Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts.     

Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions.

Using the relations between pellet structure and electric properties derived from the preceding paper, the responses of rabbit erythrocyte pellets to osmotic or colloidal-osmotic effects from exchanged supernatants and from electroporation were investigated. Changing the ionic strength of the supernatant, or replacing it with dextran or poly(ethylene glycol) solutions, caused changes of Rp according to the osmotic behavior of the pellet. Rp was high and ohmic before electroporation, but dropped abruptly in the first few microseconds once the transmembrane voltage exceeded the membrane breakdown potential. After the initial drop, Rp increased as a result of the reduction of intercellular space. Rp increased regardless of whether the pellets were formed before or immediately after the pulse, indicating that porated cells experienced a slow colloidal-osmotic swelling. The intercellular or intermembrane distances between cells in a pellet, as a function of osmotic, colloidal-osmotic, and centrifugal pressures used to compress rabbit erythrocyte pellets, were deduced from the Rp measurement. This offered a unique opportunity to measure the intermembrane repulsive force in a disordered system including living cells. Electrohemolysis of pelleted cells was reduced because of limited swelling by the compactness of the pellet. Electrofusion was observed when the applied voltage per pellet membrane exceeded the breakdown voltage. The fusion yield was independent of pulse length greater than 10 microseconds, because after the breakdown of membrane resistance, voltage drop across the pellet became insignificant. Replacing the supernatant with poly(ethylene glycol) or dextran solutions, or coating pellets with unporated cell layers reduced the colloidal-osmotic swelling and hemolysis, but also reduced the electrofusion yield. These manipulations can be explored to increase electroloading and electrofusion efficiencies.     

Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols.

Rabbit erythrocyte ghosts were fused by means of electric pulses to determine the electrofusion thresholds for these membranes. Two protocols were used to investigate fusion events: contact-first, and pulse-first. Electrical capacitance discharge (CD) pulses were used to induce fusion. Plots of fusion yield vs peak field strength yielded curves that intersected the field strength axis at positive values (pseudothresholds) which depended on the protocol and decay half time of the pulses. It was found that plots of pseudothreshold vs reciprocal half time were linear for each protocol; when extrapolated to reciprocal half time = 0 (i.e., t----infinity), these lines intersected the ordinate at values of the field strength considered to be the true electrofusion thresholds. In this fashion, the contact-first protocol gave an electrofusion threshold of 46.5 +/- 11.5 V/mm for hemoglobin-free ghosts (white ghosts) and 40.9 +/- 8.8 V/mm for ghosts with fractional hemoglobin (pink ghosts), while the threshold for the pulse-first protocol applied to pink ghosts was determined to be 93.4 +/- 11.0 V/mm. Although the thresholds depended on the electrofusion protocol, plots of critical field strength vs reciprocal time had the same slopes, i.e., approximately 24 Vs/mm. The results suggest that the fusogenic state induced by an electric pulse in either the contact-first protocol or the pulse-first protocol (long-lived fusogenic state) may in fact share a common mechanism, if the two states are not actually identical.     

A delay in membrane fusion: lag times observed by fluorescence microscopy of individual fusion events induced by an electric field pulse

Low light level video microscopy of the fusion of DiI- (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeled rabbit erythrocyte ghosts with unlabeled rabbit erythrocyte ghosts, held in stable apposition by dielectrophoresis in sodium phosphate buffers, showed reproducible time intervals (delays) between the application of a single fusogenic electric pulse and the earliest detection of fluorescence in the unlabeled adjacent membranes. The delay increased over the range 0.3-4 s with a decrease in (i) the electric field strength of the fusion-inducing pulse from 1000 to 250 V/mm, (ii) the decay half-time of the fusogenic pulse in the range 1.8-0.073 ms, and (iii) the dielectrophoretic force which brings the membranes into close apposition. A change in the buffer viscosity from 1.8 to 10 mP.s caused the delay to increase from 0.36 to 3.7 s (in glycerol solutions) or to 5.2 s (in sucrose solutions). The delay decreased 2-3 times with an increase in temperature from 21 to 37 degrees C. It did not differ significantly for "white" ghosts [0.013 mM hemoglobin (Hb)] or "red" ghosts (0.15 mM Hb) or buffer strength over the range 5-60 mM (sodium phosphate, pH 8.5). The calculated activation energy, 17 kcal/mol, does not depend on the field strength. The yield of fused cells was high when the delay was short. The delay in electrofusion resembles the delays in pH-dependent fusion of vesicular stomatitis viruses with erythrocyte ghosts [Clague, M. J., Schoch, C., Zech, L., & Blumenthal, R. (1990) Biochemistry 29, 1303-1308] and of fibroblasts expressing influenza hemagglutinin and red blood cells [Morris, S. J., Sarkar, D.P., White, J. M., & Blumenthal, R. (1989) J. Biol. Chem. 264, 3972-3978].(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0. We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0 We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

细胞电融合法生产杂交瘤

细胞电融合过程包括使用交流电场使悬浮在缓冲液中的细胞排列成串,然后高压脉冲电流作用使细胞膜发生瞬时破裂,相邻细胞的细胞膜融合,继续受交流电场作用完成细胞融合。已经证明细胞电融合技术可用于杂交瘤生产,单克隆抗体生产以及细胞表面标记物细胞间的传递过程。     

Membrane electroporation--fast molecular exchange by electroosmosis.

Human and rabbit erythrocyte ghosts loaded with FITC-dextran (mol. mass = 10 kDa) and NBD-glucosamine (mol. mass = 342 Da) in buffers of different ionic strength and composition were subjected to electric pulses (intensity 0.7 kV/mm and decay half-time 1 ms) at 7-10 degrees C and 20-24 degrees C. The transfer of the fluorescent dyes from the interior of the ghosts through the electropores was observed by low light level video microscopy. The pulses caused the fluorescence to appear outside the membranes as a transient cylindrical cloud directed toward the negative electrode during the first video frame (17 ms). It was similar in both rabbit and human erythrocyte ghosts and at both temperatures but differs for the two dyes, the fluorescence cylinder is long and tall for the FITC-dextran and relatively short and thick for the NBD-glucosamine. The molecular exchange was 2-3 orders of magnitude faster within the first 17 ms after the pulse than the diffusional exchange. It decreased with increasing ionic strength. Formulae for the transfer of molecules by electroosmotic flow through the pores are in agreement with these observations. They allow estimation of the total area of pores with radii larger than that of the fluorescent dye during the pulse. The major conclusion is that electroosmosis is the dominating mechanism of molecular exchange in electroporation of erythrocyte ghosts.     

The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer

The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells’ response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41?±?9?% yield, while in isotonic buffer 32?±?11?% yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1?% in isotonic buffer to 10?±?4?% in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.     

Fusion events and nonfusion contents mixing events induced in erythrocyte ghosts by an electric pulse.

The mechanism of membrane fusion was studied by using human erythrocyte ghosts held in close contact by alternating current-induced dielectrophoresis and inducing fusion with a single electric field pulse. Individual fusion events were followed visually using either 1,1'-dihexadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate as a membrane-mixing label or 10-kD fluorescein isothiocyanate-dextran as a contents-mixing label. However, over a range of variables, the number of contents-mixing events usually considerably exceeded the number of membrane-mixing events, although the discrepancy was less at higher ionic strength. However, when the dielectrophoretic force holding the membranes in contact was turned off after the pulse, Brownian motion caused some of the groups of ghosts in which contents mixing occurred to eventually separate from one another, showing that they could not represent fusion events. Separate experiments showed, conversely, that fusion did occur in the groups that did not separate after the dielectrophoresis was turned off.     

Plasma membrane voltage changes during nanosecond pulsed electric field exposure

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of approximately 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat cells to a pulse, the voltage across the membrane at the anodic pole of the cell reached values of 1.6 V after 15 ns, almost twice the voltage level generally required for electroporation. Voltages across the membrane on the side facing the cathode reached values of only 0.6 V in the same time period, indicating a strong asymmetry in conduction mechanisms in the membranes of the two opposite cell hemispheres. This small voltage drop of 0.6-1.6 V across the plasma membrane demonstrates that nearly the entire imposed electric field of 10 V/mum penetrates into the interior of the cell and every organelle.     

High yields of specific hybridomas obtained by electrofusion of murine lymphocytes immunized in vivo or in vitro

We have studied the use of electrofusion to obtain hybridomas producing antigen-specific antibodies after immunization of murine lymphocytes in vitro. Under optimal conditions fusion frequencies of the order of magnitude of 10(-3) were obtained, which is approximately 80-fold higher than the mean value obtained with fusion induced by polyethylene glycol. The number of antigen-specific hybridomas was also increased in a comparable way. The high yields of specific hybridomas observed with electrofusion were independent of the immunization procedure, the antigen or the hapten of interest, or the sources of the lymphocytes. The data presented in this paper indicate that electrofusion may be an extremely attractive alternative method for immortalization of human lymphocytes following immunization in vitro.     

Low concentrations of macromolecular solutes significantly affect electrofusion yield in erythrocyte ghosts

Electrofusion yields in rabbit erythrocyte ghosts containing various amounts of hemoglobin, bovine serum albumin, or dextran at low concentrations were measured as a function of pulse field strength and pulse decay half-time. The presence of any of the macromolecules in low concentrations caused fusion yields to be significantly higher than when the ghosts were white (i.e., containing only buffer). The fusion yield enhancement was also critically dependent on the parameters of the electric field pulse. The fusion yield was also significantly affected by small changes in the concentration of hemoglobin when it was present outside the ghost membranes in the suspension buffer.     

In vivo cell electrofusion

In vitro electrofusion of cells brought into contact and exposed to electric pulses is an established procedure. Here we report for the first time the occurrence of fusion of cells within a tissue exposed in vivo to permeabilizing electric pulses. The dependence of electrofusion on the ratio of applied voltage to distance between the electrodes, and thus on the achievement of in vivo cell electropermeabilization (electroporation) is demonstrated in the metastasizing B16 melanoma tumor model. The kinetics of the morphological changes induced by cell electrofusion (appearance of syncytial areas or formation of giant cells) are also described, as well as the kinetics of mitosis and cell death occurrence. Finally, tissue dependence of in vivo cell electrofusion is reported and discussed, since electrofusion has been observed neither in liver nor in another tumor type. Particular microenvironmental conditions, such as the existence of reduced extracellular matrices, could be necessary for electrofusion achievement. Since biomedical applications of in vivo cell electropermeabilization are rapidly developing, we also discuss the influence of cell electrofusion on the efficacy of DNA electrotransfer for gene therapy and of antitumor electrochemotherapy, in which electrofusion could be an interesting advantage to treat metastasizing tumors.     

A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses

Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the trans-membrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.     

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.     

Electrofusion of fibroblasts on the porous membrane

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.     

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.     

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements.     

Electroacoustic fusion of millilitre volumes of cells in physiological medium

A technique is described in which erythrocytes suspended in 1.1 ml of 145 mM NaCl, have been fused by electrofusion. The cells in suspension were brought into close contact by setting up a 3 MHz ultrasonic standing wave in a cylindrical cell container. The aluminium foil base of the container served both to transmit ultrasound and as an electrode for electrofusion. The electric pulse was generated by a capacitor discharge system. The electric field strength required to fuse cells increased as the ionic strength of the cell suspending phase increased. Cells in physiological saline fused at an electric field strength of 7.3 kV/cm with a 50 microseconds pulse.