Mechanically facilitated cell-cell electrofusion.

Apparatus and methods were developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. The key component of this fusion system was a newly developed fusion chamber. The chamber was composed of two functionally identical electrodes that were housed in a multi-layer structure. The layers functioned as support for the electrodes. They also allowed adjustment of the distance between opposing electrode faces. The electrodes were constructed in a manner that allowed cells to be deposited, by vacuum, onto each face. Electrode faces were positioned at a predetermined distance from each other to mechanically force cell-cell contact between the deposited cells. Fusion was induced by delivering direct current pulses to the juxtaposed cells. Fusion products were detected and quantitated by flow cytometry. Details of the chamber design and a protocol for using the fusion chamber are given. Mechanically facilitated cell-cell electrofusion was demonstrated by using the chamber to produce fusion products from like fusion partners. The practical applicability of the chamber was demonstrated by fusing unlike cell types. Mechanically facilitated cell-cell electrofusion is not specific to the cells used in this study; the chamber can be adapted for use with other cell types.     

Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions

Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids. The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature. Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l. Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8. Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory. The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber). This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion. It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures. At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied. Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion. Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession. Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established. This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process. The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins. Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.     

Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs

In this paper, we describe the procedure of large-scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30-mm length, 1-mm width, and 2-mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 μsec twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD-1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm. The proportion of development to blastocyst in the eggs fused with the pronuclear karyoplast derived from F1 (75 and 71%) was twice that of the eggs fused with the pronuclei derived from CD-1 strain (25 and 37%). After transfer to recipients, live young were obtained from both the eggs fused with karyoplast following one or two electrofusion exposures.     

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.     

Hybridoma cells produced by electrofusion in a homogeneous electric field

In the customary technique will be used a fusion chamber with an inhomogeneous electric field. The disadvantage is that the fusion conditions are not constant in the chamber. Yet for di-electrophoretic collection of the cells their own field-inhomogenizing influence is sufficient that is, the electrofusion can proceed also in very simple chambers with homogeneous fields.     

Quantitative studies on the viability of yeast protoplasts following dielectrophoresis

Abstract Protoplasts from Saccharomyces cerevisiae and Saccharomyces diastaticus were collected in a non-homogeneous alternating electric field. The dependence of the viability of the protoplasts on different conditions of collection was tested by determining the regeneration rates in each case. The parameters varied in collection were the field strength (0.33 kV/cm–6.67 kV/cm), the frequency of the alternating field (1–2 MHz) and the collection time (2–10 min). The introduction of a new type of fusion chamber (meander chamber) permitted, for the first time, quantitative exposure of protoplasts to the electric field as well as their complete transference into the regeneration medium. The regeneration rates of yeast protoplasts collected under those conditions employed for electrofusion did not differ from those of protoplasts which had been maintained under the same experimental conditions but were not subject to the influence of an alternating electric field. The two yeast strains were fused together (collection 1 kV/cm; pulse 15 kV/cm; duration of pulse 40 μs) and the fusion products were introduced into a selection medium for regeneration. The fusion rate was about 4.8 × 10⁻⁴; on average 272 colonies grew on the selection medium for each chamber filling.     

空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。     

A single-cell surgery microfluidic device for transplanting tumor cytoplasm into dendritic cells without nuclei mixing

This study aimed to demonstrate the feasibility of generating tumor cell vaccine models by single-cell surgery in a microfluidic device that integrates one-to-one electrofusion, shear flow reseparation, and on-device culture. The device was microfabricated from polydimethylsiloxane (PDMS) and consisted of microorifices (aperture size: ∼3 μm) for one-to-one fusion, and microcages for on-device culture. Using the device, we could achieve one-to-one electrofusion of leukemic plasmacytoid dendritic cells (DC-like cells) and Jurkat cells with a fusion efficiency of ∼ 80%. Fusion via the narrow microorifices allowed DC-like cells to acquire cytoplasmic contents of the Jurkat cells while preventing nuclei mixing. After fusion, the DC-like cells were selectively reseparated from the Jurkat cells by shear flow application to generate tumor nuclei-free antigen-recipient DC-like (tarDC-like) cells. When cultured as single cells on the device, these cells could survive under gentle medium perfusion with a median survival time of 11.5 h, although a few cells could survive longer than 36 h. Overall, this study demonstrates single-cell surgery in a microfluidic device for potential generation of dendritic cell vaccines which are uncontaminated with tumor nucleic materials. We believe that this study will inspire the generation of safer tumor cell vaccines for cancer immunotherapy.     

A biophysical approach to the optimisation of dendritic-tumour cell electrofusion

Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and electrorotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%, as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells.     

Fusion of Erwinia chrysanthemi spheroplasts in an electric fields

A new approach has been elaborated for electrofusion of Erwinia chrysanthemi spheroplasts. The new approach consists of superimposition of high voltage impulses on the pellet of tightly contacting cells in the course of centrifugation. The mixture of spheroplasts of two genetically marked strains was placed into the special centrifuge chambers and spinned for 15 min at 2500 g to get a compressed pellet between chamber electrodes. Three successive pulses of 6.6 kv/cm amplitude and 30 microseconds duration were applied to spheroplast pellet during centrifugation. Fusion products were viable and after plating on the surface of hypertonic medium regenerated to the rod forms. As a result, the hybrid clones carrying the markers of both parents were isolated.     

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.     

Modulation and direction of the electrofusion response in plant protoplasts

In experiments on electrofusion of protoplasts (from Solanum brevidens, S. tuberosum, Nicotiana plumbaginifolia) the presence of divalent cations (Ca²⁺) at 1 mM in the fusion medium was found to increase the yield of hybrids observed directly after fusion and decrease the duration of pulse needed for fusion. Pretreatment of protoplasts with the polyamine spermine also enhanced fusion yield, and when combined with 1 mM Ca²⁺ the effects were additive. The improvement in fusion yield (2–4 fold) was most marked for protoplast populations (e.g. from suspension cultured cells) that were least responsive to electrofusion in mannitol alone. Short term viability, judged from FDA fluorescence was found to be high at these increased fusion levels. Optimum fusion parameters for electrofusion thus may be determined from short term experiments. Attempts to direct to fusion response between populations of protoplasts of identical properties by pretreatment of one fusion partner with spermine were inconclusive.     

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.     

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.     

Nuclear membrane fusion in electrofused mammalian cells

Fusion of nuclei was studied in electrofused cells using staining procedures and DNA flow cytometry. Homogeneous and heterogeneous electrofusion of Ehrlich ascites tumor cells. Muntjac cells and V79-S181 cells were performed in balanced-salt solutions at low temperature. Incubation of the cells subjected to electrofusion in fusion media for about 2 h was required to complete cell fusion and, in particular, nuclear membrane fusion. Under optimum electrofusion conditions it was found that fusion of nuclei is a very frequent event. Half of the fused cells (about 30 to 50% of the field-exposed cells) underwent nuclear membrane fusion. It is shown that the high frequency of nuclear membrane fusion in electrofused, unsynchronised cells resulted from intracellular dielectrophoresis occurring during cell alignment. In accordance with theory, maximum nuclear membrane fusion was observed using alignment fields of between 1 and 4 MHz (depending on the cell species), that is above the frequencies at which the plasmalemma capacity no longer shielded the cell interior from participation in the conduction process. In this frequency range a potential difference can be built up across the nuclear membrane leading to repositioning of the nuclei into the contact zone of the plasmalemmas of two attached cells. This intracellular dielectrophoresis apparently facilitated fusion of nuclei once intermingling of the plasma membranes had occurred. It was further demonstrated that exponentially growing cells showed higher cell fusion rates than cells taken from the unfed plateau phase. One, but not the only reason, might be the higher ATP content of exponentially growing cells compared to cells of the plateau phase. Addition of external ATP to plateau phase cells during electrofusion resulted, in accordance with this assumption, in an increase of fusion frequency, whereas ATP had apparently no effect on the fusion yield of exponentially growing cells. G1 cells obtained by mitotic selection after nocodazole-induced blockage in metaphase also showed higher cellular and nuclear membrane fusion yields than exponentially growing cells. Most importantly, it could be demonstrated both experimentally and theoretically that electrofusion of cells in a dielectrophoretically aligned chain is controlled by a simple law of probability resulting predominantly in fusion of two cells independent of the number of cells in the chain. The likelihood of fusion of various numbers of cells in a chain is given by the appropriate power of the probability of two-cell fusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of X-ray induced aberrations in mammalian chromosomes by electrofusion induced premature chromosome condensation

Summary Premature chromosome condensation (PCC) was induced by electrofusion of metaphase cells of an Ehrlich ascites tumor cell line with interphase cells of a Muntjac cell line or of a Chinese Hamster subline. Electrofusion was performed by cell alignment in a weakly inhomogeneous a.c. field of 200 V/cm amplitude (peak-to-peak value) and of 1.7 MHz frequency, followed by the application of a series of breakdown (fusion) pulses of 5 kV/cm strength and 15 µs duration. Most of the PCC's were of the G2 type despite the large proportion of G1 and S cells in the suspension. The number of chromatid aberrations observed in electrofused cells which had not been subjected to irradiation was not significantly above the spontaneous level. This indicates that electrofusion, at least as used here, did not lead to lesions expressed as structural aberrations. When interphase cells were irradiated by X-ray doses below 3 Gy before electrofusion PCC analysis showed chromosome damage consisting mainly of breaks and gaps. The frequency of aberrations recorded by PCC was 6 to 40 fold larger than that seen in conventional metaphase analysis. This large increase probably arose because of an effective suppression of the G2 repair of chromosomal lesions by the fast condensation process which took place within about 30 min. This assumption was supported by PCC experiments in which the time between X-irradiation and fusion with subsequent chromosome condensation was varied. The results demonstrated that G2 repair of chromosomal lesions was not detectable until 20 min after fusion with a half-time of the repair kinetics of about 1.5 h. The selectivity of premature chromosome condensation in G2 cells is discussed in terms of the differences between electrofusion and chemically or virally induced fusion. It is assumed that the concentration and the transfer rate of the chromosome condensation factor from the metaphase to the interphase cell are the limiting factors in achieving PCC. This is because the localised permeabilisation of the membrane and the dominance of two-cell fusions are characteristic of electrofusion.     

Ionic-strength modulation of electrically induced permeabilization and associated fusion of mammalian cells

Application of a high electric field to cells in culture has been shown to make them both permeable and fusogenic. The molecular events involved in the phenomenon are still poorly understood. In this study we investigated the effects of the ionic strength of the pulsing buffer on the electropermeabilization and electrofusion of Chinese hamster ovary cells. Increasing the ionic strength of the pulsing medium results in an increase in sieving of transient permeant structures, but decreases the fusion index. Treatment of cells with trypsin or pronase before application of the pulses abolishes the ionic modulation of both electropermeabilization and electrofusion. A similar rate of expansion of permeabilization is obtained whatever the ionic content of the pulsing buffer, and cells fuse even at high ionic strength. This observation lends support to our hypothesis that membrane proteins play a role in electrofusion.     

Electrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process,fusion of sperm cells with synergids and central cells,and cell reconstitution in maize

Summary Fusion products were created by the electrofusion of single sperm cells with single synergids and central cells. The synergid was also fused with the sperm cell, occasionally in the presence of adhering second synergids, egg cells, and central cells. Single egg cells were fused with single sperm cells in the presence of adhering synergids and the central cell. Cytoplasmic organelles were transmitted through the fertilization process by electrofusion using cytoplasts of maize mesophyll cells. Cell reconstitution was achieved by fusion of one or two sperm cells with single enucleated protoplasts, thus creating a haploid or a diploid cell.     

Hybridization frequencies of different mammalian cell types by electrofusion

The efficiency of electrofusion of four types of cells: CHO, HeLa, mouse melanoma cells and human skin fibroblasts has been studied. The frequencies of fusion products were determined 1) directly in a closed flow-through fusion chamber after dielectrophoresis and pulsation; 2) after short-term postfusion cultivation period of 5 to 10 minutes; and 3) in various intervals up to 30 hours after fusion induction. No substantial differences were found in the rates of formation of heterokaryons and synkaryons between the individual cell types, and this confirmed the uniformity of the effects of electric fields on diverse cell membranes. After 5 hours of culture the yield of fusion products reached 15 to 35% in various cell combinations and the frequencies of synkaryons reached up to 7% in almost all the combinations studied 24 to 30 hours after fusion.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.