Electromanipulation of mammalian cells:: Fundamentals and application

Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian tells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of tells, Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permeabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermeabilization in response to a breakdown pulse, Transient stretching force assumes a maximum value in low-conductivity pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible yields of(genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed about 100 mu s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivity. Such media are also beneficial because field-induced apoptosis does not occur under these conditions tin contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: I) to high incorporation rates of plasmids CDNA) or artificial chromosomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising agents for cancer treatment, as shown by first clinical trials.