Study type: Medical/biological study (experimental study)

Electric-field-induced permeabilization and fusion of embryonic amphibian cells. tech./dosim.

Published in: Exp Cell Res 1989; 184 (1): 207-218

Aim of study (acc. to author)

To determine adequate experimental conditions that allow electropermeabilization without damage to embryonic amphibian cells.

Background/further details

The technique of electropulsation has been shown to be highly efficient in promoting penetration of exogenous molecules into living cells, transfection, and cell fusion in different vegetal, animal, and bacterial cell systems. Introduction of such exogenous compounds, i.e., plasmids, into living cells is of interest for embryological studies.
Embryonic amphibian ectodermal cells from Pleurodeles waltl gastrulae were used.

Endpoint

Exposure

Exposure Parameters
Exposure 1: 1 Hz
Modulation type: pulsed
Exposure duration: 5 pulses
Exposure 2: 1 Hz
Modulation type: pulsed
Exposure duration: 2 sets (5 s interval) of 3 pulses

Exposure 1

Main characteristics
Frequency 1 Hz
Type
Exposure duration 5 pulses
Modulation
Modulation type pulsed
Pulse width 100 µs
Pulse type rectangular
Exposure setup
Exposure source
  • electropulsator connected to a pair of parallel stainless steel electrodes
Setup The electrodes (5 or 10 mm apart) were dipped in half diluted Holtfreter's solution used as pulsating medium (1 mm thick, 4°C) and seeded against the bottom of the culture dish.
Additional info The whole procedure was performed on ice at 4°C.
Parameters
Measurand Value Type Method Mass Remarks
electric field strength 140 kV/m maximum measured - -

Exposure 2

Main characteristics
Frequency 1 Hz
Type
Exposure duration 2 sets (5 s interval) of 3 pulses
Modulation
Modulation type pulsed
Pulse width 100 µs
Exposure setup
Exposure source
Additional info The electrodes were rotated 90° between the two sets of pulsation.
Parameters
Measurand Value Type Method Mass Remarks
electric field strength 190 kV/m maximum measured - -

Exposed system:

Methods Endpoint/measurement parameters/methodology

Investigated system:
Time of investigation:
  • after exposure

Main outcome of study (acc. to author)

The data demonstrated that embryonic gastrula cells from the amphibian P. waltl, either freshly dissociated or cultured for 5 days, could be permeabilized when submitted to an external electric field using high-voltage square wave pulses: 500 V/cm for isolated spherical cells and 150-200 V/cm for plated cultured cells.
With the application of higher field intensities (600 V/cm for freshly dissociated cells and 300 V/cm for cultured cells) cell fusion and syncytial structures could also be obtained.
Isolated spherical cells had 100% viability immediately after being pulsed at intensities up to 600 V/cm. Cell lysis was revealed above this value, although the nonlysed cells were observed to spread on a substrate and differentiate normally.
For the cultured plated cells, cell viability fell with increasing electric-field strength. Nevertheless, for electric-field intensities less than or equal to 300 V/cm, 100% of the cells remained attached to the substrate and differentiated normally over the following 5 days.

Study character:

Study funded by