Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions

Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization

“…[23][24][25][26] One of the earliest approaches to improve electrofusion efficiency was the use of hypotonic electrofusion buffer that resulted in the considerable fusion efficiency increase. [27][28][29][30][31][32][33][34] To ensure the improvement of fusion efficiency in hypotonic buffer the duration and the osmolarity of the hypotonic treatment has to molarity of solutions was determined with Knauer vapor pressure osmometer K-7000 (Knauer, Wissenschaftliche Gerätebau, Germany). Buffers pH was 7.2.…”

“…susceptibility due to the increase in cell size. 16,28,[30][31][32][33]39,52 In conclusion, cell size dynamics should be carefully analyzed and observed for each cell line in order to obtain all potential benefits of using hypotonic buffer for electrofusion. Electroporation should be performed when cells are close to their maximal size i.e.…”

Cell electrofusion using nanosecond electric pulses

“…They found that 1–3 pulses of such duration and with appropriate pulse amplitude (2–3 kV/cm) are enough for efficient electroporation of both fusion partners23. With some variations this protocol has, indeed, proved to be optimal in many cases1524252627 and also found its way into manuals of some commercial electrofusion systems (e.g. Eppendorf).…”

“…The efficiency of electrofusion, nevertheless, not only depends on the pulse parameters, but also on the composition of the fusion medium, osmolarity of the fusion medium, temperature, post pulse incubation of cells and other factors1314152223242526272829. However, despite many efforts for improving electrofusion of cells with different size, higher susceptibility of large cells to electric pulses remained one of the obstacles, and the number of viable hybridomas obtained with respect to the number of input B lymphocytes reached only ~1% or less132630.…”

Prospects for monoclonal antibodies in the diagnosis and treatment of bacterial infections

“…The present fusion method requires over 105 copies of one specific B-cell to obtain one specific hybridoma. All B-cells present in the fusion mixture with a lower copy number are lost during the fusion process, a drawback which can possibly be overcome by the use of novel fusion procedures such as electrofusion (15). Human monoclonal antibodies must also be examined for safety as they may crossreact with tissue antigen, or the human monoclonal antibody preparation may be contaminated with viruses or bacteria, etc.…”