Breakthroughs and Applications of Organ-on-a-Chip Technology

Round 1

Reviewer 1 Report

This review compiles information on lung-on-a-chip, heart-on-a-chip, kidney-on-a-chip, liver-on-a-chip, gut-on-a-chip, and skin-on-a-chip devices.  The review also includes a cursory review of machine learning to support data acquisition and analysis from organ on a chip devices.   The overview of companies working globally in this space was particularly useful.   Overall, the manuscript is written in an accessible manner for individuals not familiar with the OOAC field, is well organized, and fairly well written.  The figures support the manuscript but could use some further development.  The tables are highly informative. 

Addressing the following items will improve the manuscript:

• It appears that a number of the author’s notes to themselves while writing remain in the manuscript. For example, on line 373, the text reads, “human pluripotent stem cells can be a gamechanger (in what), allowing for…”  I do agree that greater depth and detail would strengthen this portion of the manuscript. 
• There were also items tagged as (ref) and a few typos.
• Section 6: Challenges and Future Perspective and Section 7: Conclusions were both weakly developed. Topics were mentioned without sufficient context and supporting details.  The organization was also not intuitive.  For example, the discussion on sensor integration into machine learning that is well contextualized in the relevant physiology would be highly meaningful and useful for the readers.

In summary, this work compiles information on a wide variety of OOACs; it is a valuable review article for the field.  However, I recommend edits to improve the manuscript as noted above. 

Author Response

Addressing the following items will improve the manuscript:
“It appears that a number of the author’s notes to themselves while writing remain in the manuscript. For example, on line 373, the text reads, “human pluripotent stem cells can be a gamechanger (in what), allowing for…”  I do agree that greater depth and detail would strengthen this portion of the manuscript.” 
Needful has been done.

“There were also items tagged as (ref) and a few typos.”
These concerns have been taken care in the revised manuscript.

“Section 6: Challenges and Future Perspective and Section 7: Conclusions were both weakly developed. Topics were mentioned without sufficient context and supporting details.  The organization was also not intuitive.  For example, the discussion on sensor integration into machine learning that is well contextualized in the relevant physiology would be highly meaningful and useful for the readers.”
We agree with the reviewer and would like to thank the reviewer for taking time to read the manuscript and proving suggestions. We have incorporated all the points, including re-writing portions of the Sections 6 & 7.

Reviewer 2 Report

Koyilot et al. presented a review of breakthroughs and applications of organ-on-a-chip technology. The field of Organ-on-a-chip is rapidly growing with new technological advancements to address the critical need for modelling human diseases and testing drugs rather than using live animals. Therefore, this review is valuable in terms of its target to highlight important steps and applications within the field of organ-on-a-chip. The review is well-organized. Table 2 is very useful to give a comprehensive summary of important organ-on-chip platforms. Overall, the manuscript is well-written.  I have the following specific comments.

*More discussion on the existing commercial OOAC platforms may be included. 

*Future perspective may be improved to give insight for the future of OOAC platforms.  

Author Response

*More discussion on the existing commercial OOAC platforms may be included. 

*Future perspective may be improved to give insight for the future of OOAC platforms.  

We would like to thank the reviewer for taking out time to read out Manuscript and provide suggestions for improvement. We have updated the text to include the suggested changes.

We took a lot of effort to put together the current improvements and advancements in OOAC technology, including identifying the commercial efforts in this space and presented it as Table 2. The text leading to the table has been separated to identify major governmental efforts in this regard as well.

We have also expanded and re-written portions of the future perspectives to include anticipated changes in the near future.

Reviewer 3 Report

The authors have provided a solid review for the Organ-on-a-Chip applications. The topic is interesting and the manuscript is well written. Also, good informative tables are provided. But I have some concerns and here I provide a few comments for the authors:

• Please mention what highlights this review, when other good reviews in this field are already available. For example, see DOI: 1038/s41576-022-00466-9.
• Important recent works such as DOIS: 10.1038/s41467-022-29562-4, 1038/s41576-022-00466-9, and 10.1039/D2LC00069E are missing.
• The tables play important roles in this manuscript; however, a table/tables comparing the advantages/disadvantages of the similar organ-on-a-chip devices is/are missing.
• I believe Figure 2 need to be modified. Does it show the different steps in fabricating and testing the chip? If so, the last part (organ on a chip) is not actually the last section, but it is composed of the whole chart. You may modify the chip such that it depicts the output would be a chip, but now it is not showing like that. Also, shear stress is not necessarily involved in all organ on a chip experiment.

All in all, the work is promising and if the authors choose to answer the comments, I would be happy to re-read it.

Author Response

•    Please mention what highlights this review, when other good reviews in this field are already available. For example, see DOI: 1038/s41576-022-00466-9.

This is a rapidly evolving field where new breakthroughs are happening constantly and it warrants a constant update of the literature review to facilitate new researchers and update those interested in the field. To make it more integrative, we have in addition, provided a list of commercial efforts in the field and put together a list of available technologies with a description of the system novelty, unique features and product name. This, we hope will benefit common readers and entrepreneurs as well.

•    Important recent works such as DOIS: 10.1038/s41467-022-29562-4, 1038/s41576-022-00466-9, and 10.1039/D2LC00069E are missing.

We would like to thank the reviewer for pointing out these omissions. Both the research studies are major breakthroughs in the field and they have been added into the text in appropriate places. We also have referred the review suggested in section 6 as an additional resource for the readers interested in the field.

•    The tables play important roles in this manuscript; however, a table/tables comparing the advantages/disadvantages of the similar organ-on-a-chip devices is/are missing.

Thank you for this suggestion. We did discuss about putting a similar table but all these are emerging fields which are still developing and there are many challenges and we really can’t state advantages of one chip over the other as all of them are in different stages of development. We have tried to include major challenges in section 6. For systems that are more commonly used, we put together Table 2 that describes commercial availability of the technologies.

•    I believe Figure 2 need to be modified. Does it show the different steps in fabricating and testing the chip? If so, the last part (organ on a chip) is not actually the last section, but it is composed of the whole chart. You may modify the chip such that it depicts the output would be a chip, but now it is not showing like that. Also, shear stress is not necessarily involved in all organ on a chip experiment.

We would like to thank the reviewer for pointing this out. We have modified the figure to incorporate the suggestions. We do agree that shear stress might not be relevant to all organ on a chip, but since were going to discuss relevant systems with flowrates such as Kidney-on-a-chip and lung-on-a-chip, we decided to include it here as it does play a significant role. For other systems, although not as relevant from fluidics point of view shear stress could be involved in cell-cell interactions.

•    All in all, the work is promising and if the authors choose to answer the comments, I would be happy to re-read it.

We would like to sincerely thank the reviewer for critically reading the manuscript and providing immensely helpful suggestions. We have addressed all the points suggested, including adding the references suggested with contextual material in the right sections.

Reviewer 4 Report

This review article summarized recent advances of organ-on-a-chip technology. This topic is interesting, and this paper is well-organized. However, there are many typos and mistakes (see comments). In addition, some of points should be modified (see comments). The reviewer would recommend minor revision.

 

Comments

1. The affiliations 4 and 5 are strange. Are they correct?
2. Many review articles have been already published. Please add a short comment on the novelty of the author's paper.
3. “embryonic stem cells (ESC)” may be changed to “embryonic stem cells (ESCs)”.
4. Some spaces have been gone. For example, “Table1” should be changed to “Table 1”.
5. “smooth muscle cells (SMC)” might be changed to “smooth muscle cells (SMCs)”.
6. “epithelial cells (EC)” might be changed to “epithelial cells (ECs)”.
7. “extra cellular matrix” should be changed to “extracellular matrix”.
8. The authors should consider the necessity of Fig. 2, because a sentence seems to be enough instead of the illustration.
9. Were Refs. 13-15 correctly cited In Table 1? These references do not describe lung-on-a-chip.
10. In Table 1, “Lithography” may be changed to “Soft lithography”.
11. “,,” of “(20-22),,.” should be deleted.
12. In Table 2, “Mimetic” might be changed to “Mimetas”.
13. What does “human emulation system” mean in Table 2?
14. The copyright information of Fig. 3C may be added. For example, “Reproduced from Ref. 42. Copyright © 2016, Zhou et al.” may be added. Please checked the guideline of the journals.
15. There is no description on what “GFM” in Fig. 3C is.
16. There is no information of copyright and permission for Fig. 3D.
17. Although the authors describe that Fig. 3C was modified from Ref. 42, this reviewer disagrees to it. This is the same of the original.
18. “Top Channel” may be changed to “Top channel”.
19. “Bottom Channel” may be changed to “Bottom channel”.
20. In Table 2, the contents of Technology are a little strange. Are they “technology”?
21. “iPSC have” may be changed to “iPSCs have”.
22. “endothelial cells (EC)” may be changed to “endothelial cells (ECs)”.
23. “(ref)” should be corrected.
24. “polydimethylsiloxane (PDMS)” in the section of 5.4 should be changed to “PDMS”.
25. The authors should standardize the writing of author names. For example, “Lin Niu and co-workers” may be changed to “Niu and co-workers”.
26. “et. al” should be changed to “et al.”.
27. The format of the reference section should be corrected.
28. “PDM” might be changed to “PDMS”.

Author Response

“The reviewer would recommend minor revision.
1.    The affiliations 4 and 5 are strange. Are they correct?
Corrected, and updated
2.    Many review articles have been already published. Please add a short comment on the novelty of the author's paper.
This is an excellent point, and we want to address it separately. Since this is a rapidly evolving field where new breakthroughs are happening constantly, it warrants a constant update of the literature review to facilitate newer researchers and update those interested in the field. To make it more integrative, we have in addition, provided a list of commercial efforts in the field and pulled out a list of available technologies with a description of the system novelty, unique features and product name. This, we hope will benefit common readers and entrepreneurs.
3.    “embryonic stem cells (ESC)” may be changed to “embryonic stem cells (ESCs)”.
Updated
4.    Some spaces have been gone. For example, “Table1” should be changed to “Table 1”.
Updated
5.    “smooth muscle cells (SMC)” might be changed to “smooth muscle cells (SMCs)”.
Updated
6.    “epithelial cells (EC)” might be changed to “epithelial cells (ECs)”.
Updated
7.    “extra cellular matrix” should be changed to “extracellular matrix”.
Updated
8.    The authors should consider the necessity of Fig. 2, because a sentence seems to be enough instead of the illustration.
Corrected, and updated
9.    Were Refs. 13-15 correctly cited In Table 1? These references do not describe lung-on-a-chip.
Verified and updated.
10.    In Table 1, “Lithography” may be changed to “Soft lithography”.
Updated
11.    “,,” of “(20-22),,.” should be deleted.
Deleted
12.    In Table 2, “Mimetic” might be changed to “Mimetas”.
Updated
13.    What does “human emulation system” mean in Table 2?
With Human Emulation System, what we intended to say is it is something that replicated the chemical response and biological functioning of different organs/organ systems in the human body
14.    The copyright information of Fig. 3C may be added. For example, “Reproduced from Ref. 42. Copyright © 2016, Zhou et al.” may be added. Please checked the guideline of the journals.
Updated
15.    There is no description on what “GFM” in Fig. 3C is.
Updated
16.    There is no information of copyright and permission for Fig. 3D.
Updated
17.    Although the authors describe that Fig. 3C was modified from Ref. 42, this reviewer disagrees to it. This is the same of the original.
Updated
18.    “Top Channel” may be changed to “Top channel”.
updated
19.    “Bottom Channel” may be changed to “Bottom channel”.
Updated
20.    In Table 2, the contents of Technology are a little strange. Are they “technology”?
Changed to 'System'
21.    “iPSC have” may be changed to “iPSCs have”.
Updated
22.    “endothelial cells (EC)” may be changed to “endothelial cells (ECs)”.
Updated
23.    “(ref)” should be corrected.
Updated
24.    “polydimethylsiloxane (PDMS)” in the section of 5.4 should be changed to “PDMS”.
Updated
25.    The authors should standardize the writing of author names. For example, “Lin Niu and co-workers” may be changed to “Niu and co-workers”.
Edited and maintained a uniform approach throughout the article
26.    “et. al” should be changed to “et al.”.
Updated
27.    The format of the reference section should be corrected.
Corrected, and updated
28.    “PDM” might be changed to “PDMS”.
Updated

We would like to sincerely thank the reviewer for critically reading the manuscript and providing immensely helpful suggestions. We have addressed and incorporated all of the suggestions. A thorough re-reading of the manuscript was also done to check for typos and references.

Reviewer 5 Report

The authors describe an overview of OOC technology; listing projects and companies in the field. An appreciated look is towards Artificial Intelligence world applied to manage and process huge quantity data and images.

 

Please add citation to this review https://spj.sciencemag.org/journals/research/2022/9869518/ in the machine learning section

Some grammatical errors/typos are present in the text

Line 17 Replace microfluids with based on microfluidic platforms for in  vitro cell culture that have promising future in the healthcare industry

Table 1 about fabrication technologies  Soft Lithography not Lithography

In the companies table, Mimetas not mimetic

 

Author Response

We have revised the manuscript in light of Reviewer # 5. The response is given below:

Reviewer 5

 

“Please add citation to this review https://spj.sciencemag.org/journals/research/2022/9869518/ in the machine learning section”

We have included the citation in the revised manuscript.

“Some grammatical errors/typos are present in the text”

We have corrected the errors.

“Line 17 Replace microfluids with based on microfluidic platforms for in vitro cell culture that have promising future in the healthcare industry”

As suggested, we have replaced microfluids with microfluidic platforms.

“Table 1 about fabrication technologies  Soft Lithography not Lithography”

Lithography has been replaced by Soft Lithography.

“In the companies table, Mimetas not mimetic”

mimetic has been replaced Mimetas.

 

Round 2

Reviewer 3 Report

Authors have provided some answers to my comments, and I appreciate it. However, the manuscript is not revised well. My suggestion in the last review report was “major revision”; but the authors have only added a few citations to the work and revised a figure a little bit (not completely), which is not convincing.

• In my initial review report, I asked why we need this extra review, while we have some good reviews already available in this field. The authors, unfortunately, failed to answer this important question well. Authors mention the field is rapidly moving; however, the available reviews I mentioned are published just recently, in 2022 (see the work by Prof. Ingber, for example). Thus, this answer does not convince me. Then authors mention the inclusion of the commercial efforts, which may highlight the current review. But then they mention they cannot provide any comparison table, since these are mostly emerging and not developed yet. These two do not agree. If we are talking about the commercialized products, they need to be fully compared. If we are talking about the technologies under development, good solid recent reviews are already available, and please mention in the introduction, clearly, why we need a new one. This explanation needs to convince the readers.
• The sample articles I mentioned are cited, which is good. But, as mentioned above, a comprehensive comparison table, mentioning advantages and disadvantages of the works is needed. If not, I cannot find much different information in the current manuscript, compared to the ones already published.
• In figure 2, I should politely say that, the shear stress box, really is not relevant. There are many different phenomena happening in these systems, during the “experiments” section. Showing one of them only out of the box here does not make sense.

I still believe this work has good potentials; however, currently it is not ready yet. The main problem with this work is the lack of enough explanation in the introduction to exactly show why this review work is needed.

Author Response

Response to reviewers:

•  
• In my initial review report, I asked why we need this extra review, while we have some good reviews already available in this field. The authors, unfortunately, failed to answer this important question well. Authors mention the field is rapidly moving; however, the available reviews I mentioned are published just recently, in 2022 (see the work by Prof. Ingber, for example). Thus, this answer does not convince me. Then authors mention the inclusion of the commercial efforts, which may highlight the current review. But then they mention they cannot provide any comparison table, since these are mostly emerging and not developed yet. These two do not agree. If we are talking about the commercialized products, they need to be fully compared. If we are talking about the technologies under development, good solid recent reviews are already available, and please mention in the introduction, clearly, why we need a new one. This explanation needs to convince the readers.
• The sample articles I mentioned are cited, which is good. But, as mentioned above, a comprehensive comparison table, mentioning advantages and disadvantages of the works is needed. If not, I cannot find much different information in the current manuscript, compared to the ones already published.
• In figure 2, I should politely say that, the shear stress box, really is not relevant. There are many different phenomena happening in these systems, during the “experiments” section. Showing one of them only out of the box here does not make sense.

 

Below is a detailed response to the reviewer’s comments. The same has been updated in the accompanying MS (with track change enabled).Based upon the reviewer’s suggestion “. If we are talking about the commercialized products, they need to be fully compared”, (the new) Table 3 has been expanded to include limitations of the various commercial products.

 

Table 3:

Company	System	Selected Products	Features	Limitations	Region	References
Mimetas	- PhaseGuideTM technology, cells are free to interact and migrate, supporting cell-cell interaction, imaging, and quantification.	- OrganoPlate®️ 2-lane 96 -OrganoPlate®️  Graft -OrganoFlow®️	- Layered tissue without artificial membranes - Automated imaging - Robotic liquid handling equipment	- Chips are not reusable after washing - Cell culture can be retained for up to 2 weeks only - OrganoPlates are non-compatible under electron microscopy	The Netherlands	[77-80]
Emulate	Human emulation system to culture multiple organs	- Brain chip - Kidney chip - Liver chip - Lung chip	- Stretch parameters to emulate peristalsis, breathing - Can culture up to 12 organ chips	- Chances of test material interaction with the chip can alter the output of the experiment [81]	USA	[82-85]
AxoSim	Nerve-on-a-chip	- NERVESIMTM - BrainSIMTM	- Cultures iPSCs in a 3D environment	- Nerve conduction velocity for the developed platform is only about 0.13-0.28 m/s [86] - Limited automation on existing models	USA	[87,88]
TARA Biosystems	Heart-on-a-chip	- BiowireTM II platform - Cardiotype	- Can develop disease models from patients	- Improvisation is needed to develop a closer physiologically biomimetic model. [89]	USA	[90,91]
AlveoliX	Lung-on-chip	- AXLung-on-chip system	- Recreates air - blood barrier with ultra-thin membrane	- Since these utilize Collagen-Elastin (CE) membrane, the flexibility of the membrane depends on the ratio between both. - Gelation temperature has a direct impact on the mechanical properties of the membrane. [54,92]	Switzerland	[93,94]
TissUse	Human-on-a-chip	- HUMIMIC Chip 2 - HUMIMIC Chip 3 - HUMIMIC Chip 4 - HUMIMIC Chip           XX/XY	- Can mimic biological barriers while integrating multiple organs on a chip - Long term performance	- Single-use devices. - Chips can be stored only for 7 days. - For longer use the buffer solution must be changed, which voids guarantee.	Germany	[95-98]
CN Bio Innovations	- Single organ-on-a-chip - Multiple organs-on-a-chip	- PhysioMimixTM Liver-on-a-chip (MPS-LC12)	- Recirculating fluid flow to deliver essential materials - Inter and Intra organ-specific flow rate can be adjusted	- Still a high-level system has to be developed to replicate multi-organs-on-a-chip to mimic all the physiological function of organ systems [99]	UK	[100]
Kirkstall	- QuasiVivo®️ , an interconnected cell culture flow system for growth of cell	- QV500 - QV600 - QV900	- They are flexible and long-term culture is possible	- The chambers are made of PDMS and there are chances of components getting absorbed which can alter experimental outcomes.	UK	[101,102]
SynVivo	3D tissue and OOAC model	- SynTumour 3D Cancer model - SynALI Lung model - SynBBB Blood-Brain Barrier model	- Quantitative real-time visualization is possible	- Low throughput system - Require improvised design for the chip to enhance the seeding capacity [103]	USA	[104]
Hesperos Inc.	- Multi-organ micro physiological system	- Heart-liver two organ model - Neuromuscular junction two organ model	- Uses a serum-free cell medium - Posse’s gravity flow system	- Studies are conducted in monoculture and co-cultures studies need to be conducted for reliability [105].	USA	[105]
InSphero	- Organ-on-a-chip system - Production of microtissues using 3D SelectTM process	- 3D Insight tumor microtissues - 3D Insight Islet microtissues - 3D Insight liver microtissues	- Can capture long term drug effect - In vivo like morphology and functionality	- Further studies have to be conducted on human iPSCs to understand predictive power of assays [106]	Switzerland	[106,107]
Nortis Bio	- Organ-on-a-chip - Perfusion system platform	- ParVivo Chips	- Vascularization of tissues - Produce tumor microenvironment	- ParVivo Chips have 96-well plate footprint and are 2 inches high limiting their compatibility to specialized microscopes.	USA	[108]

 

 

As for “please mention in the introduction, clearly, why we need a new one“, the following paragraph replaces the original one.

In this perspective we have examined the different types of OOAC platforms and their application including an overall discussion on the major advancements in certain chip platforms. We have discussed different fabrication techniques involved in design and development, such as those developed to replicate the anatomical and physiological structure and function of organs, and models for diseases associated with certain organs, along with some of the pros and cons of these platforms. It is highly evident that OOAC technology has major potential in the healthcare sector and integration of multiple recent technologies will help to develop a highly advanced system. Hence, we emphasized the need of integrating AI into these systems, identified crucial government-supported projects in OOAC and the leading manufacturers that are contributing to developing new OOAC technologies. Potential barriers that need to be carefully addressed before the technology can be transferred from the laboratory to the clinical market are also mentioned. In the end we conclude with the challenges facing these platforms that needs to be addressed to develop a more reliable and accurate system.

 

• The sample articles I mentioned are cited, which is good. But, as mentioned above, a comprehensive comparison table, mentioning advantages and disadvantages of the works is needed. If not, I cannot find much different information in the current manuscript, compared to the ones already published.

 

Type of OOAC	Advantages	Disadvantages
Lung-on-a-chip	Lung-on-a-chip are an excellent replacement to fill the gaps during the transition of test results from an in vitro model to an in vivo environment. One of the pioneering developments in lung chip platform was that the ability to replicate breathing mechanism [17]. Second-generation lung-on-a-chip studies are aiming to replicate alveolar network, including physical and biochemical characteristics of alveolar basal membrane by developing stretchable models [56].	Despite the advancements lung-on-a-chip, the functional timeline of the chips last up to four weeks only, limiting modelling of chronic conditions. In addition, other characteristics such as cell-to-liquid ratio has to be addressed properly in order to avoid the dilution of metabolites, proteins and other substances [56].
Heart-on-a-chip	Heart-on-a-chip system help conduct studies on various cardiac diseases, drug screening and testing. Several associated platforms reviewed in this article have shown high throughput, portability, and the ability to replicate the cardiac system’s physical, electrical, biomechanical characteristics.	Since heart is a complex structure, it is comparatively difficult to recreate an environment which consist of different types of cells with properties such as polarization and electric impulses to manage contraction of heart chambers, and alignment of these cells and providing external stimuli. Efforts are being made to overcome this by adopting different fabricating techniques such as 3D scaffolds and micropattern substrates [57].
Kidney-on-a-chip	Biomimetic kidney-on-a-chip have a significant role in drug toxicological and filtration studies. Various kidney chip models discussed in this article are improvising on each stage and can retain highly relevant renal characteristics.	Some of the challenges in kidney-on-a-chip, includes occurrence of bubbles because of smaller dimensions of the chip, degradation of matrix which can impact cell viability and optimization of high throughput system. It is highly challenging to maintain consistency in cell seeding as it determines the chip’s characteristics. As mentioned above for other OOAC platforms, the viability and functionality may vary from 3 to 4 weeks [58].
Liver-on-a-chip	It is evident from the various studies that we reviewed on liver-on-a-chip have high reproducibility and can highly correlate chemical and toxicological testing. Some of the developed architecture of chip designs can replicate in vivo physiological environment of lung more closely.	Despite major advancements, there are still discrepancies on usage of cell sources. For example, stem cell induces hepatocytes and have stable function including albumin secretion and urea production. But they require specific induction factors and are costly. Primary hepatocytes can express liver intrinsic characteristic; but are difficult to isolate and incompatible for long term usage. Based on this, biomarker-values vary along with discrepancies in the metabolic functions of these cells [59]. Liver chip platforms have low throughput which limits them for large scale industrial applications [60].

A new table 2 provides the advantages and disadvantages of the various OOAC systems discussed in the MS.

• In figure 2, I should politely say that, the shear stress box, really is not relevant. There are many different phenomena happening in these systems, during the “experiments” section. Showing one of them only out of the box here does not make sense.

 

Figure has been updated thusly:

 

 

Author Response File: Author Response.pdf