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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Electrofusion Pulse
The function of this waveform is to form pathways in the cell
membranes to allow the cytoplasm to mix and the cells to fuse.
One or more high electric field pulses are needed. This process
is electroporation and is illustrated in Figure 21. An electric
field is applied that is high enough to induce a voltage across
the cell membrane that induces pore formation. The minimum
transmembrane voltage is approximately one volt. This requires
applying electric fields of hundreds of volts/cm to achieve the
at least one volt across the cell membrane. Small diameter cells
require higher electric fields than do large diameter cells.
Theoretically, the formation of a pore in one cell increases the
likelihood that a pore will form in an adjacent cell membrane in
the same location as the original pore. This is because pores in cell
membranes conduct electricity through the pore.
Whatever the actual mechanism of formation, pores in cells whose
membranes are touching can re-seal in one of two ways. One is that
the pore will simply close in the cell membrane. Another is that
adjacent pores in the membranes of two touching cells will fuse
together, combining the cell membranes of two cells.
Post Fusion Pulse AC Sine Wave
The objective of this function is to hold in place, the cells that
have been fused together, to allow them to mature. Immediately
stopping the AC sine wave causes the cells to break apart by
thermal motion. The same AC Amplitude functions described in
Pages 39–41 can be used. Experience has shown that a simple
decreasing Linear Ramp Amplitude (k=1) has proven to be the
most effective.
Parameter Range of Hybrimune
™
Voltage Waveform
Generator
The following parameters may be programmed in the waveform
generator.
Figure 21: Electric Field Pulse Forms Pathways
AC Before
Fusion Pulse
and
AC After
Fusion Pulse
Amplitude
5 to 75 v peak, 5 volts steps
Amplitude
function
Constant Linear increase or
decrease (k=1)
Step Ramp k = 0.1 to 20
Frequency
0.2 to 2.0 MHz
in 0.2 MHz steps
Duration
0 to 60 seconds in
1 second steps
Set zero inhibits AC
Fusion Pulse
Amplitude
100 to 1000 volts
in 5 volt steps
Width
20 to 1000 µs in 10 µs steps
Interval
0.125 seconds
in 0.001 second steps
Number
0 to 10 in steps of 1
0 inhibits pulse
Cell Electrofusion Tutorial