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The Journal of Membrane Biology

, Volume 236, Issue 1, pp 107–116 | Cite as

Cell–Cell Electrofusion: Optimization of Electric Field Amplitude and Hypotonic Treatment for Mouse Melanoma (B16-F1) and Chinese Hamster Ovary (CHO) Cells

  • Marko Ušaj
  • Katja Trontelj
  • Damijan Miklavčič
  • Maša Kandušer
Article

Abstract

Efficient electroporation of cells in physical contact induces cell fusion, and this process is known as electrofusion. It has been shown that appropriate hypotonic treatment of cells before the application of electric pulses can cause a significant increase in electrofusion efficiency. First, the amplitudes of the electric field were determined spectrofluorometrically, where sufficient permeabilization in hypotonic buffer occurred for B16-F1 and CHO cells. In further electrofusion experiments 14 ± 4% of fused cells for B16-F1 and 6 ± 1% for CHO was achieved. These electrofusion efficiencies, determined by double staining and fluorescence microcopy, are comparable to those of other published studies. It was also confirmed that successful electroporation does not necessarily guarantee high electrofusion efficiency due to biological factors involved in the electrofusion process. Furthermore, not only the extension of electrofusion but also cell survival depends on the cell line used. Further studies are needed to improve overall cell survival after electroporation in hypotonic buffer, which was significantly reduced, especially for B16-F1 cells. Another contribution of this report is the description of a simple modification of the adherence method for formation of spontaneous cell contact, while cells preserve their spherical shape.

Keywords

Hypotonic buffer Electroporation Electrofusion Cell survival Fluorescence microscopy Spectrofluorometer B16-F1 cell CHO cell 

Notes

Acknowledgements

This research was supported by the Slovenian Research Agency (ARRS).

References

  1. Abe S, Takeda J (1988) Effects of La3+ on surface charges, dielectrophoresis, and electrofusion of barley protoplasts. Plant Physiol 87:389–394CrossRefPubMedGoogle Scholar
  2. Abidor IG, Barbul AI, Zhelev DV, Doinov P, Bandrina IN, Osipova EM, Sukharev SI (1993) Electrical properties of cell pellets and cell electrofusion in a centrifuge. Biochim Biophys Acta 1152:207–218CrossRefPubMedGoogle Scholar
  3. Ahkong QF, Lucy JA (1986) Osmotic forces in artificially induced cell fusion. Biochim Biophys Acta 858:206–216CrossRefPubMedGoogle Scholar
  4. Barrau C, Teissie J, Gabriel B (2004) Osmotically induced membrane tension facilitates the triggering of living cell electropermeabilization. Bioelectrochemistry 63:327–332CrossRefPubMedGoogle Scholar
  5. Blangero C, Rols MP, Teissié J (1989) Cytoskeletal reorganization during electric-field-induced fusion of Chinese hamster ovary cells grown in monolayers. Biochim Biophys Acta 981:295–302CrossRefPubMedGoogle Scholar
  6. Castro-Garza J, Barrios-Garcia HB, Cruz-Vega DE, Said-Fernandez S, Carranza-Rosales P, Molina-Torres CA, Vera-Cabrera L (2007) Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains. J Med Microbiol 56:733–737CrossRefPubMedGoogle Scholar
  7. Cegovnik U, Novakovic S (2004) Setting optimal parameters for in vitro electrotransfection of B16F1, SA1, LPB, SCK, L929 and CHO cells using predefined exponentially decaying electric pulses. Bioelectrochemistry 62:73–82CrossRefPubMedGoogle Scholar
  8. Cemazar M, Jarm T, Miklavcic D, Macek-Lebar A, Ihan A, Kopitar NA, Sersa G (1998) Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro. Electromagn Biol Med 17:263–272CrossRefGoogle Scholar
  9. Chen EH, Grote E, Mohler W, Vignery A (2007) Cell–cell fusion. FEBS Lett 581:2181–2193CrossRefPubMedGoogle Scholar
  10. Escoffre J-M, Portet T, Wasungu L, Teissie J, Dean D, Rols M-P (2009) What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol 41:286–295CrossRefPubMedGoogle Scholar
  11. Evans EA, Parsegian VA (1986) Thermal–mechanical fluctuations enhance repulsion between bimolecular layers. Proc Natl Acad Sci USA 83:7132–7136CrossRefPubMedGoogle Scholar
  12. Finaz C, Lefevre A, Teissie J (1984) Electrofusion. A new, highly efficient technique for generating somatic cell hybrids. Exp Cell Res 150:477–482CrossRefPubMedGoogle Scholar
  13. Foung S, Perkins S, Kafadar K, Gessner P, Zimmermann U (1990) Development of microfusion techniques to generate human hybridomas. J Immunol Methods 134:35–42CrossRefPubMedGoogle Scholar
  14. Gabrijel M, Repnik U, Kreft M, Grilc S, Jeras M, Zorec R (2004) Quantification of cell hybridoma yields with confocal microscopy and flow cytometry. Biochem Biophys Res Commun 314:717–723CrossRefPubMedGoogle Scholar
  15. Gabrijel M, Kreft M, Zorec R (2008) Monitoring lysosomal fusion in electrofused hybridoma cells. Biochim Biophys Acta 1778:483–490CrossRefPubMedGoogle Scholar
  16. Gabrijel M, Bergant M, Kreft M, Jeras M, Zorec R (2009) Fused late endocytic compartments and immunostimulatory capacity of dendritic–tumor cell hybridomas. J Membr Biol 229:11–18CrossRefPubMedGoogle Scholar
  17. Gillies RJ, Didier N, Denton M (1986) Determination of cell number in monolayer cultures. Anal Biochem 159:109–113CrossRefPubMedGoogle Scholar
  18. Golzio M, Mora M-P, Raynaud C, Delteil C, Teissié J, Rols M-P (1998) Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J 74:3015–3022CrossRefPubMedGoogle Scholar
  19. Gong J, Avigan D, Chen D, Wu Z, Koido S, Kashiwaba M, Kufe D (2000) Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells. Proc Natl Acad Sci USA 97:2715–2718CrossRefPubMedGoogle Scholar
  20. Gottfried E, Krieg R, Eichelberg C, Andreesen R, Mackensen A, Krause SW (2002) Characterization of cells prepared by dendritic cell–tumor cell fusion. Cancer Immun 2:15PubMedGoogle Scholar
  21. Hayashi T, Tanaka H, Tanaka J, Wang R, Averbook BJ, Cohen PA, Shu S (2002) Immunogenicity and therapeutic efficacy of dendritic–tumor hybrid cells generated by electrofusion. Clin Immunol 104:14–20CrossRefPubMedGoogle Scholar
  22. James K, Bell GT (1987) Human monoclonal antibody production. J Immunol Methods 100:5–40CrossRefPubMedGoogle Scholar
  23. Jantscheff P, Spagnoli G, Zajac P, Rochlitz C (2002) Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells. Cancer Immunol Immunother 51:367–375Google Scholar
  24. Jaroszeski MJ, Gilbert R, Fallon PG, Heller R (1994) Mechanically facilitated cell–cell electrofusion. Biophys J 67:1574–1581CrossRefPubMedGoogle Scholar
  25. Jaroszeski MJ, Gilbert R, Heller R (1995) Cytometric detection and quantitation of cell–cell electrofusion products. In: Nickoloff JA (ed) Animal cell electroporation and electrofusion protocols. Humana Press, Totowa, NJ, pp 355–363CrossRefGoogle Scholar
  26. Kanduser M, Sentjurc M, Miklavcic D (2006) Cell membrane fluidity related to electroporation and resealing. Eur Biophys J 35:196–204CrossRefPubMedGoogle Scholar
  27. Knutton S, Jackson D, Graham JM, Micklem KJ, Pasternak CA (1976) Microvilli and cell swelling. Nature 262:52–54CrossRefPubMedGoogle Scholar
  28. Kotnik T, Bobanovic F, Miklavcic D (1997) Sensitivity of transmembrane voltage induced by applied electric fields—a theoretical analysis. Bioelectrochem Bioenerg 43:6CrossRefGoogle Scholar
  29. Lambert IH (2007) Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells. Am J Physiol Cell Physiol 293:C390–C400CrossRefPubMedGoogle Scholar
  30. Lee WT, Shimizu K, Kuriyama H, Tanaka H, Kjaergaard J, Shu S (2005) Tumor–dendritic cell fusion as a basis for cancer immunotherapy. Otolaryngol Head Neck Surg 132:755–764CrossRefPubMedGoogle Scholar
  31. Macek-Lebar A, Miklavcic D (2001) Cell electropermeabilization to small molecules in vitro: control by pulse parameters. Radiol Oncol 35:10Google Scholar
  32. Mally MI, McKnight ME, Glassy MC (1992) Protocols of electroporation and electrofusion for producing human hybridomas. In: Chang D, Chassy B, Saunders J, Sowers A (eds) Guide to electroporation and electrofusion. Academic Press, San Diego, pp 507–522Google Scholar
  33. Martens S, McMahon HT (2008) Mechanisms of membrane fusion: disparate players and common principles. Nat Rev Mol Cell Biol 9:543–556CrossRefPubMedGoogle Scholar
  34. Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, Billard V, Geertsen PF, Larkin JO, Miklavcic D, Pavlovic I, Paulin-Kosir SM, Cemazar M, Morsli N, Rudolf Z, Robert C, O’Sullivan GC, Mir LM (2006) Electrochemotherapy—an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. EJC Suppl 4:3–13Google Scholar
  35. Matibiri EA, Mantell SH (1995) Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts. Plant Cell Tissue Organ Culture 40:125–131CrossRefGoogle Scholar
  36. McIntosh TJ, Advani S, Burton RE, Zhelev DV, Needham D, Simon SA (1995) Experimental tests for protrusion and undulation pressures in phospholipid bilayers. Biochemistry 34:8520–8532CrossRefPubMedGoogle Scholar
  37. McIntosh TJ, Kulkarni KG, Simon SA (1999) Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations. Biophys J 76:2090–2098CrossRefPubMedGoogle Scholar
  38. Mekid H, Mir LM (2000) In vivo cell electrofusion. Biochim Biophys Acta 1524:118–130PubMedGoogle Scholar
  39. Miklavcic D, Towhidi L (2010) Numerical study of the electroporation pulse shape effect on molecular uptake of biological cells. Radiol Oncol 44:8Google Scholar
  40. Mir LM (2009) Nucleic acids electrotransfer-based gene therapy (electrogenetherapy): past, current, and future. Mol Biotechnol 43:167–176CrossRefPubMedGoogle Scholar
  41. Mizukami Y, Kito H, Okauchi M (1993) Factors affecting the electrofusion efficiency of Porphyra protoplasts. J Appl Psychol 5:29–36Google Scholar
  42. Neil GA, Zimmermann U (1993) Electrofusion. Methods Enzymol 220:174–196CrossRefPubMedGoogle Scholar
  43. Neumann E, Sowers AE, Jordan CA (1989) Electroporation and electrofusion in cell biology. Springer-Verlag, New YorkGoogle Scholar
  44. O’Hare MJ, Ormerod MG, Imrie PR, Peacock JH, Asche W (1989) Electropermeabilization and electrosensitivity of different types of mammalian cells. In: Neumann E, Sowers AE, Jordan CA (eds) Electroporation and electrofusion in cell biology. Plenum Press, New York, pp 319–330Google Scholar
  45. Ohno-Shosaku T, Okada Y (1985) Electric pulse-induced fusion of mouse lymphoma cells: roles of divalent cations and membrane lipid domains. J Membr Biol 85:269–280CrossRefPubMedGoogle Scholar
  46. Pucihar G, Miklavcic D, Kotnik T (2009) A time-dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells. IEEE Trans Biomed Eng 56:1491–1501CrossRefPubMedGoogle Scholar
  47. Ramos C, Bonenfant D, Teissie J (2002) Cell hybridization by electrofusion on filters. Anal Biochem 302:213–219CrossRefPubMedGoogle Scholar
  48. Rols MP, Teissie J (1989) Ionic-strength modulation of electrically induced permeabilization and associated fusion of mammalian cells. Eur J Biochem 179:109–115CrossRefPubMedGoogle Scholar
  49. Rols MP, Teissie J (1990) Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure. Biochemistry 29:4561–4567CrossRefPubMedGoogle Scholar
  50. Rubinsky B, Onik G, Mikus P (2007) Irreversible electroporation: a new ablation modality–clinical implications. Technol Cancer Res Treat 6:37–48PubMedGoogle Scholar
  51. Salomskaite-Davalgiene S, Cepurniene K, Satkauskas S, Venslauskas MS, Mir LM (2009) Extent of cell electrofusion in vitro and in vivo is cell line dependent. Anticancer Res 29:3125–3130PubMedGoogle Scholar
  52. Salvi A, Quillan J, Sadée W (2002) Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein. AAPS J 4:21–28Google Scholar
  53. Schmitt JJ, Zimmermann U (1989) Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions. Biochim Biophys Acta 983:42–50CrossRefPubMedGoogle Scholar
  54. Schnettlera R, Zimmermann U (1992) Zinc ions stimulate electrofusion of Hansenula polymorpha protoplasts. FEMS Microbiol Lett 106:47–51CrossRefGoogle Scholar
  55. Scott-Taylor TH, Pettengell R, Clarke I, Stuhler G, La Barthe MC, Walden P, Dalgleish AG (2000) Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines. Biochim Biophys Acta 1500:265–279PubMedGoogle Scholar
  56. Sowers AE (1986) A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses. J Cell Biol 102:1358–1362CrossRefPubMedGoogle Scholar
  57. Sowers AE (1989) Evidence that electrofusion yield is controlled by biologically relevant membrane factors. Biochim Biophys Acta 985:334–338CrossRefPubMedGoogle Scholar
  58. Stenger DA, Kubiniec RT, Purucker WJ, Liang H, Hui SW (1988) Optimization of electrofusion parameters for efficient production of murine hybridomas. Hybridoma 7:505–518CrossRefPubMedGoogle Scholar
  59. Stenger DA, Kaler KV, Hui SW (1991) Dipole interactions in electrofusion. Contributions of membrane potential and effective dipole interaction pressures. Biophys J 59:1074–1084CrossRefPubMedGoogle Scholar
  60. Stevens RH, Macy E, Morrow C, Saxon A (1979) Characterization of a circulating subpopulation of spontaneous antitetanus toxoid antibody producing B cells following in vivo booster immunization. J Immunol 122:2498–2504PubMedGoogle Scholar
  61. Stuhler G, Walden P (1994) Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes. Cancer Immunol Immunother 39:342–345 CrossRefPubMedGoogle Scholar
  62. Sukharev SI, Bandrina IN, Barbul AI, Fedorova LI, Abidor IG, Zelenin AV (1990) Electrofusion of fibroblasts on the porous membrane. Biochim Biophys Acta 1034:125–131PubMedGoogle Scholar
  63. Sukhorukov VL, Arnold WM, Zimmermann U (1993) Hypotonically induced changes in the plasma membrane of cultured mammalian cells. J Membr Biol 132:27–40PubMedGoogle Scholar
  64. Sukhorukov VL, Reuss R, Zimmermann D, Held C, Müller KJ, Kiesel M, Geßner P, Steinbach A, Schenk WA, Bamberg E, Zimmermann U (2005) Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion. J Membr Biol 206:187–201CrossRefPubMedGoogle Scholar
  65. Sukhorukov VL, Reuss R, Endter JM, Fehrmann S, Katsen-Globa A, Gessner P, Steinbach A, Muller KJ, Karpas A, Zimmermann U, Zimmermann H (2006) A biophysical approach to the optimisation of dendritic–tumour cell electrofusion. Biochem Biophys Res Commun 346:829–839CrossRefPubMedGoogle Scholar
  66. Teissie J, Blangero C (1984) Direct experimental evidence of the vectorial character of the interaction between electric pulses and cells in cell electrofusion. Biochim Biophys Acta 775:446–448CrossRefPubMedGoogle Scholar
  67. Teissie J, Ramos C (1998) Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes. Biophys J 74:1889–1898CrossRefPubMedGoogle Scholar
  68. Teissie J, Rols MP (1986) Fusion of mammalian cells in culture is obtained by creating the contact between cells after their electropermeabilization. Biochem Biophys Res Commun 140:258–266CrossRefPubMedGoogle Scholar
  69. Teissie J, Knutson VP, Tsong TY, Lane MD (1982) Electric pulse-induced fusion of 3T3 cells in monolayer culture. Science 216:537–538CrossRefPubMedGoogle Scholar
  70. Trontelj K, Rebersek M, Kanduser M, Serbec VC, Sprohar M, Miklavcic D (2008) Optimization of bulk cell electrofusion in vitro for production of human–mouse heterohybridoma cells. Bioelectrochemistry 74:124–129CrossRefPubMedGoogle Scholar
  71. Urano N, Higashikawa R, Hirai H (1998) Effects of mitochondria on electrofusion of yeast protoplasts. Enzyme Microbial Technol 23:107–112CrossRefGoogle Scholar
  72. Usaj M, Trontelj K, Hudej R, Kanducer M, Miklavcic D (2009) Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization. Radiol Oncol 43:108–119CrossRefGoogle Scholar
  73. Vienken J, Zimmermann U (1985) An improved electrofusion technique for production of mouse hybridoma cells. FEBS Lett 182:278–280CrossRefPubMedGoogle Scholar
  74. Wang J, Lu C (2006) Microfluidic cell fusion under continuous direct current voltage. Appl Physics Lett 89:234102–234103CrossRefGoogle Scholar
  75. Wu Y, Montes JG, Sjodin RA (1992) Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols. Biophys J 61:810–815CrossRefPubMedGoogle Scholar
  76. Yu X, McGraw PA, House FS, Crowe JE Jr (2008) An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies. J Immunol Methods 336:142–151CrossRefPubMedGoogle Scholar
  77. Zheng Q, Chang DC (1991) Reorganization of cytoplasmic structures during cell–cell fusion. J Cell Sci 100:431–442PubMedGoogle Scholar
  78. Zimmermann U (1982) Electric field–mediated fusion and related electrical phenomena. Biochim Biophys Acta 694:227–277PubMedGoogle Scholar
  79. Zimmermann U, Neil GA (1996) Electromanipulation of cells. CRC, Boca Raton, FLGoogle Scholar
  80. Zimmermann U, Friedrich U, Mussauer H, Gessner P, Hamel K, Sukhoruhov V (2000) Electromanipulation of mammalian cells: fundamentals and application. IEEE Trans Plasma Sci 28:72–82CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Marko Ušaj
    • 1
  • Katja Trontelj
    • 1
  • Damijan Miklavčič
    • 1
  • Maša Kandušer
    • 1
  1. 1.Faculty of Electrical EngineeringUniversity of LjubljanaLjubljanaSlovenia

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