Fig. treat different diseases, like diabetes1,2,3, regenerate axons of the central nerve system4, and produce cells with desired properties, such as in cell vaccines for cancer immunotherapy5,6,7. However, the first and most known application of cell fusion is production of monoclonal antibodies in hybridoma technology, where hybrid cell lines (hybridomas) are formed by fusing specific antibody-producing ADP B lymphocytes with a myeloma (B lymphocyte cancer) cell line8,9. Myeloma cells were selected for their ability to grow in culture, since B lymphocytes do not survive outside their natural environment. Initially, in hybridoma technology polyethylene glycol (PEG) was used for cell fusion, and in some laboratories it is still the most preferable fusogen10. Nevertheless, cell fusion based on cell membrane electroporation C electrofusion C was suggested as a more efficient technique11,12,13. Electrofusion in comparison to PEG fusion improved not only the number of fused cells obtained (i.e. fusion yield), but also the hybridoma growing rate; electrofused cells grew more vigorously than the ones fused with PEG11. Electrofusion also holds great promise for the use of hybridomas in clinical environment, since the method does not require viral or chemical additives. By definition, electrofusion is a two-condition process: (i) close physical contact between cells has to be established, and (ii) cell membranes have to be brought into fusogenic state14. A physical contact between cells can be achieved in various ways, though the most widely used is dielectrophoresis, where cells are aligned in pearl chains using alternating electric field15. Dielectrophoresis is most frequently used especially in the field of hybridoma technology and production of cell vaccines, since it enables establishing contacts between cells in suspension. The second condition for electrofusion, the membrane fusogenic state, is achieved by electric pulse application resulting in structural rearrangement of the lipid bilayer. It is generally accepted that the transmembrane voltage, which is induced on the cell membrane during exposure to high electric fields, reduces the energy barrier for formation of hydrophilic pores in the lipid bilayer16, although other explanations are also plausible17. The phenomenon is termed electroporation and is related to experimentally observed dramatic increase in membrane permeability16,17. At the same time, membrane fusogenicity correlates with electroporation18. Both, the extent of electroporation and the fusion yield, can be controlled by the amplitude, duration, and number of the applied pulses; namely, increasing any of the pulse parameters mentioned leads to a higher level of membrane ADP electroporation and consequently higher number of fused cells18. However, parameters of the electric pulses must be carefully chosen as to ensure that electroporation is reversible, i.e., cells survive. Failing to respect this leads to irreversible cell electroporation, thereby reducing cell survival and consequently reducing the yield of viable fused cells. At a given electric field strength the extent of membrane electroporation further depends on the cell size16,19. One of the major advantages of electrofusion is the possibility of optimizing electroporation conditions for each cell line individually. Unfortunately, there is a Rabbit Polyclonal to PPP1R2 substantial challenge in fusing cell lines that differ considerably in their size. Electric pulses that are usually used for electrofusion range from 10 to 100?s, which ensures that cell membranes become fully charged during their exposure to electric pulse. Under such conditions, the induced transmembrane voltage is proportional to the cell radius, which means that small cells are electroporated (i.e. brought into fusogenic state) at higher electric field strengths19. ADP Applying pulses that effectively electroporate small cells, thus inevitably leads to excessive electroporation and consequently death of large fusion partner cells. An example where a difference.