The Electrofusion of Cells POHL

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The Electrofusion of CellsHERBERT A. POHL, K. POLLOCK, AND H . RIVERA

Department of Physics, Oklahoma State University, Stillwater, Oklahoma 74078

AbstractMany laboratories studying genetic engineering are using cell fusion to aid their research. The

new and highly efficient method of using electrical fields to induce cell-to-cell fusion, or cell-to-particle fusion, or to induce entry of D N A or other compounds into cells opens up rapid ways toaccomplish these ends.

IntroductionMembrane fusion, now an art controllable by man, has great potential inmembrane, genetic and cell research. Somatic hybridization and genetic engi-neering have the means to modify plant and animal cells, which in turn offerways to improve crops, treat disease, and improve medical science. In this reviewwe describe a new electrical procedure of outstanding efficiency for obtainingcell-to-cell, cell-to-vesicle, vesicle-to-vesicle fusion, and of introducing chemicalagents, DNA, etc., into membrane bound particles such as cells, protoplasts, orvesicles.Membrane fusion is a natural phenomenon, widely observable in nature. Itis, for example, involved in fertilization, endocytosis, exocytosis, in musclefiber formation, in secretion, in pinocytosis, in the formation of secondary ly -sosomes, and in the conjugation of bacteria and protozoans. Cell-to-cell fusion

in v im , opens vistas in membrane research, in genetic mapping, and in theformation of cells with new properties. The much desired production of newplants resistant to drought, high salinity, insect pests, insecticides, or fungi offersnew challenges to the bioscientists. In addition, the in vitro modifications openthe way to create crops able to directly assimilate nitrogen or to produce inhigher yield. On the medical side, the hybridization of a permanent cell line(oncogenic cell) with antibody-producing or hormone-producing cells offers ex-traordinarily efficient means to produce monoclonal antibodies against selectedcompounds, toxins, bacteria, viruses, or even cancer cells. The new techniqueof electrofusion opens time-saving short cuts to these goals. It is an importantnew tool of research and production.In the past decade, a variety of biochemical methods have been developed toinduce cell-to-cell fusion. These include the use of virus particles or of variouschemical agents such as dimethyl sulfoxide or polyethylene glycol (PEG). Suchfusion procedures can usually only be obtained by the application of membranedisrupting agents or by procedures which are so unphysiological as to minimizeviability. The research in such chemically and viral-induced fusion has been well

International Journal of Quantum Chemistry: Quantum Biology Symposium 1 I , 327-345 (1984)0 1984 by John Wiley & Sons, Inc. CCC 0360-8832/84/010327-19$04.00


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ELECTROFUSION OF CELLS 32 9cells [6-111. Giant cells, i.e., those made by electrofusing many cells to forma giant entity, have been prepared from yeast mammalian red blood cells, Indianmuntjac fibroblasts, and cow lung macrophage cells [10,l I ] . The resulting giantcells respond favorably to vital stain (methylene blue) tests for vitality. It isreported, however, that giant cells containing more than about four original cellsdo not survive attempts at division upon further culture [6].

Electrofusion of cells in vitro was first observed by Pohl and Buckner in 1972during studies [12] on the dielectrophoresis (DEP) of canine erythrocytes andthrombocytes. Pohl also noted that human erythrocytes suspended in deionized5% sucrose solution, and then subjected to DEP at 2.5 MHz coalesced to forma single cell following the application of a brief pulse of 200 V peak-to-peak.Similar results on electro-coalescence were obtained with canine thrombocytessuspended in 0.25M sucrose on using 1 MHz in pulses. The first publishedmorphological evidence for electrically mediated fusion of the protoplasts ofdifferent plants was that of Senda et al. [131. Neumann, Gerisch, and Opatz [7]first described electrofusion using high voltage pulses, as applied to the eucaryoticmicroorganisms, Dictyosteliurn discoideurn. In Europe, electrofusion done withthe aid 13DEP was, one author (H . Pohl) was told, first observed by MajaMischel [141, an observation that was quickly recognized for its value by Lam-precht and Zimmermann. This was followed by a spate of research on electro-fusion. The publications of Neumann et al. [7], Berg et al. [6], and Zimmermannet al. [8,10,14] stimulated much interest in electrofusion and its implications forthe interactions of electromagnetic fields with living systems.

The electrofusion procedure is a relatively gentle one, provides a sharp syn-chrony of the onset of fusion, and can lead to a high yield (20-80%) of fusedcells. The presently preferred technique is one combining DEP [5,15-181 toobtain placement of cells into close contact by the formation of pearlchains orstacks, and of controlled fusion following the application of dc pulses [7-111.

DielectrophoresisDielectrophoresis, first theoretically recognized, quantitatively analyzed, and

named by Pohl [15], is the translational motion of neutral matter induced by theaction of a nonuniform electric field (see Fig. 1). It can be considered to arisein two steps. First the electric field causes the material to be polarized, creatingan electric dipole. The two equal charge distributions of this material dipole are,however, in a nonuniform field. Since those charges in the stronger field regionexperience a stronger local force [ F = q X E (local)] than their opposite num-bers in the weaker field, a net force arises which tends to propel the matter intothe stronger field. It is called dielectrophoresis (DEP) since it depends upon thedielectric properties, the polarizability. The action of an electric field on chargedparticles, i.e. , those carrying free and excess charge, is by convention calledelectrophoresis.

The phenomenon of DEP is an ancient one. It was doubtless through it thatThales of Miletus in about 600 B.C. is reported to have observed the attraction


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330 POHL, POLLOCK, A ND RIVERA

Figure 1. Comparison of the behaviors of neutral and charged bodies in an alternatingnonuniform electric field. (a) The positively charged body moves toward the negative elec-trode. The neutral body is polarized, then is attracted towards the region of strongest field.This is because the two charge regions on the neutral body are equal in their amount ofcharge, but one of the charge regions is in a stronger field. Th e force on a charge is proportionalto the local field. (b) H ere the direction of the field is reversed. T he positively charged objectagain moves toward the negative electrode. The neutral body is again polarized, but doesnot reverse its direction although the field is reversed, for i t still seeks the region of highestfield intensity.

of charged (rubbed) amber for small (neutral, we can now realize) lint particles,and discovered electricity [ 5 ] .

The distinction between dielectrophoresis and electrophoresis is important.When an ac field is applied, for instance, DEP tries at each field reversal todrive the particle into the region of higher field strength, for the induced dipolereverses each time. The particle then tends to go into the region of stronger fieldcontinually. On the other hand, during electrophoresis, the charged particle ispulsed towards the electrode of opposite charge sign. At high frequencies itmerely shudders and makes little net motion towards either electrode. DEP, then,can act to separate neutral from charged particles. Neutral particles such as cells,can be made to collect into the region of strongest field while charged ones aremore or less ignored. A caveat is necessary here. Since one uses DEP onsuspension of cells or particles in a fluid medium, there is necessarily a com-petition of DEP forces acting upon the material of the medium and that of thesuspended particles. If the polarizability of the particles exceeds that of themedium at the field frequency chosen, the particles will tend to be collected inthe region of strongest field. This is positive DEP. If conversely the mediumis the more polarizable, then the particles will be pushed into the region ofweaker field. This is negative DEP. The procedure is quite analogous to thefamiliar floating or sinking as in gravitational fields.

If the particle is asymmetrical, or inhomogeneously made, the electrical po-larization can produce a torque so as to realign the particle in the field lines and


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ELECTROFUSION OF CELLS 33 Iminimize its energy. Thus dielectrophoretic effects can produce both a purelytranslational and an orientational result. The term electrostriction is nowadaysreserved to describe only the distortional effects of fields, such as would arisein the piezoelectric effect, the Kerr effect, or bulk distortions of matter.

A further fundamental difference between electrophoresis and DEP requiresmention. In mixtures such as suspensions of cells in aqueous media, successfulDEP requires a substantial difference between the permittivities (total polariz-abilities) of the medium and particles. The dielectric properties of living cellsand their parts change with frequency. There are four or more distinct regionsof this relaxation response, reflecting, as the frequency increases, the variouscomponents of the cell (see Fig. 2 ) . At the frequency range of about 100-10,000Hz, the very loosely bound counterions of the outer layer of the ionic doublelayers associated with membranes and cell walls strongly respond. At about 10-100 kHz the more tightly bound counterions of the ionic double layers respond.If associated with the outer layers of spherical particles, for example, theseresponse frequencies vary as the inverse of the square of the particle radius. Atabout 100-10 MHz, the predominent response is that due to the bulk-bulkinterfacial (Maxwell-Wagner-Sillars) polarization [ 5 ] . In this range, the DEPresponse of all cells is a common occurrence, reflecting the common presenceof membranal interfaces with the medium. It is probably the most useful fre-quency range for producing the cell-to-cell stacking and assuring membrane-to-membrane contact. In the frequency range above about 3 MHz the polarizationresponses of dissolved proteins, DNA, RNA, and other polar molecules can be

\ B ELI log,, F R E Q U E N C Y HzFigure 2. A simulated DEP force, or effective dielectric constant spectrum such as observedin living cells. The regions of frequency below which the various polarization mechanismsare active are: (A) responses of loose counterions of the ionic double layers; (B) responsesof tightly bound counterions in ionic double layers; (C ) Maxwell-Wagner, bulk-bulkinterfacial response region; (D ) molecular dipolar responses, of large macromolecules; (E)molecular dipolar responses, of small mo lecules.


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332 POHL, POLLOCK, AND RIVERAobserved. For further details on these points, the reader is referred to othersources [18,201.

Mutual DielectrophoresisUp to this point the possible interactions of particles in an electric field havenot been discussed. The cells normally have a different polarizability than themedium, and therefore perturb the field. A second cell will therefore be subjectto that field nonuniformity and be impelled toward the region of highest fielddensity , at the end of the first cell. T he net result is alinement of the cells alongthe field lines in a little pearl chain (see Fig. 3). Th e sam e situation occursif the polarizability of the cells are less than that of the medium for then themedium seeks minimum energy and forms regions lined up along the field,driving the cells into pearl chains by default. There is one exception. If twofreely suspended particles of approximately equal size are present, one with ahighe r, on e with a lower permittivity than the fluid me dium , then the two particlestend to form a short pearl chain sitting at right angles to the field lines.

The formation of the stacks of cells by D EP is quite useful for the process ofelectrofusion, for it puts the cells in contact as desired. The attractive force ofmutual DEP now can help overcome the natural repulsion between the adjacentmembrane surfaces due to their hydration and to their ionic double layers. Themutual DEP and cell-stacking is best done with the use of high frequency acfields. As mentioned above there is an almost universal positive DEP responseof cells in the frequency range of about 200 to 900 kHz due to the bulk-bulkinterfacial (Maxwell-Wagner) polarization resp ons e, so this is the frequencyusually used to ev ok e the cell-to-cell contact prior to the application of the highervoltage dc pulse to produce the fusion.

DEP and its concommitant cell stacking normally has to be done in mediawhich are nearly non-conducting (specific conductivity less than about 0.0001mho/cm or S / c m ) so as to avoid undue heating, turbulen ces, and other deleteriou sconsequences for the cells and the electrofusion process [ 5 ] . The use of suchmedia with low ionic content poses little or no problem for those cells havingwalls such as bacteria, yeasts, and algae. But then the electrofusion, which

Figure 3 . Diagram of pearl chaining by cells or other particles produced by the action ofDEP is due to an ac field arising between parallel wire electrodes.


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ELECTROFUSION OF CELLS 333requires intimate membranal contact is also hindered. The matter is eased bythe use of the cells in protoplast form with the medium now made properlyisoosm otic by the use of nonionizing solutes such as sucrose , glucose, m annose,ma nnitol, o r sorbito l, or of the zwitterionic m aterials such as histidine, or glycine.Exp erim ents with m am ma lian, avian , yeast protoplasts, or with plant protoplastsshows them to experience but a few deleterious side effects on their viabilityupon the brief exposure (ca. 2-30 min) to these conditions during the entireprocedure required for electrofusion.

Membrane Puncture and FunctionThe application of a strong electrical field across a bilayer membrane cancause it to be puncture d. This poration as it is sometimes called can be

temporary (reversible), or can be so severe as to induce irreversible breakdownand disruption of the whole membrane-supported assembly. In the process ofelectrofusion, we shall be most interested in the gentler, reversible type ofmembrane puncture. It provides the condition from which fusion between closelypositioned sim ilar structures can follow. T he more damag ing irreversible cellrupture is also of interest, for although it can cause trouble in the normal elec-trofusion operations, it can also be used to selectively destroy cells.

The type of membranal puncture one obtains is governed largely by the am-plitude of the field, and its duration. It is also strongly affected by the chemicalcomposition of the membrane region. The presence, for instance, of pronase isreported to improve the durability [8] of the membranes to overvoltage duringthe electrofusion process. A s representative of the effects of field amplitude andduration on the electrofusion of erythrocytes, a look at Figure 4 shows that forshort dc pulses applied to the cells formed into stacks or pearl cha ins by a gentleac field, there is little observable effect by pulses shorter than about 25 ps inthe low fields used here, but there is more and more effect as the field durationof the pulse lengthens. There is also observable a range of field amplitudes inwhich the desired fusion takes place, and a range, which if exceeded, causesalmost total destruction (lysis) of the cells. It is this type of information whichneed s to be dete rmin ed in each case as o ne studies the electrofusion of differingtypes of cells. There are some guiding principles, however.Fo r one , the potential required to induce the reversible pun cture of most cellularmem branes is known to be about 0.85 V for pulse lengths of about 20-100 ps .It was also shown by Fricke in 1924 [18,19] that the potential across the cellularmembranes due to the application of a static external field acting on a sphericalcell modelled a s a con duc tive region inside a nonconductive mem brane and lyingin a rather conductive medium should follow the relation:

V , = 1.5Er,where V , is the potential across the membrane region, E is the external fieldwhich would pertain if the cell were absent, and r is the radius of the sphericalcell. To accou nt for ang ular variations of the potential around the cell, one need


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LY02+A-i4Lyu73n.

POHL, POLLOCK, AND RIVERA

K Vcm-o l t s-150 - 6

- 5\ - 4\ \\.

\ - --2-- -- - 5 3' *---loo\\ C E L L E L E C T R O - F U S I O N.----\ -- -- ---50 21

I I2 0 4 0 60 80P U L S E D U R A T I O N ( P S e c )

Figure 4. Comparison of the fields able to evoke fusion (lower curve) and able to evokelysis (upper curve) of rabbit erythrocytes. The dc pulse amplitudes versus duration are shownas the cells were gently held in pearl chains by a small ac field. Along the lower curve,fusion of the cells occurs in about 5-15% of the cells. The electric field is that due to parallelR wires of 250-pm diameter separated by 120 pm. he cells are pearl-chained away fromthe electrodes, while on the floor of the chamber, and are in 0.3M sucrose containing addedCO, at pH 4.8.

only add a multiplying factor of the cosine of the angle 0 , away from the axisprovided by the field line going into the middle of the cell, to the formula, viz.(see Figs. 5 and 6).V,,, = 1.5Er cos0. ( 2 )

To take into account the time variations requires more com plicated analysis. Inthe Debye format, [201 one simply inserts a factor of (1 + j w T ) in the denom-inator and then obtains for the real part:(3), = (1.5Er cos0)/(1 + w 2 T 2 ) ,

Figure 5 . Diagram of cell in a field showing the coordinates of interest for pulsing fields.


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336 POHL, POLLOCK, AND RIVERA

of, say, 10 pm in our fusion chamber having a spacing of d = 200 pm, thesimple formula suggests a voltage of some 12 V. The actual value in practicewill vary from this, of course, for the real conditions will not be so ideal, butthe suggested value is helpful as an initial guide.One of the factors determining the actual field at the cells to be electrofusedis the electrode shape and spacing. A helpful guide in this is the calculations ofLafon and Pohl [23] for the field in tensities about electrodes of the sphere-sphereand wire-wire types. For the cylinder-cylinder pair, the field, E9Z) along theplane connecting the centers is shown in Figure 7. Here the field with 1 V appliedacross the electrode pair is shown for a variety of electrode dimensions. Thecalculation is based upon the relation(8)1+ -R , - Z

V 12 In ( R , I R )Z - R 2 / R ,E ( Z ) =

where V is the applied voltage, R , is the radius of the electrode wire, Z is thedistance between electrode centers,2 = R , + R 2 / R I , (9)

E. v l mFigure 7 . The electric field along the line between centers of a pair of cylindrical and parallelelectrodes between which a potential of 1 V is applied. Here, R is the electrode radius, andthe field (in V /m ) is shown as a function of the distance between the electrodes closestapproach. Also indicated are the critical fields, which if just the 1 V is applied across theelectrodes, might be expected to evoke appreciable poration of the cell of the diameterindicated.


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ELECTROFUSION O F CELLS 331is the cente r-to-center cylinder separa tion, and R , is the radius of the cylinders.To use the chart, simply multiply the values by the voltage to be used, if it isnot to be just 1 V.

To aid the experimenter wishing to use electric fields to handle living cells,we have also calculated the critical field required by relation (3 , nd assumingthe breakdown voltage V,, to be 1 O V, and ha ve plotted these values for severalfrequently encountered cell sizes (diameters) as shown in Figure 7. If, for ex-am ple, it is desired to merely handle and exa mine cells in an electric field withoutsubjecting them to critical fields liable to cause disruptive puncture, then theappropriate conditions can be read off from the graph.

Aligning CellsThe genius of electrofusion consists of bringing cells into intimate contact,then applying a short sharp pulse of field to evoke small breaks in the contacted

areas following which fusion across the temporary ruptures occurs. Membranecon tact between the properly prepared cellu lar surfaces is best achieved by D EPand mutual surface interactions in a mild ac field. The field nonuniformitiesDE P force upon an object depends upon how al l the charges in the object andin its surrounding medium respond to the applied nonuniform field. This meansthat not jus t the normal polarization (dielectric con stan t), but also the conductionresponses are involved. As a con seq uen ce, one must look after the conductivitiesto achieve maximal success. In a nutshell, successful DEP of cells and otherbiological particles requires the use of aqueous media of low conductivity. Nor-mally a sp ecif ic conductivity of about 3 x mhoicm or less is required(i.e., a specific resistivity of about 30,000 ohm cm or higher). Best results areobtained w ith specifi c conductivities below about 5 x mho/cm (i .e . , spe-cific resistivities higher than 200,000 ohm cm).

A feel for the relative importance of the polarization and conductivity factorscan be had from looking at the equation for the DEP force upon a simple spheresuspended in a fluid while in a nonuniform electric field. One relation derivedsome t ime ago [ 5 ] is

F = ( V / 2 )Re{e&T(K2 - K I ) / ( K 2+ 2 K , ) / V &I2}, (10)where V is the volume of the sphere, en is the permittivity of free space,(8.854 X lo - F / m or C /V m), K , and K 2 are the complex relative dielectricconsta nts of the medium and the sphe re respectively, (see below ). Re refers totaking the real part of what follows, and V (En) is the gradient of the square ofthe electric field which would be at the center of the sphere were it absent. Amore exact expression due to Prof. Dr. Freidrich A . Sauer of the Max-PlanckInstitute for Biophysics in Frankfurt, Germany, and derived not on the lessprecise basis of energy conservation as was the above expression, but on theconservation of momentum so as to better account for frictional losses, etc., is

F = ( 3 V / 1 6 ) [ 6 * / ( 3+ b* ) + b l ( 3 + b ) ] ( K T + KI) (EO) , (11)


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338 POHL, POLLOCK, AND RIVERAwhere b = (K& - Kl,tot)/Kl,,,,,, ,, , = K + j s / ( e , W ) , the complex relativedielectric constant, b* is the complex conjugate of b, and s is the specificconductivity. The two expressions agree fairly well for situations in which theconductivity is not too high. As can be seen, however, both the polarization(dielectric constant) and the conduction factors are important. Said another way, .the presence of minute ionic conduction (e.g., ca. 0.0005M salt) in water dras-tically raises its effective dielectric constant when considering DEP forces. Asa consequence, the effective dielectric constant of the suspended cell s, and theirDEP attractions to each other or to the desired electrode positions can be over-ridden by the high effects of effective dielectric constant of the conductivemedium, resulting in poor dielectrophoretic control of the cells. This emphasizesthe need for maintaining low conductivity in the aqueous medium. Another goodreason for maintaining low conduction in the support medium lies in the needto minimize Joule heating with its consequent thermal upsets, stirring effects,and outright danger to the cells.Dielectrophoresis and the pearl-chaining of cells, then, usually must be donein nearly nonconductive media, but where the osmotic problems are met withby using nonconductive solutes such as glucose, mannose, sucrose, histidine,and the like.

Mechan ism of ElectrofusionA brief comment on the way in which electrofusion is thought to occur is inorder. From the electrical point of view, the thin membrane is a good insulatorsituated between two conductors, i.e., the aqueous inner and outer media. Afield imposed across the membrane-water interfaces will therefore produce itsmajor potential drop (field strength and electric stress) across the membraneitself. When the cells are brought into contact, as by DEP, the ensuing strong(dc) electric pulse produces small punctures in the membrane regions, especiallywhere the field is most intense, at the poles of the cells along the lines of thefield, i.e., toward the electrode surfaces, or toward the usual contact areas of

the cells in the pearl-chains. In living cells there already exists a strong fieldacross the outer membrane (plasmalemma), amounting to about 100,000 V/cm.As the external field pulse is applied in a particular direction, this adds to thefield across one of the two membranes in contact, and subtracts from the other.The membrane with the greater field is thought to then break down first. Thepores and defects in its surface then act to focus the remaining field on thesecond membrane causing it to develop punctures in the locale. Th e time requiredfor this early ev ent is judg ed to be in the ord er of a small fraction of microsecon d.The e nsuing time of the pulse duration of some 10-100 k s , sa y, is then involvedin holding open the communa l intercellular puncture while the new edges kn itor seal to each other. Following this the field is turned off and the newly sealededges develop under surface forces to form part of a single unitized membraneenveloping the original cells.

An example of the sequence of events is shown in Plates 1-6. Here murine


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Plate 1

3 3 9

Plate 2


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340 POHL, POLLOCK, AN D RIVERA

Plate 3

Plate 4


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Plate 5

341

Plate 6


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ELECTROFUSION OF CELLS 343

electrode. In Plates 1-3 even a larger number of cells is seen electrofusing. InPlate 1, cells are aligned into a pearl chain by dielectrophoresis before the fusionpulse. After the fusion pulse Plates 2 and 3 show the evolution of the fusionprocess. The cells of the Plates 1-6 are myeloma cells P 3 x 63-Ag8-653.

Electrofusion ExperimentsTypically, the electrofusion is carried out in electrode chambers in which the

fusion operation can be observed under the microscope. The commercially avail-able ones (D.E.P. Systems, Inc., 4350 Barber Road, Metamora, MI 48455) areof two basic types: open top and closed. The open top chambers are typicallyused for small batch type experiments. They permit easy loading and access tothe active fusion area between the platinum electrodes and are used where sterileconditions can be relaxed, although their operation can be done with sterileconditions readily maintained, as in a proper hood, etc. The closed chamber canbe operated in a semi-continuous manner to fuse cells. Here the maintenance ofsterile conditions is somewhat easier.

In a typical experiment, the cells to be fused, or to be subject to field pulsesto help drive in desired agents (e.g., DNA) from the surrounding medium, arefirst removed from their medium by gentle centrifugation, resuspended in deion-ized and isoosmotic sucrose, say, and again spun out. This process of spinningout and resuspending in the deionized and isoosmotic medium is repeated untilthe conductivity of the suspension is less than about 0.00001 mho/cm (10 ps/cm). If it is desired to pretreat the cells with proteases or cellulases to decorticatethem and produce protoplasts, this can also be done at this time. Following this,the cells (o r perhaps now protoplasts) can be injected into the fusion chamberand the fusion carried out. The cells are first gathered onto each other with gentleDEP by the application of the ac field, then subject to the dc pulse(s) of pre-selected amplitude and duration. In a typical experiment, an ac voltage of some7 V at 250 kHz is used to gather the cells and produce the desired interfacialcontact, following which a pulse of, say, 60 V lasting for 40 p s is made. (It isusually desirable to reduce the ac chaining voltage to about f value duringthe application of the dc pulse.) If this process of DEP plus a pulse had producedthe desired fusion result, the fused cells would be let sit for a few moments soas to permit the fused portion to grow and knit the cells together. A gentleinflux of medium may be injected to help induce better overall shaping of thefused cells. The fused cells are then drawn out, the chamber cleaned and readiedfor the next use.

Fusion can be used as a technique to introduce DNA, plasmids, enzymes,proteins, markers, etc., inside cells. In a preliminary step the molecules of interestare absorbed from solution on the surface of the cells. Then the cells are broughtinto contact by DEP. If the molecule of interest is trapped in the cell-to-cellcontact zone it will be introduced by the fusion process inside the new envelope


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[I81 R. Pethig, Dielectric and Electronic Properties o Biological Materials (Wiley, New York,[I91 H. ricke, Phys. Rev. 24, 575 (1924).[20] H. ricke, Phys. Rev. 24, 678 (1924).[21] A . H. on Hippel, Dielectrics and Waves (Wiley, New York, 1954). p. 175.[22] H. . Schwan, Adv. Biol. Med. Phys. 5, 147 (1957).1231 H. . Schwan, Ann. N.Y. Acad. Sci. 103, 198 (1977).1241 E. E. Lafon and H. . Pohl, J . Electrostat. 9, 209 (1981).1251 F. A. Sauer (personal communication, 1982).

1979).

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The Electrofusion of Cells POHL

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