Squeeze-E squeeze-E

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Science Translational Medicine  22 Mar 2017:
Vol. 9, Issue 382, eaan0766
DOI: 10.1126/scitranslmed.aan0766


Adding an electrical pulse to a microfluidic device that squeezes cells through narrow channels efficiently delivers DNA to the nucleus.

Cell engineers face a formidable foe: the cellular membrane. Whether constructing cancer-fighting T cells or regenerative cell–based therapeutics, the quest to alter cell behavior requires efficient methods for introducing large biomolecules, such as DNA, across cell barriers into the nucleus. Physical and biological methods developed for this purpose suffer from low efficiency, poor scalability, or high toxicity. However, investigators recently developed a mechanical squeeze-loading method that leverages the ability of cell membranes to heal after disruption. In this approach, cells flow through narrow microfluidic constrictions that cause deformation and reversible membrane disruption, allowing entry of extracellular cargo before membrane healing. Unfortunately, although the approach efficiently delivered proteins into the cytosol, it did not enable delivery of DNA into the nucleus, likely because of DNA’s high molecular weight and charge.

Ding et al. now overcome this problem with “disruption-and-field-enhanced (DFE) delivery,” which combines mechanical squeeze-loading with electroporation, a method that uses brief electrical field exposure to transiently permeabilize the nuclear membrane and drive nucleic acids into the nucleus. The team designed a continuous-flow silicon-based microfluidic device consisting of 75 parallel channels with constrictions for squeeze-loading and planar electrodes for electric field application. By varying the applied voltage and the flow rate, investigators controlled the electrical pulse strength and duration seen by the cells. To characterize the device, they mixed HeLa cells with plasmid DNA encoding green fluorescent protein (GFP) and evaluated loading efficiency by measuring cellular fluorescence with flow cytometry after DFE delivery and culture. Balancing design trade-offs enabled improved loading efficiency while maintaining comparable viability to electroporation alone. Perhaps most interesting was that DFE delivery led to near maximal GFP expression after only 1 hour compared with 24 to 48 hours required for lipofection or electroporation alone. This high-delivery efficiency rivals that of low-throughput microinjection but does so at the tremendous throughput of millions of cells per minute.

Some of the most promising applications for cell engineering involve cell-based therapeutics. Therefore, it will be important to determine whether DFE delivery of DNA can be adapted to primary cells, which are notoriously fragile. Nevertheless, based on results expressing fluorescent proteins, the future of cellular engineering looks bright.

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