經(jīng)過改造的噴墨式打印機打印一個成纖維細胞。觀察細胞內(nèi)部表明熒光標記的肌動蛋白單體已經(jīng)成功地整合到細胞骨架中。圖片版權歸The Journal of Visualized Experiments所有,。
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來自美國克萊姆森大學的研究人員開發(fā)出一種方法：利用標準噴墨式打印機HP DeskJet 500在活細胞的膜上產(chǎn)生臨時性的孔。相關研究結(jié)果于2012年3月16日發(fā)表在Journal of Visualized Experiments(JoVE)期刊上,。
“當我們想要可視化觀察在細胞上施加力量而導致的細胞骨架排布變化時,，我們便閃現(xiàn)開發(fā)這種方法的念頭”，論文通訊作者Delphine Dean博士說,。
她說,，其他研究人員一直使用這種方法在載玻片上打印細胞，但是他們最近發(fā)現(xiàn)耗時幾個小時打印細胞會破壞它們的細胞膜,。因此創(chuàng)建臨時性孔可允許研究人員將一些通常不能進入細胞的分子導入細胞內(nèi)部,，然后研究這些細胞如何作出反應。
“作者們使用一種完全創(chuàng)新性的方法來對細胞進行生物打印(bioprint),。而且,，除細胞打印之外，這種方法也能夠有很多其他方面的應用”,，JoVE期刊科學編輯Nandita Singh博士說,，“利用這種技術能夠?qū)⒒|(zhì)蛋白(matrix protein)打印在底板上從而實現(xiàn)細胞圖形化(cell patterning)。這篇JoVE論文將使得這種方法變得更加簡單和便于使用,，并且也能夠讓其他實驗室重復這種實驗流程,。”
所用的打印機經(jīng)過改造：移除進紙裝置(paperfeed mechanism)，同時添加一個“操作臺(stage)”以便放入載玻片,。用細胞溶液取代油墨,，這樣細胞就被直接打印在載玻片上。
利用這種方法,，研究人員在幾分鐘內(nèi)能夠處理上千個細胞,。Dean博士領導的研究小組利用產(chǎn)生的臨時性孔引入熒光分子從而照亮整個細胞骨架。
“我們確實對細胞遭受擠壓時的細胞力學(cell mechanics)感興趣,。這種方法允許我們按壓細胞,，然后輕松地觀察細胞作出的反應”,，Dean博士說，“我們對心血管細胞以及它們在遭受機械壓力時作出的反應感興趣,。”
鑒于JoVE是唯一的得到同行評價,、被PubMed收錄而且發(fā)表以文字和視頻形式存在的所有內(nèi)容的期刊，所以Dean博士選擇把她開發(fā)出的方法提交給該期刊,。根據(jù)她的說法,，“除非你眼見為實，否則你很難理解這種過程,。” (生物谷：towersimper編譯)
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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.
