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Electropermeabilization of dense cell suspensions

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Abstract

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.

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Abbreviations

ΔΨ :

induced transmembrane voltage, V

R :

cell radius, m

E :

applied electric field, V/m

ϕ:

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

ϕ c :

critical angle where permeabilization occurs, °

E S :

critical amplitude of the electric field, V/m

A perm :

permeabilized surface of the membrane, m2

A tot :

total area of the membrane, m2

α:

angle, °

Φ :

flow of molecules, mol/s

P :

permeability coefficient, m/s

ΔS :

concentration difference of the molecule S, mol/m3

F(t):

fraction of membrane defects in the permeabilized region, –

F *(N,T):

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

t :

time, s

N :

number of pulses, –

T :

pulse duration, s

k :

resealing rate, 1/s

F NC :

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

c :

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

c 1 :

concentration of PI in external medium, mol/m3

V :

total volume (cells + external medium), m3

V 1 :

volume of external medium, m3

EMEM:

eagle’s minimum essential medium, –

PI:

propidium iodide, –

CHO:

Chinese hamster ovary cells, –

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Acknowledgments

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.

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Authors and Affiliations

  1. Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, 1000, Ljubljana, Slovenia

    Gorazd Pucihar, Tadej Kotnik & Damijan Miklavčič

  2. IPBS, CNRS, UMR 5089, 205 Route de Narbonne, Toulouse Cedex, France

    Justin Teissié

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  1. Gorazd Pucihar
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  2. Tadej Kotnik
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  4. Damijan Miklavčič
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Correspondence to Justin Teissié.

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Pucihar, G., Kotnik, T., Teissié, J. et al. Electropermeabilization of dense cell suspensions. Eur Biophys J 36, 173–185 (2007). https://doi.org/10.1007/s00249-006-0115-1

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  • DOI: https://doi.org/10.1007/s00249-006-0115-1

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