European Biophysics Journal

, Volume 36, Issue 3, pp 173–185 | Cite as

Electropermeabilization of dense cell suspensions

  • Gorazd Pucihar
  • Tadej Kotnik
  • Justin Teissié
  • Damijan Miklavčič
Original Paper


This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 106 cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 106 to 400 × 106 cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 106 and 400 × 106 cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.


Electroporation Cell pellets Propidium iodide Membrane resealing 

List of symbols


induced transmembrane voltage, V


cell radius, m


applied electric field, V/m


angle between E and the normal vector to the membrane, °


critical angle where permeabilization occurs, °


critical amplitude of the electric field, V/m


permeabilized surface of the membrane, m2


total area of the membrane, m2


angle, °


flow of molecules, mol/s


permeability coefficient, m/s


concentration difference of the molecule S, mol/m3


fraction of membrane defects in the permeabilized region, –


fraction of membrane defects immediately after the onset of permeabilizing pulse, –


time, s


number of pulses, –


pulse duration, s


resealing rate, 1/s


fraction of permeabilized surface where cell contacts are not present, –


total concentration of PI after permeabilization (cells + external medium), mol/m3


concentration of PI in external medium, mol/m3


total volume (cells + external medium), m3


volume of external medium, m3


eagle’s minimum essential medium, –


propidium iodide, –


Chinese hamster ovary cells, –



The author (G. P.) would like to thank Dr. M. Golzio, Dr. M. P. Rols and Dr. B. Gabriel for their valuable discussions during the experiments, and Ms. C. Millot for her help with cell cultures. This work was supported by the Ministry of the Higher Education and Science of the Republic of Slovenia. G. P. was also a recipient of a scholarship from the French government. The two institutes are partners in a Slovenian-French CNRS PICS program.


  1. 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 1152:207–218CrossRefGoogle Scholar
  2. Abidor IG, Li LH, Hui SW (1994) Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions. Biophys J 67:427–435Google Scholar
  3. Barnett A, Weaver JC (1991) Electroporation: a unified, quantitative theory of reversible electrical breakdown and rupture. Bioelectrochem Bioenerg 25:163–182CrossRefGoogle Scholar
  4. Canatella PJ, Black MM, Bonnichsen DM, McKenna C, Prausnitz MR (2004) Tissue electroporation: quantification and analysis of heterogeneous transport in multicellular environments. Biophys J 86:3260–3268Google Scholar
  5. Čemažar M, Grošel A, Glavač D, Kotnik V, Škobrne M, Kranjc S, Mir LM, Andre F, Opolon P, Serša G (2003) Effects of electrogenetherapy with p53wt combined with cisplatin on survival of human tumor cell lines with different p53 status. DNA Cell Biol 22:765–775CrossRefGoogle Scholar
  6. Fattori E, Cappelletti M, Zampaglione I, Mennuni C, Calvaruso F, Arcuri M, Rizzuto G, Costa P, Perretta G, Ciliberto G, La Monica N (2005) Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates. J Gene Med 7:228–236CrossRefGoogle Scholar
  7. Golzio M, Mora MP, Raynaud C, Delteil C, Teissié J, Rols MP (1998) Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J 74:3015–3022Google Scholar
  8. Golzio M, Teissié J, Rols MP (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci 99:1292–1297CrossRefADSGoogle Scholar
  9. Gothelf A, Mir LM, Gehl J (2003) Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev 29:371–387CrossRefGoogle Scholar
  10. Grosse C, Schwan HP (1992) Cellular membrane potentials induced by alternating fields. Biophys J 63:1632–1642Google Scholar
  11. Kinosita K, Tsong TY (1977) Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature 268:438–441CrossRefADSGoogle Scholar
  12. Kotnik T, Bobanović F, Miklavčič D (1997) Sensitivity of transmembrane voltage induced by applied electric fields—a theoretical analysis. Bioelectrochem Bioenerg 43:285–291CrossRefGoogle Scholar
  13. Li LH, Ross P, Hui SW (1999) Improving electrotransfection efficiency by post pulse centrifugation. Gene Ther 6:364–372CrossRefGoogle Scholar
  14. Mir LM, Orlowski S (1999) Mechanisms of electrochemotherapy. Adv Drug Deliver Rev 35:107–118CrossRefGoogle Scholar
  15. Neumann E, Ridder MS, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845Google Scholar
  16. Neumann E, Toensing K, Kakorin S, Budde P, Frey J (1998) Mechanism of electroporative dye uptake by mouse B cells. Biophys J 74:98–108Google Scholar
  17. Neumann E, Kakorin S, Toensing K (1999) Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem Bioenerg 48:3–16CrossRefGoogle Scholar
  18. Okino M, Mohri H (1987) Effects of high-voltage electrical impulse and an anticancer drug on in vivo growing tumors. Jpn J Cancer Res 78:1319–1321Google Scholar
  19. Pauly H, Schwan HP (1959) Über die impedanz einer suspension von kugelförmigen teilchen mit einer schale. Z Naturforsch 14B:125–131Google Scholar
  20. Pavlin M, Pavšelj N, Miklavčič D (2002) Dependence of induced transmembrane potential on cell density, arrangement, and cell position inside a cell system. IEEE Trans Biomed Eng 49:605–612CrossRefGoogle Scholar
  21. Pavlin M, Kandušer M, Reberšek M, Pucihar G, Hart FX, Magjarević R, Miklavčič D (2005) Effect of cell electroporation on the conductivity of a cell suspension. Biophys J 88:4378–4390CrossRefGoogle Scholar
  22. Pucihar G, Kotnik T, Valič B, Miklavčič D (2006) Numerical determination of transmembrane voltage induced on irregularly shaped cells. Annals Biomed Eng 34:642–652CrossRefGoogle Scholar
  23. Rols MP, Teissié J (1990) Electropermeabilization of mammalian cells: quantitative analysis of the phenomenon. Biophys J 58:1089–1098Google Scholar
  24. Rols MP, Teissié J (1998) Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 75:1415–1423Google Scholar
  25. Rols MP, Dahhou F, Mishra KP, Teissie J (1990) Control of electric field induced cell membrane permeabilization by membrane order. Biochemistry 29:2960–2966CrossRefGoogle Scholar
  26. Rols MP, Delteil C, Golzio M, Teissié J (1998) Control by ATP and ADP of voltage-induced mammalian-cell-membrane permeabilization, gene transfer and resulting expression. Eur J Biochem 254:382–388CrossRefGoogle Scholar
  27. Šatkauskas S, Bureau MF, Puc M, Mahfoudi A, Scherman D, Miklavčič D, Mir LM (2002) Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis. Mol Ther 5:133–140CrossRefGoogle Scholar
  28. Šatkauskas S, Batiuškaite D, Šalomskaite-Davalgiene S, Venslauskas MS (2005) Effectiveness of tumor electrochemotherapy as a function of electric pulse strength and duration. Bioelectrochemistry 65:105–111CrossRefGoogle Scholar
  29. Schmeer M, Seipp T, Pliquett U, Kakorin S, Neumann E (2004) Mechanism for the conductivity changes caused by membrane electroporation of CHO cell-pellets. PCCP 6:5564–5574ADSGoogle Scholar
  30. Schwan HP (1957) Electrical properties of tissue and cell suspensions. Adv Biol Med Phys 5:147–209Google Scholar
  31. Serša G, Čemažar M, Miklavčič D (1995) Antitumor effectiveness of electrochemotherapy with cis-diammindichloroplatinum(II) in mice. Cancer Res 55:3450–3455Google Scholar
  32. Sixou S, Teissié J (1993) Exogeneous uptake and release of molecules by electroloaded cells: a digitized videomicroscopy study. Bioelectrochem Bioenerg 31:237–257CrossRefGoogle Scholar
  33. Susil R, Šemrov D, Miklavčič D (1998). Electric field induced transmembrane potential depends on cell density and organization. Electro Magnetobiol 17:391–399Google Scholar
  34. Teissié J, Eynard N, Gabriel B, Rols MP (1999) Electropermeabilization of cell membranes. Adv Drug Deliver Rev 35:3–19CrossRefGoogle Scholar
  35. Tsong TY (1991) Electroporation of cell membranes. Biophys J 60:297–306CrossRefGoogle Scholar
  36. Weaver JC, Chizmadzhev YA (1996) Theory of electroporation: a review. Bioelectrochem Bioenerg 41:135–160CrossRefGoogle Scholar

Copyright information

© EBSA 2007

Authors and Affiliations

  • Gorazd Pucihar
    • 1
  • Tadej Kotnik
    • 1
  • Justin Teissié
    • 2
  • Damijan Miklavčič
    • 1
  1. 1.Faculty of Electrical EngineeringUniversity of LjubljanaLjubljanaSlovenia
  2. 2.IPBSCNRS, UMR 5089Toulouse CedexFrance

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