Possibilities of cell fusion and transformation by electrostimulation

Bioeiectrochemistry and Bioenergetics, 12 (1984) 119-133 A section of J. Electroanal. Chem., and constituting Vol. 173 (1984) Elsevier Sequoia S.A., Lausanne -. Printed in The Netherlands

119

664-POSSIBILITIES OF CELL FUSION AND TBANSFOBMATION ELECIBOSTIMULATION l

HERMANN HANS-EGON

BERG, KURT AUGSTEN JACOB, PETER MUHLIG

l*,

ECKHARD BAUER, WALTER and HERBERT WEBER ***

FGRSTER,

Academy of Sciences of the G. D.R., Central Institute of Microbiology and Experimental Department of Biophysical Chemistry, Jena, DDR -69 Jena (G.D.R) (Manuscript

received

October

BY

Therapy,

26th 1983)

SUMMARY A survey of three electrostimulation techniques: (A) the macro-technique with agglutination of cells, (B) the micro-technique with dielectrophoretic collection of cells, (C) the micromanipulator technique with needle electrodes for contacting cells, is given, illustrated by examples of fusion between protoplasts of microbiological mutants, or between animal cells on the one hand and the enhancement of the transformation rate with plasmid DNA on the other. For an explanation of pore enlargement and membrane breakdown, and electrodichroitic model is described. From these results it appears that this new bioelectrochemical method will become a powerful tool for gene and biotechnology.

INTRODUCTION

At the 5th Symposium on Bioelectrochemistry (1979), the first evidence of electrostimulated cell fusion (ECF) by two different techniques of single electric field pulses was presented on a poster [l] and during the general discussion [2]. Nowadays, three types of techniques are widely used: (A) the macro-technique with agglutinating substances; (B) the micro-technique with dielectrophoresis for cell collection; and (C) the micromanipulator technique with needle electrodes used for the compression of two cells. Now numerous phenomena of ECF for plant protoplasts ‘and animal cells have been described [3,4]. Typical examples for each of the above-mentioned techniques will be illustrated together with the possibility of an electrostimulated transformation. In every case, an enhancement of pores and (or) a destabilization (breakdown) of membrane structure occur depending on the intensity

Presented at the 7th International Symposium 1983. l * Department of Immunopathology. *** Department of Genetics of Yeast.

l

0302-4598/84/SO3.00

Q 1984 Elsevier Sequoia

on Bioelectrochemistry,

S.A.

Stuttgart

(F.R.G.),

18-22

July

120

of the pulse. For every cell, an optimal intensity of the pulse exists leading to subsequent resealing and re-establishment of the membrane, which has to be determined for each case. EXPERIMENTAL

In order to attain fusion of the cell suspension in the range of cubic centimeters we used macro-vessels (Fig. 1) connected to different capacitors of our temperaturejump apparatus (Fig. 2). According to technique A, the agglutination of cells by polyethylene glycol (PEG), dextran, etc. prior to the pulse is necessary [3]. Both techniques B and C allow the pathway of fusion to be observed microscopically and can be combined with a Quantimet 720M image analyzer (Cambridge Instruments). The micro-vessel of technique B consisted of two parallel platinum wires at a distance of 2 0.1 mm, connected to a device producing a high-frequency voltage for dielectrophoretic collection [4] and single rectangular pulses for fusion (Fig. 2). Working with this sophisticated technique, a medium of low conductance is the prerequisite, nevertheless, some organic substances can promote the fusion process. Technique C is shown in Fig. 3 with a two-cell blastomere as the target between two

Fig. 1. Macro-vessels of technique A for lateral filling and withdrawing: side, constant volume. Electrodes (black) can have different surfaces.

left side, variable volume; tight

121

pulse

L lo-7ov

2 - 100~s

Fig. 2. Electrostimulated fusion techniques: chains. On the right: pulse types.

(upper

series)

for cell suspension;

(lower

series) for cell

platinum needle electrodes movable by a micromanipulator. In the original version of Senda et al. [2], both silver electrodes were shielded by thin glass capillaries filled with 3 M KCl.

I

Fusion

Generotor

I

Fig. 3. The electrostimulated fusion technique C with a scheme of the fusion generator Between the needle electrodes is a two-cell blastomere.

of our institute.

122

The ECF method is suitable for enhancement of the penetration and also for local disruption of rigid cell walls by higher pulses. In this way, the incorporation of drugs is possible and repair processes are measurable [5,6]. The preparation of cells and protoplasts has been described elsewhere [7-111. Further treatment depends on the properties of the target. Technique A

(1) The pulsed yeast protoplast suspension from the macro-vessel was incubated for 30 min at 28 “C and afterwards plated in a Petri dish on solidified minimal medium. Prototrophic colonies were selected after further incubation and regeneration of fused protoplasts as a result of complementation of auxotrophic mutants. Back-mutations did not occur during 20 generations. (2) The fusion products of mutants of Bat. thuringiensis protoplasts were identified by colonies with kanamycine resistance (plasmid pUB110) and the formation of a brown pigment [ll]. (3) For macrophages, the following fusion procedure without PEG was possible. The murine peritoneal exudate cells were obtained from the peritoneal cavity by washing with 2 cm3 phosphate-buffered saline (PBS), pH 7.4. The animals were sacrificed and the fluid with cells was removed with a syringe and treated in a discharge macro-vessel with a single electric field pulse. The untreated and treated cells were allowed to set on glass during incubation for 10 min at 37 “C in a moist chamber. Thereafter, the cells were fixed in 0.75 % glutaraldehyde in PBS for 1 h at 4 “C, dehydrated in a graded series of ethanol solutions, and allowed to dry in air. Specimens were examined in a Joel JSM 35 scanning electron microscope at 25 kV without tilting. (4) The transformation of the plasmid pUBll0 DNA carrying kanamycine resistance to Bat. cereus protoplasts was tested against this antibiotic drug [lo]. Technique B

In this case, the pathways of cell movements and membrane fusion were followed photographically or by video recording for automatic image analysis of the changes in the fusing region between both cells. Technique C The main aim with pulsed zygotes and blastomeres (inside the tona pellucida) was the test of viability after implantation and the course of further embryonic development [12]. RESULTS

As an introduction, tion technique.

some basic results will be mentioned for each electrostimula-

123

(A) The macro-technique

with agglutinating substances

In order to agglutinate cells, mostly PEG (molecular weight about 4000-6000) was used. Although its mechanism is not fully understood, the main effects are dehydration, membrane bridging and even membrane destruction probably by its impurities. For DNA this dehydration leads to conformational changes and to compaction of the chain molecule [13] facilitating permeation of the membrane. (Al) Fusion of yeast mutants as protoplasts [I, 7-91 The following auxotrophic strains were fused: Sacch . lipolytka

Met, Ade

Sacch . lipolyticaArg,

Ade

Sacch. cerevisiae Ade, His, Lys, Thr, Arg

+

Sacch . lipolytica Ile, Lys

+

Lodderom . elongisporus Ade, His

+

Sacch . cerevisiae His, Thr, Leu

with a marked enhancement of colony formation on the selected minimal medium, depending on the field strength E of the pulse (Table 1). Table 1 gives different optimal E-values and different stabilities and Fig. 4 shows the corresponding prototrophic colonies on minimal medium.

Fig. 4. Formation of prototrophic colonies on minimal medium without (control) and after increasing the electric field pulses in the presence of PEG. Parental strains: Saccharomycopsis Iipolytica S 113 and 26-10

[71.

124

TABLE 1 Relative colony number from fused cells on minimal medium in dependence

of E

E (kV/cm)

S. cerev.

s. lip.

S. lip - L.. el.

0 (control)

1 122 233 50 30

1 1 8 36 10

-

-

78 20 8

1.25 2.5 3.75 5.0 10.0 15.0 20.0

-

1

(A2) Fusion of Bacillus thuringiensis mutants as proiopkasts [I l] Up to now the fusion of Bacillus thuringiensis protoplasts has only been successful

with the aid of U.V. irradiation [14]. For electrostimulated fusion, however, three subsequent pulses (At = 3 s) of 14 or 20 kV/cm (time constant 5 ps) in the presence of 40 S PEG were applied to the mixture of kanamycine-resistant protoplasts and pigment-forming protoplasts. After this treatment and 60 min incubation in a water bath at 30 OC, the suspension was plated for screening on a selective medium (nutrient broth with 100 pg kanamycine/c&). In this case, PEG has only a agglutinating and not a fusing effect. An electric field pulse of 14 kV/cm did not succeed in fusion whereas with 20 kV/cm recombinants were detected by kanamytine resistance and the formation of a brown pigment with a fusion frequency of 10m3 (recombinants/regenerated colonies). These stable recombinants produce a crystalline protein which is responsible for the toxic action against insects, and they possess the plasmid pUBll0 as well as several minor plasmids of the parental strain, as was shown by agarose-gel electrophoresis. In this way, new -strains of B. thuringiensis with a higher toxicity against harmful insects may be constructed. (A3) Fusion of macrophages

In this case, fusion may be electrostimulated without PEG too. The reason why is illustrated in Fig. 5. The morphology of untreated peritoneal macrophages is characterized by many microvilli and thread-like extensions projecting from the cell surface along a substrate (so-called fihpodis). Contacts between two macrophages are made by such filipodis (Fig. 5a). A single’electric field pulse of 5 kV/cm and of 5 ps duration induced a cobblestone effect at the surface and a reduction of these microvilli and filopodis. All these structures appeared to be dumpy, shortened, and broad-based compared to the control macrophages. The connections between the cells consisted of some thick ropes, usually originating from the peripheral edge of a spread cell (Fig. 5b). It can be seen that the contact zone is built up of many thick ropes and that the other part of the surface is characterized by craters and roughly perpendicular membrane extensions, occasionally appearing as ridges, veils or folds with lengths up to 5 nm and more.

125

Fig. 5. Scanning electron microscope text).

photographs

of macrophages

(a) before and (b) after the pulse (see

(A4) Transformation of Bacillus cereus protoplasts by the plasmid p UBI IO [l OJ Bacillus cereus does not possess plasmids and has no resistance against kanamytin, whereas its sensitivity to electric field pulses up to 20 kV/cm is very low [lo]. For the transformation of protoplasts (4 X 108/cm3) with plasmid DNA (50 pg/cm3, 40 % PEG), a field strength of 10 or 14 kV/cm was applied with the result of a ten-fold enhancement of the transformation rate in the latter case: 1.1 X 10v3 (number of kanamycin-resistant colonies on the selective medium/number of protoplasts used), however, there was a lack of crystals of the toxic protein.

0 About 10% OF protoplasts are fused l All protoplasts

are Fused

0 0

,

10 Fig. 6. Electrofusion length (linearized).

r/p5

,

100

of protoplasts of barley (Hordeurn

uulgure): the relation field strength E and pulse

60s

Fig. 7. Electrofusion

of two barley protoplasts by a pulse of 1.8 kV/cm

and 1.3 cs.

127

(B) The micro-technique

with dielectrophoresis

for cell collection

The main advantage of this technique [4] is the immediate observation of a series of simultaneously fusing cells. The time course of fusion depends on the pulse height, the pulse duration and the physiological properties. For barley protoplasts the corresponding linearized relation is given in Fig. 6. Such a fusion process itself is indicated in Fig. 7 for two protoplasts collected by dielectrophoresis (151. The increase in the contact area, measured two-dimensionally as optical density by the Quantimet image analyzer, proceeds stepwise according to the curve in Fig. 8 and finally the new membrane surface is lowered by 26 % compared to the extent of both original cells. During such processes many new effects can be observed: -rotation of single cells depending on the frequency and the voltage of the alternating field for dielectrophoresis; -changes in the density distribution of the chloroplasts and sometimes exocytosis of some of them, or even eruptive loss of pieces of membrane; -oscillations of both connected cells shortly after the pulse. In this case (Fig. 9), the fast fusing cells exhibit an ellipsoidal shape after rapid elongations in the field direction when the pulse is applied. Similar results have been obtained with cancer cells and combination with spleen cells for hybridoma production [16].

20

-

A

10 0

k after

5, L

50

pulse (5)

t

I

100

150

I 200

Fig. 8. Time course of fusion measured by L, the width of contact area (optical density). The minimum indicates the ejection of membrane material from the area.

128

15s

301

901

180

240

Fig. 9. Electrofusion

of two barley protoplasts

by a pulse of 0.45 kV/cm

and 17.5

129

(C) The micromanipulator

technique with needle electrodes

The metallic needle electrodes, combined with one or two suction capillaries were successful in several respects: -manipulating large cells (about 100 pm) for pulse application between the tips; -subsequent introduction of one or both needles into the fused cell for fusing the nuclei; -variable parallel positioning of the needles for dielectric collection of small cells, according to technique B. With the aid of this micromanipulating technique, it is now possible to fuse blastomeres, etc. within or without the zona pellucida of zygotes in order to stop the cleavage during embryonic development forming polyploidal cells. Examples are shown in Figs. lo-13 for several stages (for preparation see Ref. 12). From these experiments it can be concluded that: -without the zona pellucida the fusion proceeds by lower pulse amplitudes or with shorter pulse times; -if two double blastomeres are positioned between the electrodes in such a way that the inner membranes are perpendicular to the electrode axis, they will fuse before the outer membrane melts. In order to observe further development, it is necessary to prove the viability by implantation. Under pulse treatment of 2 kV/cm and 100 ps, about 50 % of the zygotes showed further development 11 days later [17]. This is a promising step in the direction of polyploidic zygote production. DISCUSSION

These three basic techniques for cell fusion and enhanced membrane permeation have been modified in several ways, especially for flowing samples [4]. Therefore the possibilities of electrostimulation are amazing nowadays. The only problems seem to

Fig. 10. Electrofusion of the two-cell stage without the zone pellucidrr removed by pronase occurs easily (5 kV/cm, 100 ps) (a) before, (b) 10 min and (c) 50 min after the pulse..

treatment

Fig. 12. Electrofusion of the eight-cell stage within the rona pellucida. (a) Before, (b) 10 rnin and (c) 50 min after the pulse (6 kV/cm,

100 cs),

Fig. 11. Electrofusion of the two-cell stage within the zoner pellucidaneeds a higher energy (6 kV/cm, 100 ps) and contact of the electrodes on the cell. (a) Before, (b) 45 min after the pulse, (c) disruption of the membrane by a higher field duration (5 kV/cm, 200 ps).

131

Fig. 13. Electrofusion kV/cm, 100 /AS).

of the blastocyst stage within the zona

pellucida.(a) Before, (b) after the pulse (6.5

be the finding of optimal conditions under which the viability is warranted. The new living beings must be stabilized and cultured. Besides our examples enabled by these three techniques, many corresponding results have been published recently [4] i.e. -with technique A: fusion of Dictyostelium discoideum [ 181, incorporation of plasmid DNA (TK) in mouse L cells [19], fusion in monolayer cultures [20] -with technique B: fusion of Vicia fubu protoplasts [21] as well as Arena sativa, Kalanchoe daigremontiana, etc. fusion of sea urchin eggs [22] formation of giant erythroblasts and erythrocytes [23] production of hybridoma [24] -with technique C: fusion of R serpentina protoplasts [25] exocytosis of Oxytrichiu [26]. In this fast-growing field of electrostimulation, theoretical models for breakdown or melting of bilayers and pore growth in biological membranes have also been developed [4,27-291. Finally a heuristic model shall be illustrated based on the field effect leading to the electrodichroism of rod-shaped molecules in solution measurable by spectroscopic relaxation kinetics. Fast dipole induction, molecule orientation and charge repulsion of proteins enhance the pores and disturb the bilayer structure in such a way that penetration of bulky cations, drugs [30] or even DNA occurs as shown schematically in Fig. 14. Membrane breakdown and the coincidental field orientation of DNA compacted by PEG facilitate the incorporation process. The same principle of protein orientation would trigger after the field pulse the fusion process between two cells contacted by polymers or by dielectrophoresis (Fig. 15). By such a mechanism, the opening of membranes and, moreover, cell walls [5] should be

132

Fig. 14. A membrane before the pulse with outside cations, drugs, DNA, etc. and after the the enlargement of pores, the disturbance and the breakdown of the bilayer schematically.

pulseshowing

possible in any case and therefore fusion between natural and artificial membranes of quite different origin may occur, where chemical or colloidal fusogens are ineffective or unsuitable because of toxicity. From the present results it is clearly seen that electrostimulation will become a powerful tool for gene- and biotechnology. Fusion

Fig. 15. Breakdown of the membrane by protein orientation cell fusion can occur.

and movement

in the field direction before

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