The Rapid Generation of Cell-Laden, FACS-Compatible Collagen Gels

Round 1

Reviewer 1 Report

This manuscript describes a technique to encapsulate cells within a collagen hydrogel, that is compatible for FACs sorting.

I would like to clarify some details in the manuscript, namely:

For determining the dead/ live cells, authors used calcein AM staining and observed the images obtained. I think other quantitative method should be used, or authors should determine which percentage of cells are viable and proliferating, for example using DNA quantification along the culture time. 

Authors claim: "These results confirm that 450 gels survive the sorting and that gels do not significantly stick to each other during manipulations." Did you put the sorted gels with cells back in culture? Did cells grow normally? How many passages did they survived?

I would like also to know what are the advantages of this method. Authors indicate that "Research in topics such as tissue engineering and organoid development will benefit from methods to identify and select desired cell clusters that can then be assembled into precise 3D structures". These methods already exist and there are quite simple, in fact, there are several works reporting sorting cells and placing them in culture to develop into organoids. Can you please explain why is your method more advanced, compared to others?

A final comment: cell lines are quite different from primary cultures and iPSCs. If applying the methods described to primary cells, I am not sure if they would survive the cell-laden process and the sorting. Have you tried the methods using primary cultures?

Thank you.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

See attached document: Peer-review-28223694

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript is changed accordingly and I do not have any extra comments. Thank you for considering my recommendations.

Author Response

Hello

Thanks again for your time and careful reading of the manuscript. Your comments really did improve it. We have corrected the typos and now believe it is complete. 

Regards,

John

Reviewer 2 Report

Manuscript: organoids-2317833

Title: Rapid generation of cell-laden, FACS-compatible collagen gels

Thanks for considering my suggestions and adjusting the manuscript based on my comments made in previous review. I truly believe the method that you developed of FACS-compatible collagen gels is highly valuable for the research world. I believe with some extra time to do some minor experiments and changes in some figures, the manuscript will be improved.

I appreciate the addition to the Method section to facilitate the FACS-compatible collagen gels for other cell types. The scope of Organoids MDPI include several organoid related research topics.  Addition of iPSCs in a next manuscript version with their specific microfluidic settings will ensure to fit with the scope of Organoids MDPI journal. Please add 2D and 3D collagen-I culture conditions, as essential controls, next to the FACS-sorted Collagen-I droplets to compare growth and viability of cells in sorted and non-sorted collagen-I droplets. This will strengthen your found application.

The secondary antibody problem is well-solved by re-ordering primary antibody and test all secondary antibodies in the lab to avoid confusion. Could you please show the staining results with controls in the next version of the manuscript. Please give also some extra attention to the primary antibody that is used. Anti-collagen-I antibody (Abcam, ab34710) is described to react with human collagen-I, you use Rat tail collagen-I. For sure there will be strong similarities in collagen-I amino acid sequence of human compared to rat, but to be save one could use an antibody that has been described to recognize Rat-tail collagen-I. Another option would be to add a human collagen-I scaffold as control to verify the functionality of primary antibody (Abcam, ab34710).

It is stated (figure 4) that immediately after encapsulation, cells stained bright for calcein AM fluorescence (viable cells) and no cells stained for ethidium homodimer-1 (death cells). It looks like not all cell clusters are colored for green or red in figure4. Are homodimer-I and calcein AM staining done on the same cell encapsulated collagen-I droplets on the indicated days? Could you please add stainings of  calcein AM together with homodimer-1 in one condition. You should combine calceinAM, ethidium homodomer-I and DAPI/Hoechst (nuclear staining) per collagen-I droplet immediately after encapsulation (essential control).  With these staining you could give a quantification ratio of how many cells are viable or non-viable after encapsulation. Brightfield images and a graph, representing the quantification with standard deviation, will improve the manuscript.

Thanks for reconstructing figure 6. Please have a careful look at figure 6A. I noticed that the old figure 6A is different compared to the revised version of figure 6A. The gating of the old version starts at 10⁴ and in the revised version at 10³.

Could you also explain why the gating of figure6A of the green dots is exactly the same as the gating of figure6C in the revised version.

I believe with some extra time, to carefully do some control experiments and make some clear figures, the manuscript will be improved. Reconsider also suggestions and controls made in previous review.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Great improvements with the extra work on iPSCs!

Please carefully read one more time the paper as there are still some typos in the text. An example is Hoescht as the correct version is Hoechst. 

Author Response

Hello

Thanks again for your time and careful reading of the manuscript. Your comments really did improve it. We have corrected the typos and now believe it is complete. 

Regards,

John