• Immuno-therapy – producing therapeutic hybrids by combining a tumor cell with a dendritic cellHybridoma Production – to manufacture antibodies
• Nuclear Transfer – for fertility treatment and animal propagation

Electrofusion was first presented by Zimmerman in the early 1980’s. Much of today’s work is based on those early papers.

There are a number of techniques used to fuse dissimilar cells, such as chemical, mechanical and electrical. The Cyto Pulse systems are all electric field based resulting in very high cell viability and cell fusion efficiency. There are four distinct steps:

• Align and compress the cellsFuse the cellsHold the cells in place while the membranes mature
• Proper environment and nutrients for the cells to mature

The first step is to align and compress the cells. In this step the two cell types are placed in an chamber with a low conductivity medium. This is a critical step since cells will not survive the process in typical cell culture medium such as PBS and cells would not behave properly if they did survive. Cell fusion medium has a low conductivity (high resistivity). The initial waveforms applied during the process would cause excessive heating if more conductive medium were used. Other critical components are low millimolar amounts of divalent cations and buffers.

The next step is to bring cells into contact using a process called dielectrophoresis. Dielectrophoresis is an alignment of cells by the application of non-uniform alternating electric fields.

The cartoon at the right illustrates this step. Physics says that an electric field does not apply a force on a body that is not charged. A living cell does not have a net charge; therefore a uniform electric field will apply no force to the cell. Dielectrophoresis is the process of using a non-uniform field. In this case the charges inside of the cell are separated and a dipole is formed. The positive end of the cell will then move to the negative electrode. Cells then begin to line up in a chain with the negative end of one cell aligning with the positive end of another cell. An image of this process is shown at the right.

The second step is to subject the aligned cells to one or more intense electric field pulses to initiate cell fusion. The requirements for this pulse are similar to those for electroporation. An electric field at or above threshold is required to create the required transmembrane voltage. Cell membranes of adjacent cells will begin to mix at this point.

The third step is to again apply a weak non-linear electric field to hold the cells in place while the fused membranes mature and the cytoplasm mixes. Cell electrofusion is a process that begins at the time of application of the high electric field pulses and proceeds for some time after the pulses are applied. For that reason, dielectrophoresis is used for a period of time following application of the high electric field pulses. This keeps the cells in alignment for the most critical period of cell fusion maturation. The fusion process continues to mature during the next 10-30 minutes so they are disturbed as little as possible during this time. During cell fusion maturation, components of the cell membrane and cell cytoplasm begin to mix. The following cartoon illustrates the mixing of cell cytoplasm during cell fusion. The yellow indicates the mixing of green and red dyed cells.

For the fourth step, after cell membrane fusion maturation, the cells are placed in tissue culture to promote cell viability and growth.

Mature cell fusions of K562 cells are shown below at the left. The cells were fused and allowed to mature for 45 minutes. An aliquot of cells was placed onto microscope slides using a Cytospi (Shandon, Inc.) centrifuge. Cells were stained with Hema 3 stain and photographed. The giant multinucleated cell in the center is an extreme example of what is possible. Electrical parameters can be chosen that produce fewer of the large cells. Cells with two nuclei also are present. The image at the right shows fusion of dendritic cells (red) and A549 tumor cells (brown).

Refererences, General Electrofusion

Abidor, I. G. & Sowers, A. E. (1992). Kinetics and mechanism of cell membrane electrofusion. Biophys.J., 61, 1557-1569.

Glaser, R. W., Volk, H. D., Liebenthal, C., Jahn, S., & Grunow, R. (1990). Immortalization of magnetically separated human lymphocytes by electrofusion. Hum.Antibodies Hybridomas, 1, 111-114.

Hui, S. W. & Stenger, D. A. (1993). Electrofusion of cells: hybridoma production by electrofusion and polyethylene glycol. Methods Enzymol., 220, 212-227.

Jaroszeski, M. J., Gilbert, R., & Heller, R. (1994). Detection and quantitation of cell-cell electrofusion products by flow cytometry. Anal.Biochem., 216, 271-275.

Jaroszeski, M. J., Gilbert, R., & Heller, R. (1998). Flow cytometric detection and quantitation of cell-cell electrofusion products. Methods Mol.Biol., 91, 149-156.

Klock, G., Wisnewski, A. V., el Bassiouni, E. A., Ramadan, M. I., Gessner, P., Zimmermann, U., & Kresina, T. F. (1992). Human hybridoma generation by hypo-osmolar electrofusion: characterization of human monoclonal antibodies to Schistosoma mansoni parasite antigens. Hybridoma, 11 , 469-481.

Mekid, H. & Mir, L. M. (2000). In vivo cell electrofusion. Biochim.Biophys.Acta, 1524, 118-130.

Neil, G. A. & Zimmermann, U. (1993). Electrofusion. Methods Enzymol., 220, 174-196.

Ohno-Shosaku, T. & Okada, Y. (1984). Facilitation of electrofusion of mouse lymphoma cells by the proteolytic action of proteases. Biochem.Biophys.Res.Commun., 120 , 138-143.

Ohno-Shosaku, T. & Okada, Y. Electric pulse-induced fusion of mouse lymphoma cells: roles of divalent cations and membrane lipid domains. 85, 269-280. 1985.

Schmitt, J. J. & Zimmermann, U. (1989). Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions. Biochim.Biophys.Acta, 983, 42-50.

Tomita, M. & Tsong, T. Y. (1990). Selective production of hybridoma cells: antigenic-based pre-selection of B lymphocytes for electrofusion with myeloma cells. Biochim.Biophys.Acta, 1055, 199-206.

References, Nuclear Transfer

Fulka, J., Jr., Moor, R. M., & Fulka, J. (1995). Electrofusion of mammalian oocytes and embryonic cells. Methods Mol.Biol., 48, 309-316.

Kanka, J., Fulka, J., Jr., Fulka, J., & Petr, J. (1991). Nuclear transplantation in bovine embryo: fine structural and autoradiographic studies. Mol.Reprod.Dev., 29, 110-116.

Ogura, A., Inoue, K., Takano, K., Wakayama, T., & Yanagimachi, R. (2000). Birth of mice after nuclear transfer by electrofusion using tail tip cells [In Process Citation]. Mol.Reprod.Dev., 57, 55-59.

Ouhibi, N., Fulka, J., Jr., Kanka, J., & Moor, R. M. (1996). Nuclear transplantation of ectodermal cells in pig oocytes: ultrastructure and radiography. Mol.Reprod.Dev., 44, 533-539 .

Tatham, B. G., Giliam, K. J., & Trounson, A. O. (1996). Electrofusion parameters for nuclear transfer predicted using isofusion contours produced with bovine embryonic cells. Mol.Reprod.Dev., 43, 306-312.

Tatham, B. G., Pushett, D. A., Giliam, K. J., Dowsing, A. T., Mahaworasilpa, T. L., & Trounson, A. O. (1995). Electrofusion of in vitro produced bovine embryonic cells for the production of isofusion contours for cells used in nuclear transfer. J.Reprod.Fertil.Suppl, 49, 549-553.

Tsunoda, Y. & Kato, Y. (1993). Nuclear transplantation of embryonic stem cells in mice. J.Reprod.Fertil., 98, 537-540.

Tsunoda, Y., Kato, Y., & Shioda, Y. (1987). Electrofusion for the pronuclear transplantation of mouse eggs. Gamete Res., 17, 15-20.

Willadsen, S. M. (1986). Nuclear transplantation in sheep embryos. Nature, 320, 63-65.