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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Electrofusion
The electrofusion process is done by applying a series of electrical
waveforms. These are described in this section. The physics and
theory of these processes are described in the Cell Electrofusion
Tutorial, pages 29–43. Follow the set-up procedures described on
pages 19–22.
Pre-Fusion Pulse AC
The first step is to align the cells in a chain and then compress by
increasing the force. This is done by applying a high frequency sine
wave with varying amplitude to the cells in Cytofusion Medium
immediately prior to applying the fusion pulse. Initially the cells are
aligned using a lower amplitude waveform. Once aligned, additional
force is applied to compress the cells. This provides intimate
contact of the cell membranes for optimum fusion. There are two
published approaches for this first step which are presented in the
table on page 43.
The optimum process will be dependent on cell type. The user is
encouraged to develop protocols for the specific cell line in use.
BTX recommends starting with the two level sine wave. Use a low
amplitude to align and a high amplitude to compress, Figure 19
Non-linear Step. Reasonable values are:
40 volts peak for 15 seconds at 0.8 Mhz
75 volts peak for 20 seconds at 0.8 Mhz
The 2 ml Optimization Chamber supplied with the Hybrimune
System has a clear bottom, see Figure 15. This is so a microscope
can be used to observe the cell alignment. Cell compression
cannot be observed. The cells should align in an orderly manner
and migrate toward the inner electrode. No turbulence or violent
movements should be seen. Electrically, the 9 ml chamber and
the 2 ml chamber are identical. Protocols optimized using the 2
ml Optimization Chamber can be directly used in the 9 ml Batch
Production Chamber.
The causes of turbulence, if it is observed, are listed below:
• High current due to high ion content
Dirty electrode
Contaminated or high conductivity medium
Inadequate cell wash
• Excessive force applied to cells
AC amplitude too high
AC amplitude too long
On the front of the Hybrimune voltage waveform generator there is
a bar graph labeled “Relative Load Indicator”. It will illuminate bars
during waveform delivery depending on a number of factors. See
Pages 11 and 14. Generally, the load Indicator should have green
and possibly yellow lights illuminated during sine wave delivery.
If the indicator shows red bars illuminated then the current is too
high and aqueous heating and turbulence can occur.
Fusion Pulse
The next step after aligning and compressing the cells is to apply
one or more high voltage pulses to induce pores in adjacent
cell membranes followed by fusion of the same adjacent cell
membranes. The principles of pore formation for electrofusion
are the same as those for pore formation for electroporation.
The difference is that in electrofusion, the pores are forming in
intimately touching cell membranes. This is a short but essential
step in the electrofusion process. An example of an electrofusion
pulse used for the Hybrimune System is the application of one
1000 Volt pulse for 40 microseconds.
Post-Fusion Pulse AC
After the fusion pulse has been applied, the cell fusion process
starts. The connections between cells are delicate until the fusion
process begins to mature. A post pulse AC waveform is applied
for 30 seconds to 1 minute after the pulse to hold delicately
connected cells together immediately after the pulse.
BTX recommends starting with a decreasing linear ramp, see
Figure 19. The start amplitude is lower than the ending amplitude
of the pre fusion pulse sine wave amplitude. If 70 was used then
start at 50 or 60 volts. End at 5 volts over 30 to 60 seconds. Set
frequency to 0.8 Mhz.
Producing Fusion Products Using Hybrimune
™