Single-cell electroporation

Olofsson, J., Nolkrantz, K., Ryttsen, F., Lambie, B.A., Weber, S.G., Orwar, O., 2003. Single-cell electroporation. Curr. Opin. Biotechnol. 14, 29—34. 

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Khine, M., et ah, 2007. Single-cell electroporation arrays with real-time monitoring and feedback control. Lab Chip 7, 457 62. 

Huang, Y., Rubinsky, B., Flow-through micro-electroporation chip for high efficiency single-cell genetic manipulation, Sensors Actuators A 2003, 104, 205-212. 

ES cell preparation Trypsinize subconfluent ES cells to a single cell suspension as described in Subheading 3.2.2., centrifuge the cells, resuspend the pellet in 10-mL electroporation buffer, determine the cell density, centrifuge the cells again and resuspend the pellet with electroporation buffer to 2 x 107 cells/mL. 

To address biological processes at the single cell level in a live organism, electroporation techniques have been developed [5]. They involve electroporation by injection of various molecules (e.g., DNA, RNA or morpholinos) present in a micropipette brought in the close vicinity of the targeted cell (see Fig. 16.1). The technique is invasive, and the amount of electroporated material is unknown. Moreover, because the whole tissue is displaced by the inserted micropipette, the success rate in targeting a specific cell in a live embryo is very low. It would be desirable if a noninvasive technique could be devised to control the rapid activation of a known concentration of biomolecules in a specific cell of a live organism. 

There are different techniques to overcome the cell membrane barrier and introduce exogenous impermeable compounds, such as dyes, DNA, proteins, and amino acids into the ceU. Some of the methods include lipofection, fusion of cationic liposome, electroporation, microinjection, optoporation, electroinjection, and biolistics. Electroporation has the advantage of being a noncontact method for transient permeabilization of cells (Olofsson et al., 2003). In contrast to microinjection techniques for single cells and single nuclei (Capecchi, 1980), the electroporation technique can be applied to biological containers of sub-femtoliter volumes, that are less than a few micrometers in diameter. Also, it can be extremely fast and well-timed (Kinosita et al., 1988 Hibino et al., 1991), which is of importance in studying fast-reaction phenomena (Ryttsen et aL, 2000). 

Sandberg, M., Orwar, O., 2001. Electroporation of single cells and tissues with an electrolyte-filled capillary. Anal. Chem. 73, 4469—4477. 

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Ghosh, J., Liu, X., Gillis, K. D. 2013. Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode. Lab Chip 13 2083-2090. 

Bao, N., Zhan, Y., Lu, C., 2008. Microfluidic electroporative flow cytometry for studying single-cell biomechanics. Anal. Chem. 80 (20), 7714-7719. Available at http //www.ncbi. nlm.nih.gov/pubmed/18798650. 

Furthermore, a single intratumoral injection of IL-12 pDNA followed by electroporation resulted in a significant inhibition of tumor growth as well as a reduction in the growth of a distant tumor. Tumors treated with IL-12 pDNA and electroporation were infiltrated with NK cells and CD3+ T cells and a tumor-specific CTL response was generated. 

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