Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization
Background. Various electrofusion parameters have to be adjusted to obtain the optimal electrofusion efficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treatment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and viability of four different cell lines in hypotonic buffer and to study the influence of the electroporation on the selected cell line in hypotonic buffer.
Materials and methods. Cell size dynamics of different cell lines exposed to hypotonic buffer and electroporation were analyzed by time-resolved cell size measurements. The viability of hypotonically treated or/and electroporated cells was determined 24 h after the experiment by a modified crystal violet (CV) viability assay.
Results. In our experimental conditions the hypotonic treatment at 100 mOsm was efficient for CHO, V79 and B16-F1 cell lines. The optimal duration of the treatment was between two and five minutes. On the other hand the same hypotonic treatment did not cause cell swelling of NS1 cells. Cell swelling was also observed after electroporation of B16-F1 in isotonic buffer and it was amplified when hypotonic buffer was used. In addition, the regulatory volume decrease was successfully inhibited with electroporation.
Conclusions. Cell size dynamics in hypotonic conditions should be studied for each cell line since they differ in their sensitivity to the hypotonic treatment. The inhibition of cell regulatory volume decrease by electroporation may be beneficial in achieving higher electrofusion efficiency. The hypotonic treatment in itself did not significantly affect the cell viability; however, electric field parameters for electroporation should be carefully selected taking into account the hypotonically induced volume increase of cells.
Chang DC, Chassy BM, Saunders JA, Sowers AE. Guide to Electroporation and Electrofusion. San Diego: Academic Press; 1992.
Hui SW, Stenger DA. Electrofusion of cells: Hybridoma production by electrofusion and polyethylene glycol. Method Enzymol 1993; 220: 212-27.
Zimmermann U, Friedrich U, Mussauer H, Gessner P, Hämel K, Sukhorukov V. Electromanipulation of mammalian cells: Fundamentals and application. IEEE IEEE Trans Plasma Sci 2000; 28: 72-82.
Zimmermann U. Electric field-mediated fusion and related electrical phenomena. Biochim Biophys Acta 1982; 694: 227-77.
Teissie J, Rols MP. Fusion of mammalian cells in culture is obtained by creating the contact between cells after their electropermeabilization. Biochem Biophys Res Commun 1986; 140: 258-66.
Neumann E, Kakorin S. Digression on membrane electroporation and electroporative delivery of drugs and genes. Radiol Oncol 1998; 32: 7-17.
Rudolf Z, Stabuc R, Cemazar M, Miklavcic D, Vodovnik L, Sersa G. Electrochemotherapy with bleomycin. The first clinical experience in malignant melanoma patients. Radiol Oncol 1995; 29: 229-35.
Cemazar M, Miklavcic D, Vodovnik L, Jarm T, Rudolf Z, Stabuc R, et al. Improved therapeutic effect of electrochemotherapy with cisplatin by intratumoral drug administration and changing of electrode orientation for electropermeabilization on EAT tumor model in mice. Radiol Oncol 1995; 29: 121-7.
Sersa G, Cemazar M, Miklavcic D. Tumor blood flow modifying effects of electrochemotherapy: a potential vascular targeted mechanism. Radiol Oncol 2003; 37: 43-8.
Sersa G, Cemazar M, Miklavcic D, Rudolf Z. Electrochemotherapy of tumours. Radiol Oncol 2006; 40: 163-74.
Pavselj N, Miklavcic D. Numerical modeling in electroporation-based biomedical applications. Radiol Oncol 2008; 42: 159-68.
Zupanic A, Corovic S, Miklavcic D. Optimization of electrode position and electric pulse amplitude in electrochemotherapy. Radiol Oncol 2008; 42: 93-101.
Teissié J, Rols M. An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. Biophys J 1993; 65: 409-413.
Weaver JC, Chizmadzhev YA. Theory of electroporation: A review. Bioelectrochem Bioener 1996; 41: 135-60.
Neumann E, Sowers AE, Jordan CA. Electroporation and electrofusion in cell biology. New York: Plenum Press; 1989.
Cemazar M, Jarm T, Miklavcic D, Macek-Lebar A, Ihan A, Kopitar NA, et al. Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro. Electro Magnetobiol 1998; 17: 263-72.
Canatella PJ, Karr JF, Petros JA, Prausnitz MR. Quantitative study of electroporation-mediated molecular uptake and cell viability. Biophys J 2001; 80: 755-64.
Macek-Lebar A, Miklavcic D. Cell electropermeabilization to small molecules in vitro: control by pulse parameters. Radiol Oncol 2001; 35: 193-202.
Cegovnik U, Novakovic S. Setting optimal parameters for in vitro electrotransfection of B16F1, SA1, LPB, SCK, L929 and CHO cells using predefined exponentially decaying electric pulses. Bioelectrochemistry 2004; 62: 73-82.
Kanduser M, Sentjurc M, Miklavcic D. Cell membrane fluidity related to electroporation and resealing. Eur Biophys J 2006; 35: 196-204.
Vienken J, Zimmermann U. An improved electrofusion technique for production of mouse hybridoma cells. FEBS Lett 1985; 182: 278-80.
Mally MI, McKnight ME, Glassy MC. Protocols of electroporation and electrofusion for producing human hybridomas. In: Chang D, Chassy B, Saunders J, Sowers A, editors. Guide to Electroporation and Electrofusion. San Diego: Academic Press; 1992. p. 507-22.
Scott-Taylor TH, Pettengell R, Clarke I, Stuhler G, La Barthe MC, Walden P, et al. Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines. Biochim Biophys Acta 2000; 1500: 265-79.
Hayashi T, Tanaka H, Tanaka J, Wang R, Averbook BJ, Cohen PA, et al. Immunogenicity and therapeutic efficacy of dendritic-tumor hybrid cells generated by electrofusion. Clin Immunol 2002; 104: 4-20.
Yu X, McGrawa PA, House FS, Crowe JE Jr. An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies. J Immunol Methods 2008; 336: 142-51.
Schmitt JJ, Zimmermann U. Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions. Biochim Biophys Acta 1989; 983: 42-50.
Zimmermann U, Gessner P, Schnettler R, Perkins S, Foung SK. Efficient hybridization of mouse-human cell lines by means of hypoosmolar electrofusion. J Immunol Methods 1990; 134: 43-50.
Foung S, Perkins S, Kafadar K, Gessner P, Zimmermann U. Development of microfusion techniques to generate human hybridomas. J Immunol Methods 1990; 134: 35-42.
Rols MP, Teissié J. Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure. Biochemistry 1990; 29: 4561-7.
Rehman SMM, Perkins S, Zimmermann U, Foung SKH. Human hybridoma formation by hypo-osmolar electrofusion. In: Chang D, Chassy B, Saunders J, Sowers A, editors. Guide to Electroporation and Electrofusion. Academic Press; 1992. p. 523-33.
Sukhorukov VL, Reuss R, Zimmermann D, Held C, Müller KJ, Kiesel M, et al. Surviving high-intensity field pulses: Strategies for improving robustness and performance of electrotransfection and electrofusion. J Membr Biol 2005; 206: 187-201.
Sukhorukov VL, Reuss R, Endter JM, Fehrmann S, Katsen-Globa A, Geßner P, et al. A biophysical approach to the optimisation of dendritictumour cell electrofusion. Biochem Biophys Res Commun 2006; 346: 829-39.
Kiesel M, Reuss R, Zimmermann D, Zimmermann H, Shirakashi R, Bamberg E, et al. Biophys J 2006; 90: 4720-9.
Pavlin M, Kanduser M, Rebersek M, Pucihar G, Hart FX, Magjarevic R, et al. Effect of cell electroporation on the conductivity of a cell suspension. Biophys J 2005; 88: 4378-439.
Gillies RJ, Didier N, Denton M. Determination of cell number in monolayer cultures. Anal Biochem 1986; 159: 109-13.
Sarkadi B, Attisano L, Grinstein S, Buchwald M, Rothstein A. Volume regulation of Chinese hamster ovary cells in anisoosmotic buffer. Biochim Biophys Acta 1984; 774: 159-68.
Barrau C, Teissié J, Gabriel B. Osmotically induced membrane tension facilitates the triggering of living cell electropermeabilization. Bioelectrochemistry 2004; 63: 327-32.
Reuss R, Ludwig J, Shirakashi R, Ehrhart F, Zimmermann H, Schneider S, et al. Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways. J Membr Biol 2004; 200: 67-81.
Zimmermann D, Terpitz U, Zhou A, Reuss R, Müller K, Sukhorukov VL, et al. Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system. Biochem Biophys Res Commun 2006; 348: 673-81.
Knutton S, Jackson D, Graham JM, Micklem KJ, Pasternak CA. Microvilli and cell swelling. Nature 1976; 262: 52-4.
Sukhorukov VL, Arnold WM, Zimmermann U. Hypotonically induced changes in the plasma membrane of cultured mammalian cells. J Membr Biol 1993; 132: 27-40.
Evans EA, Parsegian VA. Thermal-mechanical fluctuations enhance repulsion between bimolecular layers. Proc Natl Acad Sci USA 1986; 83: 7132-6.
McIntosh TJ, Advani S, Burton RE, Zhelev DV, Needham D, Simon SA. Experimental test for protrusion and undulation pressures in phospholipid bilayers. Biochemistry 1995; 34: 8520-32.
McIntosh TJ, Kulkarni KG, Simon SA. Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations. Biophys J 1999; 76: 2090-8.
Golzio M, Mora MP, Raynaud C, Delteil C, Teissié J, Rols MP. Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J 1998; 74: 3015-22.
Siebens AW, Spring KR. A novel sorbitol transport mechanism in cultured renal papillary epithelial cells. Am J Physiol Cell Physiol 1989; 257(6 Pt 2): F937-46.
Hall JA, Kirk J, Potts JR, Rae C Kirk K. Anion channel blockers inhibit swelling-activated anion, cation, and nonelectrolyte transport in HeLa cells. Am J Physiol Cell Physiol 1996; 271: 579-88.
Neil GA, Zimmermann U. Electrofusion. Methods Enzymol 1993; 220: 174.
Zimmermann U, Neil GA. Electromanipulation of Cells. Boca Raton: CRC Press; 1995.
Kotnik T, Bobanovic F, Miklavcic D. Sensitivity of transmembrane voltage induced by applied electric fields - a theoretical analysis. Bioelectrochem Bioener 1997; 43: 285-91.