Owczarczak Alexander B, Shuford Stephen O, Wood Scott T, Deitch Sandra, Dean Delphine
Department of Bioengineering, Clemson University, USA.
J Vis Exp. 2012 Mar 16(61):3681. doi: 10.3791/3681.
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. Cell viability after printing has been shown to be similar to standard cell plating methods. 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.
生物打印具有广泛的应用和意义，包括组织工程、直接细胞应用疗法和生物传感器微制造。最近，热喷墨打印也已用于基因转染。热喷墨打印过程显示可暂时破坏细胞膜而不影响细胞活力。膜上的瞬时孔隙可用于将原本太大而无法穿过膜的分子引入细胞质。此处展示的应用是利用热喷墨打印将荧光标记的γ-肌动蛋白单体掺入细胞。使用热喷墨打印将分子注入细胞的优点是该技术对细胞相对无害。打印后的细胞活力已证明与标准细胞铺板方法相似。此外，喷墨打印可在数分钟内处理数千个细胞，这比手动显微注射快得多。打印产生的孔隙已证明在约两小时内关闭。然而，这种打印技术产生的孔隙大小有限（约10纳米），这限制了该技术仅能将小蛋白质和/或颗粒注入细胞。对一台标准的惠普DeskJet 500打印机进行了改装以实现细胞打印。移除打印机的盖子，使用机械杠杆绕过进纸机构。搭建了一个平台，以便将显微镜载玻片和盖玻片直接放置在打印头下方。打开墨盒，清除墨水并在用于细胞之前进行清洗。使用标准绘图软件创建打印图案，然后通过简单的打印命令控制打印机。将3T3成纤维细胞培养至汇合，用胰蛋白酶消化，然后重悬于含有可溶性荧光标记γ-肌动蛋白单体的磷酸盐缓冲盐水中。将细胞悬液吸移到墨盒中，并将细胞线打印到玻璃显微镜盖玻片上。使用荧光显微镜对活细胞成像，发现肌动蛋白遍布整个细胞质。将荧光肌动蛋白掺入细胞可实现对短时间细胞骨架动力学的成像，并在广泛的应用中有用。
