Cell News 3/2013
14
Electrofusion of cells in microdevices
Christian Guernth-Marschner, Michael Kirschbaum,
Magnus S. Jaeger, Claus Duschl
Cell fusion in general
The primary driving force behind research into cell fusion is also
the economically most relevant one: the production of antibo-
dies from hybridoma cells. B lymphocytes selectively producing
specific antibodies against a given antigen can be obtained
in vertebrates by means of vaccination, or as it was originally
termed, inoculation. Famously, smallpox was the first disease
prevented thus in the early 18th century. However, efforts to
transfer the process into an in vitro environment failed, since
in the course of their maturation, B lymphocytes differentiate
terminally into plasma cells which no longer proliferate and are
not accessible to cell culture. Only in 1975, the groundbreaking
work by César Milstein, Georges Koehler and Niels Jerne opened
up a way to make antibodies in the lab - and today routinely on
an industrial scale (1). They combined the antibody-producing
ability of B lymphocytes (A) with the infinite longevity of tumor
cells (B) by fusing pairs consisting of one cell from each popu-
lation (AB) into a hybridoma cell. Serendipitously, a minuscule
proportion of these AB fusion products featured the desired pro-
perties of its precursors.
In the next step, these AB target cells had to be separated from
the overwhelming majority of surrounding cells of types A and
B. The former was easy: the unfused B lymphocytes (A) quickly
died off in cell culture. The latter was achieved by using au-
xotroph myeloma cells as the cell type B. These cells lack the
ability to synthesize purines for their nucleic acids from certain
precursors supplied in the culture medium. Consequently, when
grown in these minimal media (HAT), the myeloma cells (type B)
die unless they regained the synthesizing ability by having been
fused with a B lymphocyte. The resulting AB fusion products can
subsequently be cloned by limiting dilution, thus yielding cell
lines which produce exactly one kind of antibody.
Hybridoma-based antibody production is by far not the only
motivation for investigating cell fusion. The field has received a
strong impetus over the recent years in the context of reproduc-
tive technologies, like in-vitro fertilization. Every offspring from
sexually reproducing parents is of course the product of a cell
fusion leading to the zygote. In the process of somatic cell nu-
clear transfer (SCNT), popularly referred to simply as “cloning”,
electric pulses are regularly employed. This, however, mostly oc-
curs on a trial-and-error basis with very few groups having ever
investigated through which mechanism applying electric pulses
actually benefits their yield (and if at all!).
Methods of cell fusion
Cell fusion is a multistep process involving: (a) forming a close
physical contact between the two (or sometimes more) pro-
genitor cells, (b) fusing the two separate cell membranes into
one common envelope and (c) reorganization of the cell com-
ponents. Most reports about cell fusion focus on the second
point because it can be addressed with technical solutions, al-
though the third - more biological - one is limiting the outcome
to a much stronger degree. Cells undergoing fusion in vivo are
well prepared for it, e. g. during fertilization or the formation
of syncytia as in muscle. In contrast, cells fused in vitro have
to adjust themselves to suddenly being tetraploid (usually) and
possessing at least two centrosomes, both of which complicates
the next cell division and enhances the likelihood of errors being
introduced.
Five basic methods have been proven useful for initiating cell
fusion in vitro: (a) the oldest one being chemically induced fusi-
on, mostly mediated by polyethylene glycol (PEG), (b) electrofu-
sion, the popularity of which was also strengthened by its com-
mercial availability, (c) virus-mediated fusion, (d) laser-induced
fusion and (e) fusion by centrifugation, often combined with
PEG-mediated fusion. While evidently varying in the complexity
of the required equipment, all of the named methods have in
common that their yield is low for at least three reasons: (a)
only a fraction of the total initial populations A and B forms
cell pairs with a distance close enough for allowing fusion upon
application of the fusogenic stimulus, (b) if the cell pairs are
formed stochastically, only half of them are of the intended
AB type, with AA and BB pairs accounting for a quarter of all
formed pairs each, (c) in many configurations, the fusogenic
trigger is not of equal strength across all cell pairs formed,
meaning that for some pairs the stimulus may be too low to
induce fusion, while being excessively high - and consequently
harmful - for others. All of these pitfalls are alleviated to varying
degrees by the numerous designs of fusion devices proposed by
different research groups. As mentioned above, such optimiza-
tions only address the technical issue of how to fuse the cell
membranes. Increasing the proportion of fused cells with res-
pect to all invested progenitor cells is, however, not equivalent
to enhancing the yield of antibody-producing - or more gene-
rally, surviving - fusion products.
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