BTX ECM630 - General Electroporation Optimization; Electroporation Fundamentals

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DNA Delivery into Cells
Using Electroporaon
General Electroporaon Discussion
Electroporaon is the use of a transmembrane electric eld pulse
to induce microscopic pathways (pores) in a biomembrane. Their
presence allows molecules, ions, and water to pass from one side
of the membrane to the other. When the electric eld is applied,
the ions inside and outside the cell membrane migrate. As the
charge builds up on either side of the membrane the membrane
weakens and the pathways form perming material outside
of the cell to enter. If the electric eld is promptly removed
the pathways close and the membrane reseals. If the electric
eld duraon is too long the pathways increase and the cell is
killed. Ecient electroporaon depends on proper selecon of
electric eld waveforms. The electropores are located primarily
on the membrane areas which are closest to the electrodes. The
pathways form in about a microsecond and seal in seconds to
minutes. The duraon of the electric eld is tens of microseconds
to tens of milliseconds.
The use of electroporaon was described by Neumann in the early
1980’s. The roune use of electroporaon became very popular
with researchers through the 1980’s because it was found to be a
praccal way to place drugs, or other molecules into cells. In the
late 1980’s, sciensts began to use electroporaon for applicaons
in mul-cellular ssue.
In the early 1990’s Lluis Mir of the Instute Gustave-Roussy was
the rst to use electroporaon in a human trial to treat external
tumors.
Research has shown that the inducon of pathways is aected by
three major factors. First, cell-to-cell biological variability causes
some cells to be more sensive to electroporaon than other
cells. Second, for pathways to be induced, the product of the
pulse amplitude and the pulse duraon has to be above a lower
limit threshold. Third, the number of pathways and eecve
pathway diameter increases with the product of “amplitude” and
“duraon.” Although other factors are involved, this threshold
is now understood to be largely dependent on a fourth factor,
the reciprocal of cell size. If the upper limit threshold is reached,
pore diameter and total pore area are too large for the cell
to repair by any spontaneous or biological process, and the
result is irreversible damage to the cell or cell lysis. Because
the mechanism of electroporaon is not well understood,
the development of protocols for a parcular applicaon has
usually been achieved empirically, by adjusng pulse parameters
(amplitude, duraon, number, and inter-pulse interval).
Research shows that certain experimental condions and
parameters of electrical pulses may be capable of causing many
more molecules to move per unit me than simple diusion.
There is also good evidence (Sukharev et al., 1992) that DNA
movement is in the opposite direcon.
An addional important consideraon when the voltage pulse is
applied to the cells and medium is that the amount of current that
ows is dependent on the conducvity of the material in which
the cells are located. Some material is quite conducve and severe
heang will occur if the pulse duraon is too long. Therefore long
duraon elds will kill cells by destroying the membrane and
heang.
The electric eld in which the cells are located is produced by two
system components. The rst is the voltage waveform generator
and the second is the electrode which converts the voltage into
the electric eld.
As the charge accumulates at the membrane, which is a
capacitance, the voltage across the membrane increases:
voltage = capacitance charge
As charge accumulates at the membrane, the voltage across
the membrane increases. Neumann et al. (1989) described the
equaon that relates the transmembrane voltage (TMV) to electric
eld intensity:
where:
Pores in the membrane will begin to form as the voltage increases
from its quiescent value of a few tenths of a volt to more than 0.5
volts. To produce a TMV of 1 volt across the membrane of a cell
with 7 μm radius, the required electric eld intensity is:
The number of pores and eecve pore diameter increase as
the product of pulse amplitude and duraon increase. At the
upper limit threshold, pore diameter and total pore area become
too large for the cell to repair by any spontaneous or biological
process. The result is irreversible damage to the cell or cell lysis.
Another important point to consider is the generaon of heat
during electroporaon. Heat producon is directly related to
current intensity which is, in turn, dependent on the conducvity
of the material through which the electric eld is applied.
Standard saline soluons such as PBS and many ssue culture
media are highly conducve and thus will generate considerable
amounts of heat when used in cell electroporaon. Excessive
heang can be detrimental to cell viability. The eects of heang
can be reduced by using a low conducvity medium such as BTX’s
Cytoporaon Medium to resuspend cells prior to electroporaon.
Although electroporaon is an eecve method for introducing
macromolecules onto cells, the biological mechanisms by which
cells become electroporated are not completely understood.
Therefore, the development of specic protocols for parcular
applicaons is usually achieved by empirical adjustment of
pulse parameters (i.e. amplitude, duraon, pulse number, and
interpulse interval).
E= =
2
3
950 volts/cm
1
7 x 10
-4
*
General Optimization Guide for Electroporation