Cell–Cell Electrofusion: Optimization of Electric Field Amplitude and Hypotonic Treatment for Mouse Melanoma (B16-F1) and Chinese Hamster Ovary (CHO) Cells
- 414 Downloads
Efficient electroporation of cells in physical contact induces cell fusion, and this process is known as electrofusion. It has been shown that appropriate hypotonic treatment of cells before the application of electric pulses can cause a significant increase in electrofusion efficiency. First, the amplitudes of the electric field were determined spectrofluorometrically, where sufficient permeabilization in hypotonic buffer occurred for B16-F1 and CHO cells. In further electrofusion experiments 14 ± 4% of fused cells for B16-F1 and 6 ± 1% for CHO was achieved. These electrofusion efficiencies, determined by double staining and fluorescence microcopy, are comparable to those of other published studies. It was also confirmed that successful electroporation does not necessarily guarantee high electrofusion efficiency due to biological factors involved in the electrofusion process. Furthermore, not only the extension of electrofusion but also cell survival depends on the cell line used. Further studies are needed to improve overall cell survival after electroporation in hypotonic buffer, which was significantly reduced, especially for B16-F1 cells. Another contribution of this report is the description of a simple modification of the adherence method for formation of spontaneous cell contact, while cells preserve their spherical shape.
KeywordsHypotonic buffer Electroporation Electrofusion Cell survival Fluorescence microscopy Spectrofluorometer B16-F1 cell CHO cell
This research was supported by the Slovenian Research Agency (ARRS).
- Jantscheff P, Spagnoli G, Zajac P, Rochlitz C (2002) Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells. Cancer Immunol Immunother 51:367–375Google Scholar
- Macek-Lebar A, Miklavcic D (2001) Cell electropermeabilization to small molecules in vitro: control by pulse parameters. Radiol Oncol 35:10Google Scholar
- Mally MI, McKnight ME, Glassy MC (1992) Protocols of electroporation and electrofusion for producing human hybridomas. In: Chang D, Chassy B, Saunders J, Sowers A (eds) Guide to electroporation and electrofusion. Academic Press, San Diego, pp 507–522Google Scholar
- Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, Billard V, Geertsen PF, Larkin JO, Miklavcic D, Pavlovic I, Paulin-Kosir SM, Cemazar M, Morsli N, Rudolf Z, Robert C, O’Sullivan GC, Mir LM (2006) Electrochemotherapy—an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. EJC Suppl 4:3–13Google Scholar
- Miklavcic D, Towhidi L (2010) Numerical study of the electroporation pulse shape effect on molecular uptake of biological cells. Radiol Oncol 44:8Google Scholar
- Mizukami Y, Kito H, Okauchi M (1993) Factors affecting the electrofusion efficiency of Porphyra protoplasts. J Appl Psychol 5:29–36Google Scholar
- Neumann E, Sowers AE, Jordan CA (1989) Electroporation and electrofusion in cell biology. Springer-Verlag, New YorkGoogle Scholar
- O’Hare MJ, Ormerod MG, Imrie PR, Peacock JH, Asche W (1989) Electropermeabilization and electrosensitivity of different types of mammalian cells. In: Neumann E, Sowers AE, Jordan CA (eds) Electroporation and electrofusion in cell biology. Plenum Press, New York, pp 319–330Google Scholar
- Salvi A, Quillan J, Sadée W (2002) Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein. AAPS J 4:21–28Google Scholar
- Sukhorukov VL, Reuss R, Zimmermann D, Held C, Müller KJ, Kiesel M, Geßner P, Steinbach A, Schenk WA, Bamberg E, Zimmermann U (2005) Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion. J Membr Biol 206:187–201CrossRefPubMedGoogle Scholar
- Zimmermann U, Neil GA (1996) Electromanipulation of cells. CRC, Boca Raton, FLGoogle Scholar