Optical injection of mammalian cells using a microfluidic platform.
Optical injection of mammalian cells using a microfluidic platform.
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DOI：
10.1364/boe.1.000527
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发表时间：
2010-08-09
影响因子：
3.4
通讯作者：
Dholakia K
中科院分区：
文献类型：
--
作者：
Marchington RF;Arita Y;Tsampoula X;Gunn-Moore FJ;Dholakia K
利用聚焦的激光在细胞质膜上开一个亚微米的洞(光固化)，选择性地引入包括核酸在内的膜不透性物质(光学注射)(光学转染法)，是一种最常用于治疗单细胞的强大技术。然而，特别是对于飞秒光复制，这些研究一直局限于低通量、小规模的研究，因为它们需要单个细胞的顺序剂量。在这里，我们描述了一种用于增加产量和自动光学注入细胞的微流控光复制系统。流体动力聚焦被用来引导单列细胞流经聚焦的飞秒激光光束进行光复制。在穿过光束时，细胞外膜上可能会打开一些暂时性的毛孔，这使得周围的液体介质可以被吸收到细胞质中，也包含所选的注射剂。这个过程是完全自动化的，可以很容易地获得1个细胞/秒的速度，使得使用该系统每小时可以注射数千个细胞。在HEK293细胞中光学注射碘化丙啶的效率为42±8%，或考虑到注射后活力的要求而为28±4%，用Calcein AM进行了测试这项工作现在为将光催化与微流控分析、分类、纯化或芯片上细胞培养研究相结合开辟了道路。
The use of a focused laser beam to create a sub-micron hole in the plasma membrane of a cell (photoporation), for the selective introduction of membrane impermeable substances (optical injection) including nucleic acids (optical transfection), is a powerful technique most commonly applied to treat single cells. However, particularly for femtosecond photoporation, these studies have been limited to low throughput, small-scale studies, because they require sequential dosing of individual cells. Herein, we describe a microfluidic photoporation system for increased throughput and automated optical injection of cells. Hydrodynamic focusing is employed to direct a flow of single-file cells through a focused femtosecond laser beam for photoporation. Upon traversing the beam, a number of transient pores potentially open across the extracellular membrane, which allows the uptake of the surrounding fluid media into the cytoplasm, also containing the chosen injection agent. The process is entirely automated and a rate of 1 cell/sec could readily be obtained, enabling several thousand cells to be injected per hour using this system. The efficiency of optically injecting propidium iodide into HEK293 mammalian cells was found to be 42 ± 8%, or 28 ± 4% taking into account the requirement of post-injection viability, as tested using Calcein AM. This work now opens the way for combining photoporation with microfluidic analyses, sorting, purification or on-chip cell culture studies.