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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Cell Alignment and Compression
Pages 32–37 described the method of aligning cells using
dielectrophoresis. There were two important parameters, the
chamber configuration and the voltage amplitude. The chamber
configuration has been optimized and is fixed. However, the
voltage parameters can be set by the user. The objective of this
step in electrofusion is to align the cells in an optimum manner
and then apply the fusion pulse. There are a number of important
considerations:
1. A perfect alignment process would alternate
the two cell types to be fused. In general that
does not occur in any known process. The way
the cells align is a statistical event. If the cells all
have the same radius and equal numbers of
each are mixed, then (see Figure 17):
XY = 25%
YX = 25%
XX = 25%
YY = 25%
XY + YX = 50%
In addition not all cells fuse in pairs; some
products have 3, 4 or more nuclei. This can be
controlled but not eliminated by optimization
of waveform amplitude.
A practical result of the stochastic nature of cell
alignment is that the maximum yield is
obtained when the percent of each cell
population is 50% (shown above). However,
A practical result of the stochastic nature of cell
alignment is that the maximum yield is
obtained when the percent of each cell
population is 50% (shown above). However,
when 3 cells of X are present for every one cell
of Y (75%:25%), then the following yield is
obtained:
YX = 19%
XY = 19%
XX = 56%
YY = 6%
XY + YX = 38%
Unequal ratios of cells are sometimes used to
conserve a rare cell population. However, when
this is done, the total yield decreases.
2. As shown in the equations on pages 32–33, the force
on the cell during alignment is a function of the
cell radius cubed. The radius of the cell varies within
the same type and the mean radius of the two cell
types to be fused varies. This will affect the
number of fusion products produced.
3. Some cell types are much more difficult to fuse
than others. This is inherent in the cell
characteristics.
4. In the initial alignment process the cells will
accelerate when the force is applied. If the
acceleration is significant then, the cells will
move toward the center electrode and bounce
off like a billiard ball. It is possible to create
such acceleration that the cells begin
circulating in a “race track” pattern. High
acceleration is very detrimental to good cell
alignment and must be avoided. The principal
use of the BTX 2 ml “Optimization” Chamber is
waveform protocol optimization. The
alignment process must be optimized while
observing the cell alignment.
Figure 17: Cell Alignment Sequence
Cell Electrofusion Tutorial