Digital Magnetic Sorting for Fractionating Cell Populations with Variable Antigen Expression in Cell Therapy Process Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors proposed a digital magnetic sorting (DMS) technique that targets CD4 on human leukocytes based on antigen density levels. DMS separation characterized by flow cytometry and single-cell RNA sequencing showed differential CD4 expression between HMF and LMF fractions. DMS also demonstrated high-throughput processing and lower reagent consumption. It provides a tool for cell therapy developers to isolate low numbers of highly specialized cell types. I consider recommending publication after the authors make the following changes.

1.The author should not only provide the single cell RNA sequencing data, but also the flow cytometry analysis data of the same cell sample, which need to be discussed to confirm the sorting effect.

2.How to evaluate the sorting effect of Digital Magnetic Sorting technology for Cell Therapy Manufacturing? FACS or other method?

3.The process time of evaluation for DMS needs to be considered when the author compared the throughput between DMS and other methods.

4.The cited references are too outdated. The authors should consider adding more relevant references in recent 3 years to support their study, such as: 10.1021/acsami.2c23003.

5.Please check the correct abbreviation of journal names.

6.Please check the English language, and be consistent with tenses.

Comments on the Quality of English Language

Please check the English language, and be consistent with tenses.

Author Response

Comment 1. The author should not only provide the single cell RNA sequencing data, but also the flow cytometry analysis data of the same cell sample, which need to be discussed to confirm the sorting effect.

We thank the reviewer for the comment, and we have added supplemental files of the single cell sequencing data (Loupe Browser Files) and the FCS files for the flow cytometry. Figure A4 outlines the flow cytometry gating strategy.

We also added supplemental figure (Figure A5) which quantifies the cell sorting effect of DMS based on cell viability and cell activation.

Comment 2. How to evaluate the sorting effect of Digital Magnetic Sorting technology for Cell Therapy Manufacturing? FACS or other method?

We thank the reviewer for the comment, and we have added and additional supplemental figure (Figure A5) which quantifies the cell sorting effect of DMS based on cell viability and cell activation. We observe high viability post sort and little to no immune cell activation.

Comment 3. The process time of evaluation for DMS needs to be considered when the author compared the throughput between DMS and other methods.

 We thank the reviewer for the comment and have added additional clarifying language in Figure 6 to compare process time between DMS and FACS. Typically, a process step to confirm cell sorting viability in cell count is performed after sorting and generally takes ~5 minutes using standard hemocytometer based approaches. This would likely need to be performed after FACS sorting as well to evaluate true cell viability and total cell count post sort.

Comment 4. The cited references are too outdated. The authors should consider adding more relevant references in recent 3 years to support their study, such as: 10.1021/acsami.2c23003.

We thank the reviewer for the comment and have added additional and more up to date references.

Comment 5.Please check the correct abbreviation of journal names.

 We thank the reviewer for the comment and have confirmed abbreviation of journal names in the references.

Comment 6. Please check the English language, and be consistent with tenses

We thank the reviewer for the comment and have corrected some grammatical errors.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Digital Magnetic Sorting for Fractionating Cell Populations with Variable Antigen Expression in Cell Therapy Process Development” by Bshara-Corson et al. is an interesting contribution wich fits well to the journal Magnetochemistry.

The results are well described and look sound. The same is true for the experimental details and a possible reproducibility of the results.

The references are balanced.

There are only minor issues, otherwise I recommend to accept this article:

10M to 40M cells. You should not use this abbreviation for million since you use M for molar as well.

Caption: Figure 1a: Leur Lock is actually Luer Lock

Author Response

Comment 1: 10M to 40M cells. You should not use this abbreviation for million since you use M for molar as well.

We thank the reviewer for noticing this and have made appropriate corrections regarding cell numbers.

Comment 2: Caption: Figure 1a: Leur Lock is actually Luer Lock

We thank the reviewer for noticing this and have corrected Figure 1.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have proposed a digital magnetic sorting method to sort cells based on antigen density level. The work is solid and novel. I only have a few minor comments for the authors to help improve the quality of the manuscript.
-        The proposed technical method of cell sorting needs to be explained in more detail. Currently, it is not clear enough.
-        The figure labels need to be consistent with the captions. The labels are capitalized on figures, while they are not capitalized in some captions. Please modify them.
-        Figure 2 needs a color bar.

Author Response

Comment 1: The proposed technical method of cell sorting needs to be explained in more detail. Currently, it is not clear enough.

We thank the reviewer for the comment, and we have added and additional explanation describing ratcheting cytometry cell transport and sorting in supplementary figure A1.

Comment 2: The figure labels need to be consistent with the captions. The labels are capitalized on figures, while they are not capitalized in some captions. Please modify them.

We thank the reviewer for noticing this and have made appropriate corrections regarding figure labels.

Comment 3: Figure 2 needs a color bar.

We thank the reviewer for noticing this and have added a color bar to Figure 2 showing cell counts.