Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear delivery of plasmid DNA within 1 h after treatment—the most rapid DNA expression in a high-throughput setting (up to millions of cells per minute per device). Passing cells at high speed through microfluidic constrictions smaller than the cell diameter mechanically disrupts the cell membrane, allowing a subsequent electric field to further disrupt the nuclear envelope and drive DNA molecules into the cytoplasm and nucleus. By tracking the localization of the endosomal sorting complex required for transport III protein CHMP4B (charged multivesicular body protein 4B), we show that the integrity of the nuclear envelope is recovered within 15 minutes of treatment. We also provide insight into subcellular delivery by comparing the performance of the disruption-and-field-enhanced method with those of conventional chemical, electroporation and manual-injection systems.
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We thank P. Qi from the Division of Comparative Medicine at Massachusetts Institute of Technology (MIT) for performing the microinjection, Q. Chen from Richard Sherwood Lab at Brigham and Women’s Hospital for providing mouse embryonic stem cells, and X. Yin from the Jeff Karp Lab at Brigham and Women’s Hospital, Harvard Medical School for providing human embryonic stem cells. We thank I. Poser from Tony Hyman Lab at Max Planck Institute of Molecular Cell Biology and Genetics for supplying the CHMP4B-GFP HeLa cells. The assistance and expertise of G. Paradis and personnel in the flow cytometry core at the Koch Institute and the Microsystem Technology Laboratory at MIT are highly acknowledged. This research was supported by National Institutes of Health (R01GM101420-01A1), and device fabrication was performed at the Microsystem Technology Laboratory at MIT. M.P.S. was supported by the Swiss National Science Foundation through the advanced postdoc mobility fellowship P300P3_151179. M.P.S. acknowledges support from a Keith Murdoch Fellowship via the American Australian Association, a Life Sciences Research Foundation Fellowship sponsored by Good Ventures, and a Broadnext10 Catalytic Steps funding gift from the Broad Institute.
A.S., R.S.L. and K.F.J. have a financial interest in SQZ Biotechnologies.
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Ding, X., Stewart, M., Sharei, A. et al. High-throughput nuclear delivery and rapid expression of DNA via mechanical and electrical cell-membrane disruption. Nat Biomed Eng 1, 0039 (2017). https://doi.org/10.1038/s41551-017-0039
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