Efficient Generation of Stable Antibody Forming Hybridoma Cells by Electrofusion

Schmitt, J. J.; Zimmermann, U.; Neil, Garry A.

doi:10.1089/hyb.1989.8.107

Cited by 22 publications

(12 citation statements)

References 26 publications

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“…Data very close to ours were obtained by means of an electronic Coulter counter (32) on much larger samples (> lo5 Sp2 cells). We evaluate a mean volume of 1,350 pm3 with CVvolume -29%, corresponding to a mean radius 6.9 km and CV, -9.7%, from the cell volume histograms of Schmitt et al (32). (Fig.…”

Section: The Dashed Lines Inmentioning

confidence: 99%

“…Whatever the explanation, it is evident that the accelerated resealing of hypoosmotically swollen cells will reduce the undesirable leakage of cytoplasmic small molecules and ions after electropulsing. This may contribute to the higher yields of animal and plant hybrids that have been observed after hypoosmolar electrofusion (2,32,46).…”

Section: Resealing In Hypotonically Swollen Cellsmentioning

confidence: 99%

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Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmolkell) before electropulsation (0.5-3.0 kV/cm, 40 ps) in medium containing 25-50 pg/ml PI at 21-23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form >95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (73 of about 2 min. In 150 mOsm medium, resealing was faster (typically T~ -0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius de-10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C,), was taken into account. Evaluation of the C, values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. Key terms: Plasma membrane, electric breakdown, cytoplasm, fluorescent staining, propidium iodide, dye uptake, electrorotation, carboxyfluorescein, flow cytometry, cell viability Electropermeabilization allows the introduction of foreign substances (e.g., drugs, hormones, proteins, plasmids) into living cells (for reviews, see 27,44,45). This technique has been reported to be the most reliable for studying the effects of normally impermeant molecules on the regulation of cell metabolism. Other permeabilization methods (e.g., the use of bacterial toxins, detergents, hypertonic treatment) can markedly affect cell viability and cause undesirable leakage of the cytoplasm or extensive cell lysis ( 2 5 3 0 ) .Plasma membrane electropermeabilization within statistically representative cell samples can be assessed by measuring membrane crossing of tracer molecules in a flow cytometer. Extremely heterogeneous responses to electropulsing have been found in rather homogeneous (origin, size, morphology) cell samples despite the use of uniform electric fields (parallel plate electrodes) for several cell types (6,9,10,24). Flow cytometry of human red blood cells after electropulsation in the presence of FITCdextran (70 kDa) gave rise to at least three distinct subpopulations: ghosts with negligible upta...

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Section: The Dashed Lines Inmentioning

confidence: 99%

Section: Resealing In Hypotonically Swollen Cellsmentioning

confidence: 99%

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

et al. 1995

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“…The efficiency of electrofusion is considerably higher than that of PEG fusion. PEG fusion has an average fusion efficiency of 5 × 10 -6 , whereas previous methods of electrofusion achieved efficiencies up to 4 × 10 -4 (Foung and Perkins, 1989;Glassy, 1988;Ohnishi et al, 1987;Schmitt et al, 1989;Steenbakkers et al, 1993;Vienken and Zimmermann, 1985;Zimmermann and Vienken, 1982). Theoretically, electrofusion can be applied to induce hybrid formation between any two cells.…”

Section: Discussionmentioning

confidence: 99%

An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies

McGraw

House

et al. 2008

Journal of Immunological Methods

130

124

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We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.

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“…At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed.The treatment of cells with electric pulses is now widely used in biotechnology, medicine, and basic research for the electroinjection of foreign molecules, such as drugs, proteins, plasmids, etc., and also for elec- trofusion (2,13,19,20,27,37-39,41). Important applications are (a) the use of electrically loaded cells for target-specific drug administration, (b) somatic hybridization of plant protoplasts, and (c) the electrically mediated production of human hybridomas by immortalisation of a small numbers of lymphocytes (12,21,29,36).The number and viability of the hybrids and of the electrically loaded cells depend critically-among other parameters-on the peak field strength E, of the applied field pulse, and on the cell radius a. For pulses which are much longer than the charging time of the membrane, about 0.1 ps for lymphocytes in the "pulse medium" (conductivity 3.3 mS/cm), the induced membrane voltage is given by (18) 'Research funded by grants of the Deutsche Forschungsgemeinschaft (SFB 176B5 to U.Z.…”

mentioning

confidence: 99%

“…The treatment of cells with electric pulses is now widely used in biotechnology, medicine, and basic research for the electroinjection of foreign molecules, such as drugs, proteins, plasmids, etc., and also for elec- trofusion (2,13,19,20,27,37-39,41). Important applications are (a) the use of electrically loaded cells for target-specific drug administration, (b) somatic hybridization of plant protoplasts, and (c) the electrically mediated production of human hybridomas by immortalisation of a small numbers of lymphocytes (12,21,29,36).…”

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confidence: 99%

Effect of electric field pulses on the viability and on the membrane‐bound immunoglobulins of LPS‐activated murine B‐lymphocytes: Correlation with the cell cycle

et al. 1994

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The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of GO/G1 cells.After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant T = 5-40 ps) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4°C or 20°C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37°C for 30 min ("resealing") significantly decreased the percentage of permeabilized cells. Electropulsed GO/Gl cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing.The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, 'c = 5 ps, three pulses, 4°C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. Key terms: Cell cycle, deconvolution, electric field effects, flow cytometry, FITC, membrane breakdown, propidium iodide, resealing, trypan blueThe treatment of cells with electric pulses is now widely used in biotechnology, medicine, and basic research for the electroinjection of foreign molecules, such as drugs, proteins, plasmids, etc., and also for electrofusion (2,13,19,20,27,37-39,41). Important applications are (a) the use of electrically loaded cells for target-specific drug administration, (b) somatic hybridization of plant protoplasts, and (c) the electrically mediated production of human hybridomas by immortalisation of a small numbers of lymphocytes (12,21,29,36).The number and viability of the hybrids and of the electrically loaded cells depend critically-among other parameters-on the peak field strength E, of the applied field pulse, and on the cell radius a. For pulses which are much longer than the charging time of the membrane, about 0.1 ps for lymphocytes in the "pulse medium" (conductivity 3.3 mS/cm), the induced membrane voltage is given by (18) 'Research funded by grants of the Deutsche Forschungsgemeinschaft (SFB 176B5 to U.Z. and W.M.A.).

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Efficient Generation of Stable Antibody Forming Hybridoma Cells by Electrofusion

Cited by 22 publications

References 26 publications

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance

An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies

Effect of electric field pulses on the viability and on the membrane‐bound immunoglobulins of LPS‐activated murine B‐lymphocytes: Correlation with the cell cycle

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