BTX ECM630 - Waveforms and Pulse Parameters; Optimizing Field Strength and Parameters

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Publicaon 5423-020-REV 1.0 • www.btxonline.com
As described, electroporaon is the applicaon of controlled
direct current (DC) electrical pulses which are applied to living
cells and ssues for a short duraon of me. The pulse induces a
transmembrane potenal which causes the reversible breakdown
of the cellular membrane. This acon results in the permeaon
or “pore formaon” of the cell membrane which allows small
molecules (such as dye, oligonucleodes or pepdes) and large
molecules (such as proteins, DNA and RNA) to be introduced
into the cell. During this process the cellular uptake of the
molecules connues unl the pores close, which can take
milliseconds to minutes.
Opmizaon of the electroporaon process involves several
factors. Choosing the waveform, determining eld strength
and adjusng pulse length are just a few crical variables.
Other parameters which play a crucial role in opmizaon
include cell diameter, plasmid concentraons, temperature and
electroporaon buer.
Waveforms
Pulse shape generally falls into two categories, square wave or
exponenal decay wave:
Square Wave Pulse
Square wave pulses rise quickly to a set voltage level, maintain
this level during the duraon of the set pulse length and quickly
turn o. Square waves yields higher eciencies and viabilies
in mammalian cells. Square wave electroporaon in in vivo and
ex vivo ssues, embryos, and plant protoplast applicaons yield
beer results in comparison to an exponenal decay wave.
Exponenal Decay Wave Pulse
Exponenal decay waves generate an electrical pulse by allowing
a capacitor to completely discharge. As a pulse is discharged
into a sample, the voltage rises rapidly to the peak voltage set
then declines over me. The powerful exponenal decay wave
pulse is rounely used for transformaon of gram-negave and
gram-posive bacteria, yeast, plant ssues, insect cells and some
mammalian cells.
Field Strength
The eld strength is measured as the voltage delivered across an
electrode gap and is expressed as kV/cm. Field strength is crical
to surpassing the electrical potenal of the cell membrane to
allow the temporary reversible permeaon or “pore formaon”
to occur in the cell membrane. Three factors should be considered
for opmizing eld strength:
1. Electrode Gap Size
2. Cell Diameter
3. Temperature
Cell Type Field Strength Ranges
Bacteria/Yeast: 3 – 24 kV/cm
Mammalian: 0.25 – 3 kV/cm
Plant: 3 – 12 kV/cm
Electrode Gap Size
The distance between electrodes, or “gap size” is important when
opmizing your electroporaon experiment. Field strength is
calculated using voltage divided by gap size. For example, using a
4 mm gap cuvee with 500 V would provide a eld strength of
1.25 kV/cm. If instead of a 4 mm gap cuvee, a 2 mm gap
cuvee was used, the voltage would have to be reduced by
half or to 250 V in order to maintain the same eld strength
of 1.25 kV/cm. It is possible to derive the voltage needed to
accomplish electroporaon if the desired eld strength and
gap size are known. The calculaon for this is eld strength (kV)
mulplied by gap size (cm) equals voltage. For example, if a user
was certain that a 1.25 kV/cm eld strength was required in a
1 mm gap cuvee the calculaon would be: 1.25 kV x 0.1 cm =
0.125 kV or 125 V.
Example: A eld strength of 1.25 kV/cm
4 mm gap cuvee = 500 V
2 mm gap cuvee = 250 V
1 mm gap cuvee = 125 V
Cell Diameter
Generally, smaller cell sizes require higher voltages while larger
cell diameters require lower voltages for successful cell
membrane permeaon.
Temperature
The temperature at which cells are maintained during
electroporaon eects the eciency of the electroporaon
for several reasons. The majority of mammalian cell lines are
eecvely electroporated at room temperature. Samples which
are pulsed at high voltage or exposed to mulple pulses and
long pulse duraons can cause the sample to heat up. These
condions cause increased cell death and lower the transfecon
eciency. Maintaining the sample at lower temperatures can
diminish the heang eects on cell viability and eciency. Since
electroporaon causes the transient formaon of pores, keeping
the cells at a lower temperature following the pulse may allow
the pores to remain open longer to allow more uptake of the
exogenous molecules. Yet lower temperatures on other cell
lines can be damaging and cause high cell mortality. This eect
is specic to each cell line and should be considered during
opmizaon studies. The standard pulse voltage used for cells
at room temperature will need to be approximately doubled for
electroporaon at 4°C in order to eecvely permeate the cell
membrane.
General Optimization Guide for Electroporation