Electromanipulation of mammalian cells: Fundamentals and application

Electroinjection of membrane-impermeable xenomolecules into freely suspende d mammalian tells (so-called electroporation) and cell-to-cell electrofusio n 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 me mbrane permeability due to reversible electrical breakdown of the plasma me mbrane upon application of external high-intensity field pulses of very sho rt duration. Membrane charging and permeabilization caused by high-intensity field pulse s are preceded and accompanied by transient electrodeformation forces, whic h lead to an elongation of the cells in low-conductivity media, thus affect ing the membrane area of electropermeabilization in response to a breakdown pulse, Transient stretching force assumes a maximum value in low-conductiv ity pulse media. This facilitates incorporation of membrane-impermeable xen omolecules and field-mediated hybridization as well. Therefore, high and reproducible yields of(genetically) manipulated cells c an be expected provided that: 1) the duration of the high-intensity field p ulses does not exceed about 100 mu s and 2) that the (pulse or fusion) medi a are hypo-osmolar and exhibit a relatively low conductivity. Such media ar e also beneficial because field-induced apoptosis does not occur under thes e conditions tin contrast to highly conductive media). Indeed, electroporat ion 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 ce lls (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal ant ibodies that bind to and induce apoptosis in autologous tumor cells are pro mising agents for cancer treatment, as shown by first clinical trials.