![background image](http://html.mh-extra.com/html/harvard-bioscience/btx-hybrimune/btx-hybrimune_user-manual_601890037.webp)
37
Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
In summary, the design of the Hybrimune System has been
optimized for electrofusion and many of the variables are fixed
by system design such as the chamber configuration and the
conductivity of the medium. The one powerful tool available
to the user is the selection of the waveform protocol. The
waveform protocol sets the force on the cells for alignment and
compression, the pulse to fuse the cells, and the waveform to
stabilize the fused cells after the pulse. The next section covers the
available waveforms.
Waveforms for Electrofusion
The two most important metrics in an electrofusion system for a
given cell radius are the chamber configuration and the voltage
applied to the chamber to produce the electric field. In the last
section the chamber parameters were discussed. In this section
the voltage waveform parameters will be discussed.
There are two types of waveforms used, alternating current (AC)
in the form of a sine wave and pulsed direct current (DC). The
functions of these waveforms in the electrofusion process are:
Notes:
1. There have been optimized protocols that use
waveforms other than those shown as typical, for
example see the Trevor et. al, 2004.
2. There are many types of AC waveforms, rectangular,
square, trapezoid, triangle, and sine wave.
Hybrimune technology uses sine wave AC waveforms
due their smooth amplitude transition.
AC Waveform Amplitude
The AC waveform amplitude and duration are the only variables
under user control. The user can control force by changing
amplitude. In addition the duration of the AC waveform applies the
force for a given period of time which is also under user control.
Frequency is available but Figure 13 shows it has little effect.
Waveforms for Electrofusion are described in the next section.
A graph is presented in Figure 16 which presents the approximate
force applied to a cell as a function of cell radius of 5, 10 and 20
µm as a function of AC peak voltage. The AC peak voltage range is
that provided by the Hybrimune Waveform Generator. The effect
of the cell radius cubed is quite dramatic. This force is on a cell in
isolation.
20
10
5
Forces vs AC Peak Voltage
Cell radius= 5, 10, 20 µm
AC Peak Voltage - volts
For
ce on a cell - nano dynes
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0
5
10 15 20 25 30 35 40 45
50
55 60 65 70 75
Figure 16: Force on a Cell
Function
Waveform Type
Typical
1
Cell alignment
and compression
AC sine wave
2
Non-linear increasing
amplitude
Fusion
DC Pulse
One short pulse
Fusion partner
stabilization
AC sine wave
2
Linear decreasing
amplitude
Cell Electrofusion Tutorial