Electrofusion Method Research Articles

Controllable and Stable Fusion Strategy on Microfluidics.

This paper presents a microfluidic device with 200 cell "cage" structures. Based on this microfluidics device, a new simple and stable electrofusion method was developed. Under hydrodynamic force, the cells moved to the desired "cage" cell capture structure and efficiently formed cell pairs (∼80.0 ± 4.6%). Intimate intercellular connectivity was induced by the precise modulation of hypotonic solution substitution and the microstructure, which ensured no cell movement or displacement during the cell electroporation/electrofusion process. It also guaranteed repeated electroporation occurring in the same contact region and provided a stable cell membrane recombination and a cell fusion microenvironment. When the pulse signal was applied, a high fusion efficiency of ∼88.3 ± 0.6% was demonstrated on the microfluidic device.

In Vitro Fertilization System Using Wheat Gametes by Electric Fusion.

In vitro fertilization (IVF) systems using isolated gametes have been used to dissect post-fertilization events in angiosperms, as female plant gametophytes are deeply embedded within the ovaries. In addition, hybrid and polyploid zygotes can be produced by using IVF systems. Complete IVF systems of maize and rice, two out of three major energy-providing crops, have been established in order to acquire detailed knowledge of mechanisms of fertilization and early embryogenesis. Following in the footsteps of previous success, a wheat IVF system was developed to introduce the advantages of this technology to wheat research. Fusion of gametes was performed via a modified electrofusion method, and the zygote formed a cell wall and two nucleoli. The zygotes divided into symmetric two-celled embryos, globular-like embryos and multicellular club-shaped embryos which are mostly consistent with those in the embryos in planta. IVF-produced club-shaped embryos developed into compact embryonic calli and subsequently regenerated into fertile plants. In this chapter, we provide a detailed description of wheat IVF system that might become an important technique for generating new genotypes of wheat and/or new hybrids as well as for investigating fertilization-induced events in wheat.

Effect of microwave heating duration on the stability of the partially stabilised zirconia doped with CaO

In this paper, the stability of CaO doped partially stabilised zirconia (CaO-PSZ) prepared by electrofusion method was improved from 88.14% to 95% within 1 h using the microwave heating method. The increase of the stability rate was because of the microwave heating method's advantages, including selective heating and fast heating. Analysis techniques, namely X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Raman spectroscopy, and scanning electron microscopy (SEM), were used to analysis the influences of the holding time (1–4 h) on the CaO doped PSZ samples at a heating temperature of 1100 °C to understand the fundamental mechanism of the optimisation process. The Raman and XRD spectra were fitted by the Gaussian method to further analysis the effect of holding time on the microstructure evolution of the samples. The fitting results indicated that the sample's crystal quality and stability were improved after microwave heating, and the optimised holding time is 2 h.

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.

Mechanical properties of electro and butt fusion welded high-density polyethylene pipes

Abstract Electrofusion welding and butt fusion welding are common methods for joining polyethylene pipes used for water and gas distribution. Because of the wide use of these two methods in infrastructure engineering, welded pipe joints and pipe materials should be examined with the intent of reducing errors in projects. The purpose of this study is to increase knowledge of electrofusion and butt fusion welding methods and examine them in order to establish which offers the best performance for a specific engineering service project. Tensile and hydrostatic pressure tests are performed on un-welded and welded pipes for high-density polyethylene pipes. Additionally, a numerical analysis of the experimental results is conducted by employing the ANSYS program for tensile test samples. In tensile tests, according to the maximum average load, electrofusion welding values are larger than those of butt fusion welding and the elongations of the un-welded pipe at the fracture in butt fusion welding are greater than in electrofusion welding. For short-term hydrostatic pressure tests, the resistance in butt fusion welding for defects of roundness and alignment is better than that of electrofusion welding.

The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro.

Induced cell fusion is a powerful method for production of hybridoma in biotechnology and cell vaccines in medical applications. Among different alternatives, physical methods have an advantage, as they do not require any additives. Among them electrofusion, an electroporation-based cell fusion method holds a great promise. Electric pulses cause cell membrane permeabilization and due to pore formation bring cell membrane into the fusogenic state. At the same time, however, they compromise cell viability. We used a train of 8 × 100 µs electric pulses, delivered at 1Hz with strengths ranging from 400 to 1600V/cm. We evaluated electrofusion efficiency by dual color microscopy. We determined cell viability, because during electroporation reactive oxygen species are generated affecting cell survival. The novelty of our study is evaluation of the effect of lipid antioxidant α-tocopherol oncell fusion yield and cell viability on mouse B16-F1 cells. Pretreatment with α-tocopherol slowed down dynamic of cell fusion shortly after electroporation. Twenty-four hours later, fusion yields between α-tocopherol treated and untreated cells were comparable. The viability of α-tocopherol pretreated cells was drastically improved. Pretreatment of cells with α-tocopherol improved whole electrofusion process by more than 60%. We believe that α-tocopherol holds great promise to become an important agent to improve cell electrofusion method.

Establishment of an In Vitro Fertilization System in Wheat (Triticum aestivum L.).

In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect post-fertilization events in angiosperms, since the female gametophytes of plants are deeply embedded within ovaries. In addition, IVF systems have been used to produce hybrid and polyploid zygotes. Complete IVF systems have been established in maize and rice, two of three major crop species providing the majority of human energy intake. Among those crop species, gametes of wheat have not been used to establish a complete IVF system successfully. In this study, a wheat IVF system was developed to introduce the advantages of this technology to wheat research. Fusion of gametes was performed via a modified electrofusion method, and the fusion product, a zygote, formed a cell wall and two nucleoli. The first division of zygotes was observed 19-27 h after fusion, and the resulting two-celled embryo developed into 10-20-celled globular-like embryos and multicellular club-shaped embryos by 3 and 7-10 d after fusion, respectively. Such zygotic division profiles were mostly consistent with those in the embryo sac, suggesting that the division profile of IVF-produced early embryos reflects that of early embryos in planta. Although the IVF-produced club-shaped embryos did not develop into differentiated embryos but into compact embryonic calli, fertile plants could be regenerated from the embryonic calli, and the seeds harvested from those plants grew normally into seedlings. The IVF system described here might become an important technique for generating new genotypes of wheat and/or new hybrids as well as for investigating fertilization-induced events in wheat.

Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

Development of indirect competitive ELISA for quantification of mitragynine in Kratom (Mitragyna speciosa (Roxb.) Korth.)

Monoclonal antibody (MAb) against mitragynine (MG), an analgesic alkaloid from Kratom leaves (Mitragyna speciosa), was produced. MG was coupled to carrier proteins employing either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), a zero-length cross linker or a 5-carbon length glutaraldehyde cross linker. To confirm the immunogenicity, the hapten numbers were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Preparation of the MAb was accomplished by the electrofusion method. Hybridoma 1A6 that was constructed from the fusion between splenocytes of EDC/NHS conjugate immunized mice and SP2/0-Ag14 myeloma cells was selected, cloned twice and expanded. The cross-reactivities (CRs) of this MAb 1A6 with a series of indole alkaloids were 30.54%, 24.83% and 8.63% for speciogynine, paynantheine and mitraciliatine, respectively. Using this MAb, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a measurement range of 32.92–250μg/mL. Quantitative analysis of the MG contents in plant samples by icELISA correlated well with the standard high performance liquid chromatography method (R2=0.994). The MAb against mitragynine provided a tool for detection of MG in Kratom preparations.

SOI SUBSTRATE-BASED MICROFLUIDIC CHIP FOR CELL ELECTROFUSION

A microelectrode array-based cell electrofusion chip was fabricated by using the MEMS technique. Because of the short distance between two counter microelectrodes, the working voltage on this chip was only 1/100–1/20 as that in the traditional cell electrofusion method. Simulation method was used to analyze the on-chip electric field distribution and optimize the structure of the microelectrodes. The results showed the length and width of the microelectrode, and the distance between two microelectrodes in the horizontal and vertical direction would impact the strength and distribution of the electric field. Thus, optimized chip architecture was obtained, on which six individual chambers were integrated. At least 1680 microelectrodes were patterned within any one chamber. Alternating current signals have been used to manipulate and align cells, and most cells were aligned as cell–cell twins. High-intensity (~103 V/cm) electric pulses were used to fuse the aligned cell–cell twins. The fusion efficiency was about 40%, which was much higher than that in traditional chemical method (less than 1‰) and electrofusion methods (less than 5%).

Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model.

Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In contrast, mesenchymal stem cells (MSCs) are highly proliferative, anti-apoptotic and pluripotent to differentiate toward various cell types, promote angiogenesis and modulate inflammation, thereby studied as an enhancer of islet function and engraftment. Electrofusion is an efficient method of cell fusion and nuclear reprogramming occurs in hybrid cells between different cell types. Therefore, we hypothesized that electrofusion between MSC and islet cells may yield robust islet cells for diabetes therapy. We establish a method of electrofusion between dispersed islet cells and MSCs in rats. The fusion cells maintained glucose-responsive insulin release for 20 days in vitro. Renal subcapsular transplantation of fusion cells prepared from suboptimal islet mass (1,000 islets) that did not correct hyperglycemia even if co-transplanted with MSCs, caused slow but consistent lowering of blood glucose with significant weight gain within the observation period in streptozotocin-induced diabetic rats. In the fusion cells between rat islet cells and mouse MSCs, RT-PCR showed new expression of both rat MSC-related genes and mouse β-cell-related genes, indicating bidirectional reprogramming of both β-cell and MSCs nuclei. Moreover, decreased caspase3 expression and new expression of Ki-67 in the islet cell nuclei suggested alleviated apoptosis and gain of proliferative capability, respectively. These results show that electrofusion between MSCs and islet cells yield special cells with β-cell function and robustness of MSCs and seems feasible for novel therapeutic strategy for diabetes mellitus.

Inhibition of Gaussia luciferase Activity by a Monoclonal Antibody

A new electrofusion method has been developed with high efficiency for hybridoma formation. This method induces more positive clones than conventional fusion methods do. This has led to the successful establishment of functional monoclonal antibodies that can modulate the function of antigens. Such antibodies are difficult to obtain using existing methods. Monoclonal antibodies that inhibit the luminescent activity of the Gaussia luciferase (Gluc) have been prepared using this method. The affinities of antibodies for their antigens were assessed qualitatively by Biacore measurements. Epitope mapping experiments showed that these antibodies recognize the conformation of Gluc antigens rather than their specific sequences. The amino acid sequence of an antigen-recognition region of an antibody was also determined. The mechanism of luminescence inhibition by the monoclonal antibodies is discussed. Keywords: Gaussia luciferase, epitope, kinetic analysis, electrofusion, luminescence, monoclonal antibodies, hybridoma, affinity, ELISA, centrifugation

English

  In an attempt to construct superior Trichoderma harzianum isolates for improving β-glucosidase productivity, protoplast fusion technique was applied. After application of different mutagenic treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four antifungal agents. Out of them, four isolates were selected on the basis of their response to antifungal agents and their productivities of carboxymethylcellulase (CMCase) and β-glucosidase to be introduced into intraspecific protoplast fusion experiments using two different methods (PEG and electrofusion). Three crosses were carried out among the selected four isolates. Results showed that, the number of fusants obtained after electrofusion were more than those obtained after polyethylene glycol (PEG) method. In addition, high productivities of CMCase and β- glucosidase were obtained after electrofusion in the three crosses. The applied protoplast electrofusion method proved to be a good and effective method for obtaining T.  harzianum fusants with higher productivity of β- glucosidase enzyme.   Key words: Trichoderma, protoplast fusion, electroporation, β-glucosidase.

Cell activation by CpG ODN leads to improved electrofusion in hybridoma production

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.

A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device

A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO₂ film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (∼12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.

On the protoplast fusion of mulberry and paper-mulberry by electrofusion method

On the protoplast fusion of mulberry and paper-mulberry by electrofusion method

Chip‐Based Cell Electrofusion

AbstractCell fusion is a rapid developing cytobiological method utilizing engineering approaches to fuse two or more cells into one hybrid. The hybrid cell can be further cultivated into a new species or a cell‐engineering product. After many years of development, cell fusion is becoming a powerful tool for biological, medical and agricultural research. At present, electrofusion is widely used to achieve cell fusion due to its high efficiency and maneuverability. On‐chip cell electrofusion is receiving more and more attention as a result of its superior performance. This review will discuss the basic theory of chip‐based cell electrofusion, the pairing method, low‐ and high‐throughput cell electrofusion, and the possible ways to overcome the major limitations in existing electrofusion methods.

The fusion of somatic cells and human embryonic stem cells by electrofusion method

Generating pluripotent stem cells directly from somatic cells obtained from patients is one of the major methods to avoid tissues rejection in regenerative medicine. Beside somatic cell nuclear transfer, recent studies have shown that human embryonic stem cells (hESCs) have the ability to reprogram somatic cells after fusion by PEG. But the low efficiency of PEG induced fusion in the study might be one of the obstacles for the technology to be used practically. Electrofusion is a biomedically safe method that has been broadly used in nuclear transfer. This study is to investigate whether electrofusion would improve the fusion efficiency of hESCs with human embryonic fibroblasts (HEFs). A normal hESC line with 46, XX karyotype and a HEF line with 46, XY karyotype were used in this study. Before electrofusion, both of the cells were dissociated into single cells and were separately labeled by DIO and DID. 1106 hESCs and 1106 hEFs were fused using the Electro Cell Manipulator ECM 2001 in the optimized electrical parameters (AC:20v, 30s, DC:700v, 10 s, 2 times). The fusion efficiency was determined by fluorescence activated cell sorting (FACS) after fusion. Fused cell were seeded onto FBS-coated six-well plates in N2B27 serum free hESCs culture medium as described by others supplemented with 40 ng/mL bFGF. When hESC-like colonies appeared, the DIO and DID double positive colonies were mechanically picked up, passaged and analyzed by FISH for chromosome X and Y. The colonies contained the Y chromosome were considered as hybrid colonies. After fusion, FACS analysis showed that DID and DIO double positive cells was 21.52.0%(n = 2) which is superior to the results described before by Cowan et al. using PEG for fusion(0.95%0.03%, double positive cells with PEG), and this could be increased to 60.0% (n = 1) after FACS sorting. Approximately 2105 cells after fusion were plated in each well of six-well plate, Six days after plating, small colonies started to appear in culture. In the ninth day, there were 70-80 colonies in each well. 4 of 30 colonies analyzed by FISH contained Y chromosome which indicated that these colonies were hybrid of HES and HEF. The reprogramming characters of this reproggrammed ES-like colonies are in further analysis.

An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies

We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.

Study of high-throughput cell electrofusion in a microelectrode-array chip

A microfabricated high-throughput cell electrofusion chip with 1,368 pairs of high aspect ratio silicon microelectrodes is presented. These microelectrodes, which were distributed in six individual microscale cell-fusion chambers, were covered with titanium and gold thin film to improve their electric conductivity as well as surface hydrophobility. Six chambers having different electrode distances make the chip highly suitable for fusing cells with different sizes. A microfluidic platform was set up for flowing control, cell manipulation and also experimental observation. Cells for electrofusion were first aligned at the prearranged locations by the dielectrophoretic force between two counter-electrodes, which benefits the traverse of electric pulse through the cell–cell contacting point for electroporation. Several on-chip cell electrofusion experiments have been carried out on different kinds of animal cells and plant protoplasts. Compared with conventional electrofusion methods, higher fusion efficiency was achieved on this device for precisely forming micropores on the proximate membranes of two contacting cells, and high throughput was also obtained due to the use of a large number of microelectrodes for cell manipulation and fusion. Moreover, a much lower power supply was required for the shorter distance between two counter-electrodes.