Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.
Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.
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DOI：
10.1371/journal.pone.0106853
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Nomura SM
中科院分区：
文献类型：
--
作者：
Saito AC;Ogura T;Fujiwara K;Murata S;Nomura SM
在这里，我们报告了一种方法，通过电融合巨大的单层囊泡，将直径达一微米的大物体引入培养的哺乳动物细胞中。我们使用油包水（w/o）乳液离心法制备含有各种人造物体的GUV。将GUV和分散的HeLa细胞暴露于交流（AC）场以诱导线性细胞-GUV对准，然后施加直流（DC）脉冲以促进瞬时电融合。以均匀大小的荧光珠作为尺寸指标，我们成功有效地将直径为1 μm的珠与质粒哺乳动物表达载体一起引入活细胞中。我们的电融合不影响细胞活力。电融合后，细胞正常增殖直至达到融合，并且引入的荧光珠在细胞分裂期间遗传。共聚焦显微镜和流式细胞术分析支持这些发现。作为替代方法，我们还将设计的纳米结构（DNA折纸）引入活细胞中。我们在这里报告的结果代表了一个里程碑，设计人工共生的功能活跃的对象（如微型机器）在活细胞。此外，我们的技术可用于药物输送，组织工程和细胞操作。
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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