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Improving electrotransfection efficiency by post-pulse centrifugation

Gene Therapy volume 6, pages 364–372 (1999)Cite this article

Abstract

We have demonstrated that the viability of electrotransfected adherent CHO and suspended NK-L, K-562, L1210 and MC2 cells is improved if pelleting by centrifugation is performed immediately after pulsing. The protection effect on cell viability is cell line- and pellet thickness-dependent. For forming CHO cell pellets, centrifugation force (300–13000 g) and duration are not crucial; about five to 10 cell layers in the pellet provide the optimal protection effect. NK-L, K-562, L1210 and MC2 cell pellets are optimally formed by centrifugation at 13000 g in an Eppen- dorf desktop centrifuge. Pelleting improves the cell viability over the whole range of the NK-L, K-562, L1210 and MC2 cell concentrations studied. When this pelleting method is applied to load CHO cells with FITC-dextran (41000 MW), not only is the success rate close to 100%, but the growth rate is similar to the control, which is far better than the conventional electroporation method. Furthermore, the transfection efficiency of the five cell lines in pellet is significantly higher than that in suspension.

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References

  1. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH . Gene transfer into mouse lyoma cells by electroporation in high electric fields EMBO J 1982 7: 841–845

    Article  Google Scholar 

  2. Zimmermann U, Stopper H, Jones H . Large scale transfection of mouse L-cells by electropermeabilization Biochim Biophys Acta 1987 900: 38–44

    Article  Google Scholar 

  3. Li LH, Hensen ML, Zhao YL, Hui SW . Electrofusion between heterogeneous-size mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation Biophys J 1996 71: 479–486

    Article  CAS  Google Scholar 

  4. Tekle E, Astumian RD, Chock PB . Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3T3 cells Proc Natl Acad Sci USA 1991 88: 4230–4234

    Article  CAS  Google Scholar 

  5. Xie TD, Sun L, Tsong TY . Study of mechanisms of electric field-induced DNA transfection I Biophys J 1990 58: 13–19

    Article  CAS  Google Scholar 

  6. Hui SW, Stoicheva N, Zhao YL . High-efficiency loading, transfection and fusion of cells by electroporation in two-phase polymer system Biophys J 1996 71: 1123–1130

    Article  CAS  Google Scholar 

  7. Wolf H et al. Control by pulse parameters of electric field-mediated gene transfer in mammalian cells Biophys J 1994 66: 524–531

    Article  CAS  Google Scholar 

  8. Anderson GL, Evens GA . Optimization of electroporation for transfection of mammalian cell lines Analyt Biochem 1989 180: 269–275

    Article  Google Scholar 

  9. Chang DC, Gao PQ, Maxwell BL . High efficiency gene transfection by electroporation using a radio-frequency electric field Biochim Biophys Acta 1991 1992: 153–160

    Article  Google Scholar 

  10. Abidor IG, Li LH and Hui SW . Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions Biophys J 1994 67: 427–435

    Article  CAS  Google Scholar 

  11. Klenchin VA et al. Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis Biophys J 1991 60: 804–811

    Article  CAS  Google Scholar 

  12. Hui SW . Effects of pulse length and strength on electroporation efficiency Meth Molec Biol 1995 2: 29–40

    Google Scholar 

  13. Liang H et al. Uptake of fluorescence-labeled dextrans by 10T 1/2 fibroblasts following permeation by rectangular and exponential-decay electric field pulses BioTechniques 1988 6: 550–558

    CAS  PubMed  Google Scholar 

  14. Kubiniec RT, Liang H, Hui SW . Effects of pulse length and pulse strength on transfection by electroporation BioTechniques 1990 8: 1–3

    Google Scholar 

  15. Abidor IG, Li LH, Hui SW . Studies of cell pellets: I. Electrical properties and porosity Biophys J 1994 67: 418–426

    Article  CAS  Google Scholar 

  16. Kinosita K, Tsong TY . Voltage-induced pore formation and hemolysis of human erythrocytes Biochim Biophys Acta 1977 471: 227–242

    Article  CAS  Google Scholar 

  17. Li LH, Hui SW . Characterization of PEG-mediated electrofusion of human erythrocytes Biophys J 1994 67: 2361–2366

    Article  CAS  Google Scholar 

  18. Ohno-Shosaku T, Okada Y . Electric pulse-induced fusion of mouse lymphoma cells: roles of divalent cations and membrane lipid domains J Membrane Biol 1985 85: 269–280

    Article  CAS  Google Scholar 

  19. 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: 258–266

    Article  CAS  Google Scholar 

  20. Chernomordik LV, Sokolov AV, Budker VG . Electrostimulated uptake of DNA by liposomes Biochim Biophys Acta 1990 1024: 179–183

    Article  CAS  Google Scholar 

  21. Stenger DA et al. Optimization of electrofusion parameters for efficient production of murine hybridomas Hybridoma 1988 7: 505–518

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Membrane Biophysics Laboratory, Roswell Park Cancer Institute, Buffalo, NY, USA

    L H Li, P Ross & S W Hui

  2. Biomedical Engineering Department, Hunan Medical University, Changsha, P.R. China

    L H Li

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  1. L H Li
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  2. P Ross
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  3. S W Hui
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Li, L., Ross, P. & Hui, S. Improving electrotransfection efficiency by post-pulse centrifugation. Gene Ther 6, 364–372 (1999). https://doi.org/10.1038/sj.gt.3300828

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