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PLoS One. 2014 Sep 17;9(9):e106853. doi: 10.1371/journal.pone.0106853. eCollection 2014.

Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

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Department of Bioengineering and Robotics, Tohoku University, Aoba-ku, Sendai, Japan.
Department of Developmental Neurobiology, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Aoba-ku, Sendai, Japan; CREST, JST, Tohoku University, Sendai, Miyagi, Japan.
Department of Biosciences and Informatics, Keio University, Hiyoshi, Kohokuku, Yokohama, Japan; JSPS Research Fellow, Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo, Japan.


Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell-GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

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