part on “3. Vessel-On-Chip” needs to be reviewed and completed. In Figure 3, the authors show a diagram of the conventional microfluidic architecture to generate Vessel-On-Chip (VoC), more often referred to as “Microvascular Networks On-Chip” (MVN) in the literature. In this microfluidic design, a central gelified chamber (in blue in the diagram) supports the development of vascular networks that self-organize from a single cell suspension. Nutrients and oxygen are provided via two lateral channels (in red in the diagram) where the growth media flows. While this approach is indeed the most commonly used in the field to study vascular structures, it is not mentioned in the text of this section, which is very confusing for the reader. While it is understandable that the review cannot cover all publications over the last 15 years, I suggest mentioning this approach through pioneering papers, such as those from Roger Kamm’s group (e.g., Whisler et al., Tissue Engineering Part C: Methods 2012), the Lee group (e.g., Kim et al., Lab on a Chip 2013), the Steven George / Christopher Hughes groups (e.g., Sobrino et al., Scientific Reports 2016), and the Nashimoto / Yokokawa groups (e.g., Nashimoto et al., Biomaterials 2020). The patterned approach discussed by the authors is complementary and should not be removed from the manuscript. The chosen examples are pertinent.

 

6) There is no mention of the recent efforts to combine organoids and organs-on-chips into the so-called organoids-on-chips, especially in the context of vascularization (Homan et al., Nature Methods 2019; Quintard et al., Nature Communications 2024). Including this would be a good fit for this review, either in the section “3. Vessel-On-Chip” or in sections “3.5 System integration in microfluidic devices and VoC technology” and “3.6 Key advancements in VoC technology.”

 

7) As a general remark, the review is very long. A large part of Sections 3.2, 3.3, and 3.4 has already been described in detail in other reviews and does not focus specifically on the topic of this review, which is vascular organoids and vessel-on-chip. I suggest considering rewriting these sections in a more concise manner.

 

8) In section “3.2.1.2 Thermoplastics,” the fact that these materials are non-permeable to gas, unlike PDMS, should be discussed. The relative cost of these methods should also be addressed.

 

9) I suggest splitting Table 1 into two tables: one for the materials of the chip (PDMS and thermoplastics) and one for the components within the chip (alginate, collagen, etc.).

 

10) In section “3.4 Microfluidic strategies,” many references are incorrect. Throughout the review, some references continue to be misplaced; for instance, references 70 and 132 used in line 1277 are out of context. Another example can be found in section “4.4 Vascular lung organoids (LOs),” line 1562, where the authors refer to reference 183 (Zhang et al., Lab on a Chip 2021). While this is an important review article in the field that should be cited, it is not lung-related and therefore should not be cited here. There are many more examples, such as references 57 and 58 being identical, and references 18 and 217 being identical. I invite the authors to carefully check all the references they have used to avoid such issues.

 

11) In many sections, the authors chose to write a conclusive paragraph such as “Overall, the micro-encapsulation method represents a valuable tool for studying vascular biology and developing physiologically relevant vascular models. By leveraging the self-organizing capabilities of ECs and the precise control of microenvironmental cues, this approach holds promise for advancing VoC technology and enabling applications in disease modeling, drug screening, and regenerative medicine”. These very general sentences are highly redundant in the Review and do not add any value to it.

 

12) Similarly, in many sections, general overstatements claiming the potential of different techniques to revolutionize the field, make some parts of the Review heavy to read.

 

13)  In section “3.5 System integration in microfluidic devices and VoC technology” (l. 1145), the text refers to Quintard et al. Nature Communications 2024, but omits the reference.

 

14) In section “3.6 Key advancements in VoC technology”, l. 1253, the pioneers work of Nashimoto at al. Biomaterials 2020 should be mentioned regarding vascularized tumor-on-a-chip models. Regarding immune cells incorporation, it would be worth commenting on very recent studies such as Maulana et al. Cell Stem Cell 2024, and discuss the potential of VoC to study immunotherapies for cancer.

 

15) In section “3.6 Key advancements in VoC technology”, this is unclear which studies the authors are referring to when discussing multi-organ systems (l. 1277). Pioneer work of Skardal et al. Scientific Reports 2017, as well as more recent studies such as Herland et al. Nature Biomedical Engineering 2020, and Ronaldson-Bouchard et al. Nature Biomedical Engineering 2022, should be mentioned, probably later on in the specific section “4.6 Multiorgans-on-chip (MOoC) systems with vascular components”.

 

16) In section “4.5 Vascular pancreatic organoids (POs)”, it is probably worth mentioning the work of Rambol et al. Tissue Engineering Part A 2020, in an attempt to vascularize pancreatic islets within microfluidic chips, even though they used rat islets.

 

17) Some paragraphs do not fit well in the sections they are written. For example, in section “4.6 Multiorgans-on-chip (MOoC) systems with vascular components”, l. 1741, the sentence “Commonly used materials include PDMS, glass, and biocompatible polymers such as polyethylene glycol or poly(lactic-co-glycolic acid). Advanced techniques like soft lithography, 3D printing, and microfluidic patterning are employed to create intricate microchannel networks and tissue compartments.”, is not appropriate here.

 

Minor comments:

 

18) Lack of consistency with “s” or “z” for instance in words such as “vascularize”.

 

19) There are a few typographic mistakes, for instance: l. 360, “strictures” instead of “structures”, l. 528 and l.535, “elatiomers” instead of “elastomers”.

Comments on the Quality of English Language

English language is fine.

Author Response

Review-1:

The review by Cheruku et al. provides a comprehensive overview of the development and necessity of vascular organoids and vessel-on-chip models. Following a clear and concise introduction, the authors explore two main themes: vascular organoids (VO) and vessel-on-chip (VOC) models. The current approaches for generating these models are described in a well-structured manner. Overall, this is an interesting and valuable review article; however, there are several major issues that need to be addressed before it can be considered for publication.

Response: Thank you very much for your positive and encouraging comments about our work. Hopefully we have addressed all your concerns/criticisms in the revised manuscript.

 

Major comments:

 

1) In the Abstract and Introduction, there are several mentions of how single-cell RNA sequencing and high-resolution imaging techniques have the potential to enhance the utility of organoids and vessels-on-chips (which is true). However, these topics are not discussed later in the manuscript.

 Response: Thank you for pointing out this. We have now modified the Abstract and Introduction by removing such information from the revised manuscript.

 

2) Figure 1 is highly confusing. It should be noted that Wimmer et al. (Nature 2019) reported the first generation of vascular organoids. This protocol was also published in Wimmer et al. (Nature Protocols 2020). Werschler et al. (JoVE 2023) reported the same protocol, but with accompanying movies. Nikolova et al. (bioRxiv 2022) and Romeo et al. (Nature Communications 2023) also used the same protocol. Therefore, these four articles are based on the same protocol, and it is misleading to see four different branches in a diagram intended to show the different methods for generating vascular organoids. I suggest grouping these four approaches into one derived from Wimmer et al. (Nature 2019 and Nature Protocols 2020).

Response: Thanks for your nice suggestion. We have now revised the Figure 1 and modified its corresponding figure legend (Page 3).

 

3) In the section "Transplantation studies" (l. 232), more examples can be provided. For vascular organoids, it should be noted that Nikolova et al. (bioRxiv 2022) showed, via scRNA-seq, enhanced maturation of organoids after transplantation into the kidney capsule of mice. Examples with other organoids include Takebe et al. (Nature 2013) using pre-vascularized liver organoids, Takahashi et al. (Cell Reports 2018) using pre-vascularized pancreatic islet organoids, and Cakir et al. (Nature Methods 2019) with cortical organoids.

Response: As suggested, more studies have now been discussed in this section (Page 5, last paragraph).

 

 

4) In section “Challenges and limitations of vascular organoids” (l. 360), the sentence would be clearer with “inability to generate hierarchical vascular tree structures in vitro” instead of “inability to generate vascular structures in vitro”.

 Response: This sentence has been modified as suggested (Page 8, line-364).

 

5) The part on “3. Vessel-On-Chip” needs to be reviewed and completed. In Figure 3, the authors show a diagram of the conventional microfluidic architecture to generate Vessel-On-Chip (VoC), more often referred to as “Microvascular Networks On-Chip” (MVN) in the literature. In this microfluidic design, a central gelified chamber (in blue in the diagram) supports the development of vascular networks that self-organize from a single cell suspension. Nutrients and oxygen are provided via two lateral channels (in red in the diagram) where the growth media flows. While this approach is indeed the most commonly used in the field to study vascular structures, it is not mentioned in the text of this section, which is very confusing for the reader. While it is understandable that the review cannot cover all publications over the last 15 years, I suggest mentioning this approach through pioneering papers, such as those from Roger Kamm’s group (e.g., Whisler et al., Tissue Engineering Part C: Methods 2012), the Lee group (e.g., Kim et al., Lab on a Chip 2013), the Steven George / Christopher Hughes groups (e.g., Sobrino et al., Scientific Reports 2016), and the Nashimoto / Yokokawa groups (e.g., Nashimoto et al., Biomaterials 2020). The patterned approach discussed by the authors is complementary and should not be removed from the manuscript. The chosen examples are pertinent.

Response: Thanks for your excellent suggestion. We have now discussed these studies in our revised manuscript (Page 11, line-478-483; new refs: 64-67).

 

6) There is no mention of the recent efforts to combine organoids and organs-on-chips into the so-called organoids-on-chips, especially in the context of vascularization (Homan et al., Nature Methods 2019; Quintard et al., Nature Communications 2024). Including this would be a good fit for this review, either in the section “3. Vessel-On-Chip” or in sections “3.5 System integration in microfluidic devices and VoC technology” and “3.6 Key advancements in VoC technology.”

Response: Thanks for your helpful suggestion. We have now discussed these studies in our revised manuscript (Page 25, line-891-905; new refs: 135 & 148).

 

7) As a general remark, the review is very long. A large part of Sections 3.2, 3.3, and 3.4 has already been described in detail in other reviews and does not focus specifically on the topic of this review, which is vascular organoids and vessel-on-chip. I suggest considering rewriting these sections in a more concise manner.

 Response: Thanks for this constructive suggestion. We have now restructured these three sections into two new simpler sections (new 3.2 & 3.3) in our revised manuscript (Page 12-18, line-518-727).

 

8) In section “3.2.1.2 Thermoplastics,” the fact that these materials are non-permeable to gas, unlike PDMS, should be discussed. The relative cost of these methods should also be addressed.

 Response: As suggested, we have clearly mentioned that compared to other materials PDMS has a good gas permeability (line-527, 538, as well as in table 1). Additionally, we have also added the information regarding the relative cost of each method in Table 2 if applicable.

 

9) I suggest splitting Table 1 into two tables: one for the materials of the chip (PDMS and thermoplastics) and one for the components within the chip (alginate, collagen, etc.).

 Response: We have now made it clear by including ‘Materials’ & ‘Key gel components’ as two main catalogues in Table1.

 

10) In section “3.4 Microfluidic strategies,” many references are incorrect. Throughout the review, some references continue to be misplaced; for instance, references 70 and 132 used in line 1277 are out of context. Another example can be found in section “4.4 Vascular lung organoids (LOs),” line 1562, where the authors refer to reference 183 (Zhang et al., Lab on a Chip 2021). While this is an important review article in the field that should be cited, it is not lung-related and therefore should not be cited here. There are many more examples, such as references 57 and 58 being identical, and references 18 and 217 being identical. I invite the authors to carefully check all the references they have used to avoid such issues.

Response: Sorry for mixing-up the reference due to different version of ‘EndoNote’ being used by different authors. We have now carefully checked all the references to ensure the accuracy of all the references cited in our manuscript.

 

11) In many sections, the authors chose to write a conclusive paragraph such as “Overall, the micro-encapsulation method represents a valuable tool for studying vascular biology and developing physiologically relevant vascular models. By leveraging the self-organizing capabilities of ECs and the precise control of microenvironmental cues, this approach holds promise for advancing VoC technology and enabling applications in disease modeling, drug screening, and regenerative medicine”. These very general sentences are highly redundant in the Review and do not add any value to it.

Response: Thanks for pointing out this. We have now scrutinised our text and removed some unnecessary & redundant phrases from our revised manuscript.

 

12) Similarly, in many sections, general overstatements claiming the potential of different techniques to revolutionize the field, make some parts of the Review heavy to read.

Response: As advised, we have now tone down some statements in the revised manuscript.

 

13)  In section “3.5 System integration in microfluidic devices and VoC technology” (l. 1145), the text refers to Quintard et al. Nature Communications 2024, but omits the reference.

 Response: Sorry for the omission, it has now been added in the revised manuscript (Ref-135).

 

14) In section “3.6 Key advancements in VoC technology”, l. 1253, the pioneers work of Nashimoto at al. Biomaterials 2020 should be mentioned regarding vascularized tumor-on-a-chip models. Regarding immune cells incorporation, it would be worth commenting on very recent studies such as Maulana et al. Cell Stem Cell 2024, and discuss the potential of VoC to study immunotherapies for cancer.

Response: Thanks for your helpful suggestion. These two interesting studies have now been discussed in our revised manuscript (Page 24/25, line-871-878; new refs: 67 & 146).

 

15) In section “3.6 Key advancements in VoC technology”, this is unclear which studies the authors are referring to when discussing multi-organ systems (l. 1277). Pioneer work of Skardal et al. Scientific Reports 2017, as well as more recent studies such as Herland et al. Nature Biomedical Engineering 2020, and Ronaldson-Bouchard et al. Nature Biomedical Engineering 2022, should be mentioned, probably later on in the specific section “4.6 Multiorgans-on-chip (MOoC) systems with vascular components”.

Response: As advised, these interesting studies have now been discussed in our revised manuscript (Page 34, line-1355-1360; new refs: 225-227).

 

16) In section “4.5 Vascular pancreatic organoids (POs)”, it is probably worth mentioning the work of Rambol et al. Tissue Engineering Part A 2020, in an attempt to vascularize pancreatic islets within microfluidic chips, even though they used rat islets.

Response: As suggested, this interesting study has now been discussed in our revised manuscript (Page 32, line-1239-1244; new refs: 219).

 

17) Some paragraphs do not fit well in the sections they are written. For example, in section “4.6 Multiorgans-on-chip (MOoC) systems with vascular components”, l. 1741, the sentence “Commonly used materials include PDMS, glass, and biocompatible polymers such as polyethylene glycol or poly(lactic-co-glycolic acid). Advanced techniques like soft lithography, 3D printing, and microfluidic patterning are employed to create intricate microchannel networks and tissue compartments.”, is not appropriate here.

Response: Thanks for your suggestion, such contents have now been removed from our revised manuscript.

 

Minor comments:

 

18) Lack of consistency with “s” or “z” for instance in words such as “vascularize”.

 Response: Thanks for pointing out this. We have now carefully checked our text to ensure there is no interchange using of ‘vascularize’ and ‘vascularise’ (keep using ‘vascularize’) in the revised manuscript.

 

19) There are a few typographic mistakes, for instance: l. 360, “strictures” instead of “structures”, l. 528 and l.535, “elatiomers” instead of “elastomers”.

Response: Thanks for your careful reading. Such typos have now been corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

In the manuscript titled “Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip”, the authors summarize different types of VOs and VoCs and present an extensive overview on the generation and applications of VOs and VoCs. The authors also highlight clinical and translational challenges and future perspectives around VOs and VoCs. However, the content presented in the manuscript is repetitive and needs
alteration to streamline the narrative. Additionally, it is not clear if the authors want to focus on VOs and VoCs in CVD or just VOs and VoCs in general. To benefit readers, it would help if authors could include actual published images of VOs and VoCs from literature in addition to the schematic figures provided in the manuscript.

Here is a list of the major and minor issues that need to be addressed before acceptance:

1. If the authors want to focus on CVD as the major application of VOs and VoCs in the current manuscript, the title needs to incorporate CVD.
2. Page 4, Line 154-155, grammatical error, “Within a few hours of embedding, initial cellular will infiltration into the ECM…..”
3. Sections 2.2 Functional characteristics and physiological relevance of VOs, 2.2.1
3D Architecture, 2.2.2 Transplantation studies, 2.3 VO application, 2.3.1 Infectious disease pathogenesis, and 2.3.2 Disease modelling lacks references to critical studies from literature. The authors need to refer key literature for these sections and present essential figures from them in the current manuscript, and tabulate the key findings to benefit the readers
4. Page 12 section “3.2.1 Elatiomers and thermoplastics” and section “3.2.1.1
Elatiomers”. Elatiomers?
5. Page 13, line 565-567, “Additionally, PDMS's relatively low Young's modulus may not accurately replicate the mechanical properties of native blood vessels, potentially influencing cellular responses and tissue development.” Relatively low Young’s modulus can be misleading. Remove “relatively low” from the sentence.
6. Sections “3.2 Main materials for fabricating VoC” and the subsequent subsections are missing key publications from literature. The authors need to include additional references specifically for sections “3.2.2 Hydrogels, 3.2.2.1 Alginate, 3.2.2.2 Collagen and gelatin, 3.2.2.3 Fibrin, 3.2.2.4 Synthetic polymer, 3.2.2.5 Hybrid hydrogels”.
7. In “Table 2 Key strategies for creating VoC” the authors have provided key
advantages, disadvantages and potential solutions to limitations. However, to
benefit the readers the authors should include characteristic data of the vessels fabricated by the tabulated techniques such as vessel diameter, number of vascular branches, changes in diameter variation, etc.
8. In Section 4 the authors introduce vascularized organoids and present an extensive overview of different vascularized tissue organoids. However, in the abstract and introduction the authors have specifically focused on CVD. More emphasis should be laid on vascularized organoids in CVD in section 4. The overview of different vascularized tissue organoids, in general, may benefit the readers but it doesn’t go in line with the theme of the current manuscript.
9. Page 32, line 1430 “Vascularized cardiac OoCs”. Is it OoCs or COs?
10. It is not clear if the authors want to specifically focus on VOs and VoCs in CVD or want to just focus on VOs and VoCs in general. The abstract and introduction lays emphasis on CVD. However, the manuscript title and conclusion doesn’t not lay any emphasis on CVD. If CVD is the focus than authors need to modify the title and conclusion accordingly, if not, the abstract and introduction needs modification.

Comments on the Quality of English Language

Minor editing needed.

Author Response

Review-2:

In the manuscript titled “Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip”, the authors summarize different types of VOs and VoCs and present an extensive overview on the generation and applications of VOs and VoCs. The authors also highlight clinical and translational challenges and future perspectives around VOs and VoCs. However, the content presented in the manuscript is repetitive and needs alteration to streamline the narrative. Additionally, it is not clear if the authors want to focus on VOs and VoCs in CVD or just VOs and VoCs in general. To benefit readers, it would help if authors could include actual published images of VOs and VoCs from literature in addition to the schematic figures provided in the manuscript.

Response: Thank you very much for your constructive criticisms and suggestions about our work. Hopefully we have addressed all your concerns/criticisms in the revised manuscript. As suggested, our manuscript has now been modified by mainly focusing on VOs and VoCs in general, rather than specifically focused on VOs and VoCs in CVDs. Also, we respectfully disagreed with your suggestion that actual published images of VOs and VoCs from literature should be included into our review article due to copyright issue as well as the fact it requires long time to obtain the permission from publishers or authors to use such images.

Here is a list of the major and minor issues that need to be addressed before acceptance:

1. If the authors want to focus on CVD as the major application of VOs and VoCs in the current manuscript, the title needs to incorporate CVD.

Response: As previously mentioned, we have modified our manuscript to avoid potential false impression that this paper is specifically focused on VOs and VoCs in CVD.

2. Page 4, Line 154-155, grammatical error, “Within a few hours of embedding, initial cellular will infiltration into the ECM…..”

Response: Thanks for your careful reading. Such error has now been corrected (Page 4, line-143-144).

3. Sections 2.2 Functional characteristics and physiological relevance of VOs, 2.2.1 3D Architecture, 2.2.2 Transplantation studies, 2.3 VO application, 2.3.1 Infectious disease pathogenesis, and 2.3.2 Disease modelling lacks references to critical studies from literature. The authors need to refer key literature for these sections and present essential figures from them in the current manuscript, and tabulate the key findings to benefit the readers

Response: Thanks for your helpful suggestion. Some key references have now been added, and additional interesting studies in these aspects have also been discussed in the revised manuscript (Page 5-8, line-194-354).

4. Page 12 section “3.2.1 Elatiomers and thermoplastics” and section “3.2.1.1
   Elatiomers”. Elatiomers?

Response: Thanks for your careful reading. Such typo has now been corrected in the revised manuscript (Page 12, line-519).

5. Page 13, line 565-567, “Additionally, PDMS's relatively low Young's modulus may not accurately replicate the mechanical properties of native blood vessels, potentially influencing cellular responses and tissue development.” Relatively low Young’s modulus can be misleading. Remove “relatively low” from the sentence.

Response: Thanks for pointing out this. We have now removed “relatively low” from the sentence (Page 12, line-549/550).

6. Sections “3.2 Main materials for fabricating VoC” and the subsequent subsections are missing key publications from literature. The authors need to include additional references specifically for sections “3.2.2 Hydrogels, 3.2.2.1 Alginate, 3.2.2.2 Collagen and gelatin, 3.2.2.3 Fibrin, 3.2.2.4 Synthetic polymer, 3.2.2.5 Hybrid hydrogels”.

Response: To address the comments from another review, we have now restructured these sections to significantly shorten related contents. In the revised sections, key references have now been cited and discussed in this section as well (Page 12-14, line-518-620).

7. In “Table 2 Key strategies for creating VoC” the authors have provided key advantages, disadvantages and potential solutions to limitations. However, to benefit the readers the authors should include characteristic data of the vessels fabricated by the tabulated techniques such as vessel diameter, number of vascular branches, changes in diameter variation, etc.

Response: Thanks for this constructive suggestion. Such information has now been included into the revised Table 2 if applicable.

8. In Section 4 the authors introduce vascularized organoids and present an extensive overview of different vascularized tissue organoids. However, in the abstract and introduction the authors have specifically focused on CVD. More emphasis should be laid on vascularized organoids in CVD in section 4. The overview of different vascularized tissue organoids, in general, may benefit the readers but it doesn’t go in line with the theme of the current manuscript.

Response: As previously mentioned, we have modified our manuscript to avoid potential false impression that this paper is mainly focused on VOs and VoCs in CVD.

9. Page 32, line 1430 “Vascularized cardiac OoCs”. Is it OoCs or COs?

Response: Thanks for your careful reading. Such typo has now been corrected in the revised manuscript (Page 28, line-1036).

10. It is not clear if the authors want to specifically focus on VOs and VoCs in CVD or want to just focus on VOs and VoCs in general. The abstract and introduction lays emphasis on CVD. However, the manuscript title and conclusion doesn’t not lay any emphasis on CVD. If CVD is the focus than authors need to modify the title and conclusion accordingly, if not, the abstract and introduction needs modification.

Response: As advised, we have now modified our abstract and introduction to avoid potential false impression that this paper is specifically focused on VOs and VoCs in CVDs. Instead, we wish to focus on VOs and VoCs in general in this review article.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have effectively addressed the concerns I raised during the initial review.