经过改造的喷墨式打印机打印一个成纤维细胞。观察细胞内部表明荧光标记的肌动蛋白单体已经成功地整合到细胞骨架中。图片版权归The Journal of Visualized Experiments所有。
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来自美国克莱姆森大学的研究人员开发出一种方法：利用标准喷墨式打印机HP DeskJet 500在活细胞的膜上产生临时性的孔。相关研究结果于2012年3月16日发表在Journal of Visualized Experiments(JoVE)期刊上。
“当我们想要可视化观察在细胞上施加力量而导致的细胞骨架排布变化时，我们便闪现开发这种方法的念头”，论文通讯作者Delphine Dean博士说。
她说，其他研究人员一直使用这种方法在载玻片上打印细胞，但是他们最近发现耗时几个小时打印细胞会破坏它们的细胞膜。因此创建临时性孔可允许研究人员将一些通常不能进入细胞的分子导入细胞内部，然后研究这些细胞如何作出反应。
“作者们使用一种完全创新性的方法来对细胞进行生物打印(bioprint)。而且，除细胞打印之外，这种方法也能够有很多其他方面的应用”，JoVE期刊科学编辑Nandita Singh博士说，“利用这种技术能够将基质蛋白(matrix protein)打印在底板上从而实现细胞图形化(cell patterning)。这篇JoVE论文将使得这种方法变得更加简单和便于使用，并且也能够让其他实验室重复这种实验流程。”
所用的打印机经过改造：移除进纸装置(paperfeed mechanism)，同时添加一个“操作台(stage)”以便放入载玻片。用细胞溶液取代油墨，这样细胞就被直接打印在载玻片上。
利用这种方法，研究人员在几分钟内能够处理上千个细胞。Dean博士领导的研究小组利用产生的临时性孔引入荧光分子从而照亮整个细胞骨架。
“我们确实对细胞遭受挤压时的细胞力学(cell mechanics)感兴趣。这种方法允许我们按压细胞，然后轻松地观察细胞作出的反应”，Dean博士说，“我们对心血管细胞以及它们在遭受机械压力时作出的反应感兴趣。”
鉴于JoVE是唯一的得到同行评价、被PubMed收录而且发表以文字和视频形式存在的所有内容的期刊，所以Dean博士选择把她开发出的方法提交给该期刊。根据她的说法，“除非你眼见为实，否则你很难理解这种过程。” (生物谷：towersimper编译)
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doi:10.3791/3681 (2012)
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Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer
Alexander B. Owczarczak, Stephen O. Shuford, Scott T. Wood, Sandra Deitch, Delphine Dean
Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication. Recently, thermal inkjet printing has also been used for gene transfection. The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles.
A standard HP DeskJet 500 printer was modified to allow for cell printing. The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.
