Single cell transfection of human-induced pluripotent stem cells using a droplet-based microfluidic system

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research in fields such as physics, engineering, chemistry and biology have combined to produce innovation in microfluidics which has positively impacted diverse areas such as nucleotide sequencing, functional genomics, single-cell studies, single molecules assays and biomedical diagnostics. Among these areas, regenerative medicine and stem cells have benefited from microfluidics since these tools have had a profound impact on their applications. In this study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We will demonstrate that this system has great efficiency in single cells and captured droplets, like other microfluidic methods but with lower cost. Moreover, this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations and cell therapy, among other potential applications.

Germany) for single cell analysis. Also, the authors proposed that the microfluidic method was highly effective for single cell transfection; however, the cell viability in droplets is extremely low for long-term cell culture and the cell viability after transfection was not proven. Hence, unfortunately, this reviewer feels that the manuscript fails to make a strong case that would be suitable for publication in Royal Society Open Science. 1) (page 4, line 31) "Biocompatible oil FluoroSurfactant-HFE 7500 5%" is the name of a product, HFE 7500 oil with 5% fluorinated surfactants. Please clarify it.
2) (page4, line 35) Please provide the average size of droplets with standard deviation.
3) (page4, line 44) The microscope equipped with the camera might be used to capture images of droplets. 4) (page5, line 4) "4.1 Microfluidic system design" needs to rearrange in Materials and Methods. If the authors provide CAD designs of the microfluidic devices, it will be informative for readers. 5) (page 5, line14) The authors mentioned, "the efficiency of the device was characterized. It would be the capability f the device for generating monodispersed droplets. 6) (page 5, line 27) It was reported that the rate of single cell encapsulation in droplets is higher than 22% previously (Collins, D.J., Neild, A., DeMello, A., Liu, A.Q. and Ai, Y., 2015. The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. Lab on a Chip, 15(17), pp.3439-3459). Please clarify it. 7) (page5, line 38) After 24 hours of encapsulation, a high degree of cell death was observed; however, the authors proposed the microfluidic platform for long-term cell culture. Please explain further how the platform can be used for single cell long-term culture and transfection applications.  figure 4A. A bright-field microscopy image on the area is necessary to be provided. Also, are the two images (green and red fluorescence) combined images? In Figure 4B, the authors showed the fluorescence intensity degradation to quantify the cell viability. However, the fluorescence intensity can be decreased by photobleaching. Hence, if the authors would evaluate the cell viability as a function of encapsulation time, the y-axis of the graph should be the number of cells (fluorescence dots). 10) English should be updated. The manuscript is hard to read.

Decision letter (RSOS-201781.R0)
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Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) More information needed: -Some wording seems odd or misplaced -Lacking of reference papers -Better to say droplet than drop -Shematics of the microfluidic chips used (with dimensions and design) -Concentration of cell solution in the "Cell culture" part in Materials and Methods (poisson law distribution used?) -Probably more information on the Materials and Methods part (for exemple, what is the supplier of the surfactant?) -Fgures with more details + axes graphs illisible - Figure  Reviewer: 2 Comments to the Author(s) The manuscript describes a microfluidic approach for single cell transfection. A flow-focusing droplet generator and a droplet-storage device were interconnected for the encapsulation of hiPSCs in droplets and storing the droplets in microwells. The authors evaluated the viability of cells in droplets and compared the efficiency of the transfection of hiPSCs between the proposed microfluidic-based approach and the conventional method. However, the devices in the manuscript are well-known designs through many previous studies, and even a chip combined droplet generation and storage is commercially available (Fluidic 488, microfluidic ChipShop, Germany) for single cell analysis. Also, the authors proposed that the microfluidic method was highly effective for single cell transfection; however, the cell viability in droplets is extremely low for long-term cell culture and the cell viability after transfection was not proven. Hence, unfortunately, this reviewer feels that the manuscript fails to make a strong case that would be suitable for publication in Royal Society Open Science. 1) (page 4, line 31) "Biocompatible oil FluoroSurfactant-HFE 7500 5%" is the name of a product, HFE 7500 oil with 5% fluorinated surfactants. Please clarify it.
2) (page4, line 35) Please provide the average size of droplets with standard deviation.
3) (page4, line 44) The microscope equipped with the camera might be used to capture images of droplets. 4) (page5, line 4) "4.1 Microfluidic system design" needs to rearrange in Materials and Methods. If the authors provide CAD designs of the microfluidic devices, it will be informative for readers. 5) (page 5, line14) The authors mentioned, "the efficiency of the device was characterized. It would be the capability f the device for generating monodispersed droplets. 6) (page 5, line 27) It was reported that the rate of single cell encapsulation in droplets is higher than 22% previously (Collins, D.J., Neild, A., DeMello, A., Liu, A.Q. and Ai, Y., 2015. The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. Lab on a Chip, 15(17), pp.3439-3459). Please clarify it. 7) (page5, line 38) After 24 hours of encapsulation, a high degree of cell death was observed; however, the authors proposed the microfluidic platform for long-term cell culture. Please explain further how the platform can be used for single cell long-term culture and transfection applications. 8) (page5, line 55) The authors showed the efficiency of transfection in the proposed platform. However, cell viability after transfection is one of the most important parameters for single cell transfection applications. Could the authors evaluate the cell viability after transfection in the proposed system? 9) (Figure 4) It is not clear where droplets and cells are in figure 4A. A bright-field microscopy image on the area is necessary to be provided. Also, are the two images (green and red fluorescence) combined images? In Figure 4B, the authors showed the fluorescence intensity degradation to quantify the cell viability. However, the fluorescence intensity can be decreased by photobleaching. Hence, if the authors would evaluate the cell viability as a function of encapsulation time, the y-axis of the graph should be the number of cells (fluorescence dots). 10) English should be updated. The manuscript is hard to read.

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Comments to the Author(s)
The revised version looks good. Agree to accept.s Decision letter (RSOS-211510.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Lerner, I am pleased to inform you that your manuscript entitled "Single cell transfection of human induced pluripotent stem cells using a droplet-based microfluidic system." is now accepted for publication in Royal Society Open Science.
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Reviewer: #1
Comments to the Author(s) More information needed: 1) Some wording seems odd or misplaced

Answer:
English and grammar was checked and corrected in the manuscript.

Answer:
New reference was included in the manuscript, emphasizing the introduction, and focusing on the most current droplet storage methods and transfection techniques. Other bibliography and cited paragraphs were excluded from the text to improve reading comprehension and focus on the advantages of our method compared to those currently available. 3) Better to say droplet than drop

Answer:
This suggestion was taken and corrected in the manuscript.

4) Shematics of the microfluidic chips used (with dimensions and design)
Appendix A

Answer:
The diagram together with the measurements of both microdevices is described in Figure 1.

Answer:
To find the optimal cell concentration of our droplet forming microdevice, we applied the Poisson distribution was applied based on a cell concentration curve giving the optimal concentration for single cell encapsulation per droplet. This was also explained in the manuscript, giving the optimal concentration of 300,000 cells / ml. 6) Probably more information on the Materials and Methods part (for example, what is the supplier of the surfactant?)

Answer:
The size and resolution of the figures were modified so that the graphs and axes can be read more clearly. 8) Figure 1: Schematics or pictures of the setup with the microfluidic chips (both of them)?

Answer:
It was added in the segment of (figure 1) in materials and methods. All the details of the micro droplet formation and storage devices. 9) Figure 2: mean diameter and standard deviation of droplets size? Schematic of the droplet generation chip?

Answer:
The standard deviation (SD) of the droplets was also detailed in the materials and methods and in the results. Demonstrating the monodispersity of the droplets at the time of their formation in the microdevice. 10) Figure 3: possible to have the poucentage of encapsulation for each value: 0 cell, 1 cell, 2 cells, 3 cells?

Answer:
It is possible to have the total percentage of the wells that had cells, but it was decided to put the total of droplets that were captured in the experimental replicas. So, it was considered more appropriate to present it that way. The point raised by the Reviewer is substantially valid though, that is why we have included it as part of the discussion 11) Figure 4: possible to have a bright field image to better understand the fluorescence images?

Answer:
The Live-Death protocol is designed to view images of cells in fluorescence. The manuscript details that the cells were previously released from the droplets by (PFO) to measure their viability. 12) Figure 5: Are the droplets broken to take the C part? If yes, what is the protocol?

Answer:
It is detailed in the materials and methods segment. The paragraph is detailed below: In brief, the droplets that were in the micro storage device were collected. After this, base medium (E8-Flex) and a volume like that collected in the droplets of (PFO) was added. Using light movements, it was mixed and left to rest at room temperature for 2 minutes. After seeing the two separate phases (oil / medium), it was centrifuge with a short spin for 10 seconds. The supernatant containing the cells was taken for analysis or common plating.

Reviewer: #2
Comments to the Author(s) The manuscript describes a microfluidic approach for single cell transfection. A flow-focusing droplet generator and a droplet-storage device were interconnected for the encapsulation of hiPSCs in droplets and storing the droplets in microwells. The authors evaluated the viability of cells in droplets and compared the efficiency of the transfection of hiPSCs between the proposed microfluidic-based approach and the conventional method. However, the devices in the manuscript are well-known designs through many previous studies, and even a chip combined droplet generation and storage is commercially available (Fluidic 488, microfluidic ChipShop, Germany) for single cell analysis. Also, the authors proposed that the microfluidic method was highly effective for single cell transfection; however, the cell viability in droplets is extremely low for long-term cell culture and the cell viability after transfection was not proven. Hence, unfortunately, this reviewer feels that the manuscript fails to make a strong case that would be suitable for publication in Royal Society Open Science. 1) (page 4, line 31) "Biocompatible oil FluoroSurfactant-HFE 7500 5%" is the name of a product, HFE 7500 oil with 5% fluorinated surfactants. Please clarify it.

Answer:
Following the reviewer's recommendation, the supplier and more details of the oil used in the droplet formation experiments were added.

Answer:
The SD of the droplets was detailed in the materials and methods as well in the results. Demonstrating the monodispersity of the droplets at the time of their formation in the microdevice.