The Experts below are selected from a list of 4152 Experts worldwide ranked by ideXlab platform
Jun Yang - One of the best experts on this subject based on the ideXlab platform.
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A Cell Electrofusion Chip for Somatic Cells Reprogramming.
PloS one, 2015Co-Authors: Yuxiao Zeng, Jun Yang, Zhengqin YinAbstract:Cell fusion is a potent approach to explore the mechanisms of somatic cells reprogramming. However, previous fusion methods, such as polyethylene glycol (PEG) mediated cell fusion, are often limited by poor fusion yields. In this study, we developed a simplified cell Electrofusion chip, which was based on a micro-cavity/ discrete microelectrode structure to improve the fusion efficiency and to reduce multi-cell Electrofusion. Using this chip, we could efficiently fuse NIH3T3 cells and mouse embryonic stem cells (mESCs) to induce somatic cells reprogramming. We also found that fused cells demethylated gradually and 5-hydroxymethylcytosine (5hmC) was involved in the demethylation during the reprogramming. Thus, the cell Electrofusion chip would facilitate reprogramming mechanisms research by improving efficiency of cell fusion and reducing workloads.
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The structure and electric field distribution of cell Electrofusion chip.
2015Co-Authors: Yuxiao Zeng, Jun Yang, Zhengqin YinAbstract:(A) Cell trapping and pairing under positive-DEP force. (B) The electric field distribution in the cell Electrofusion chip. (C) A 3D schematic of the cell Electrofusion chip based on micro-cavity/ discrete microelectrode structure.
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Cell Electrofusion in microfluidic devices: A review
Sensors and Actuators B: Chemical, 2013Co-Authors: Jun Yang, Sang W. Joo, Arghya Narayan Banerjee, Shizhi QianAbstract:Abstract Cell Electrofusion in microfluidic devices attracted great attention in recent years due to its widespread applications potential in cell-based studies. In these microfluidic devices, many manipulation methods, such as chemical conjugation, electric field induced dielectrophoresis, and microfluidic controlling based on microstructure, are used to improve the pairing precision of cells, especially heterogeneous cells. High-strength electric field can produce minipores on cell membrane and induce cell fusion. It can be generated by a constricting electric field with microstructures or two microelectrodes. In comparison with the traditional Electrofusion or other cell-fusion methods, microfluidic cell-Electrofusion method has many advantages such as precise manipulation, high efficiency in cell pairing and fusion, higher cell viability, lower sample contamination and smaller Joule heating effect. In this article, the development of various microfluidic cell-Electrofusion methods is reviewed. Some important parameters affecting the cell Electrofusion are discussed in detail. Techniques that can be integrated on microfluidic devices for high-efficiency cell Electrofusion, such as on-chip cell separation and culture, are also discussed comprehensively.
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a high throughput dielectrophoresis based cell Electrofusion microfluidic device
Electrophoresis, 2011Co-Authors: Jun Yang, Zhengqin Yin, Irina Svir, Shizhi Qian, Bin Xia, Jiawen Yan, Wensheng Hou, Xiaolin ZhengAbstract: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.
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a cell Electrofusion microfluidic device integrated with 3d thin film microelectrode arrays
Biomicrofluidics, 2011Co-Authors: Jun Yang, Sang W. Joo, Shizhi Qian, Xiaolin ZhengAbstract:A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell Electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays to induce electroporation and Electrofusion. Compared to the design with thin film microelectrode arrays deposited at the bottom of the side walls, the 3D thin film microelectrode array could induce electroporation and Electrofusion under a lower voltage. The staggered electrode arrays on opposing side walls induce inhomogeneous electric field distribution, which could avoid multi-cell fusion. The alignment and pairing efficiencies of K562 cells in this device were 99% and 70.7%, respectively. The electric pulse of low voltage (∼9 V) could induce Electrofusion of these cells, and the fusion efficiency was about 43.1% of total cells loaded into the device, which is much higher than that of the convectional and most existing microfluidics-based Electrofusion devices.
Maša Kandušer - One of the best experts on this subject based on the ideXlab platform.
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The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro
The Journal of Membrane Biology, 2019Co-Authors: Maša Kandušer, Mojca Kokalj Imsirovic, Marko UšajAbstract: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 1 Hz with strengths ranging from 400 to 1600 V/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 on cell 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.
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Cell Electrofusion: past and future perspectives for antibody production and cancer cell vaccines
Expert opinion on drug delivery, 2014Co-Authors: Maša Kandušer, Marko UšajAbstract:Introduction: In the past few decades, new methods for drug and gene delivery have been developed, among which electroporation and Electrofusion have gained noticeable attention. Lately, advances in the field of immunotherapy have enabled new cancer therapies based on immune response, including monoclonal antibodies and cell vaccines. Efficient cell fusion is needed for both hybridoma production and cell vaccine preparation, and Electrofusion is a promising method to achieve this goal. Areas covered: In the present review, we cover new strategies of cancer treatment related to antibody production and cell vaccines. In more detail, cell electroporation and Electrofusion are addressed. We briefly describe principles of cell electroporation and focus on Electrofusion and its influential factors, with special attention on the fusogenic state of the cell membrane, contact formation, the effect of Electrofusion media and cell viability. We end the review with an overview of the very promising field of microfluidic devices for Electrofusion. Expert opinion: In our opinion, Electrofusion can be a very efficient method for hybridoma and cell vaccine production. Advances in the development of microfluidic devices and a better understanding of the underlying (biological) mechanisms will overcome the current limitations.
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Cell Electrofusion using nanosecond electric pulses
Scientific Reports, 2013Co-Authors: Lea Rems, Matej Reberšek, Maša Kandušer, Marko Ušaj, Damijan Miklavčič, Gorazd PuciharAbstract:Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, Electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that Electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in Electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.
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The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer
The Journal of Membrane Biology, 2012Co-Authors: Marko Ušaj, Maša KandušerAbstract: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 appli- cation of electric pulses significantly improves electrofu- sion efficiency. How hypotonic treatment improves Electrofusion is still not known in detail. Our results indi- cate that at given induced transmembrane potential elec- troporation 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.
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cell cell Electrofusion optimization of electric field amplitude and hypotonic treatment for mouse melanoma b16 f1 and chinese hamster ovary cho cells
The Journal of Membrane Biology, 2010Co-Authors: Marko Ušaj, Katja Trontelj, Damijan Miklavčič, Maša KandušerAbstract:Efficient electroporation of cells in physical contact induces cell fusion, and this process is known as Electrofusion. It has been shown that appropriate hypotonic treatment of cells before the application of electric pulses can cause a significant increase in Electrofusion efficiency. First, the amplitudes of the electric field were determined spectrofluorometrically, where sufficient permeabilization in hypotonic buffer occurred for B16-F1 and CHO cells. In further Electrofusion experiments 14 ± 4% of fused cells for B16-F1 and 6 ± 1% for CHO was achieved. These Electrofusion efficiencies, determined by double staining and fluorescence microcopy, are comparable to those of other published studies. It was also confirmed that successful electroporation does not necessarily guarantee high Electrofusion efficiency due to biological factors involved in the Electrofusion process. Furthermore, not only the extension of Electrofusion but also cell survival depends on the cell line used. Further studies are needed to improve overall cell survival after electroporation in hypotonic buffer, which was significantly reduced, especially for B16-F1 cells. Another contribution of this report is the description of a simple modification of the adherence method for formation of spontaneous cell contact, while cells preserve their spherical shape.
Marko Ušaj - One of the best experts on this subject based on the ideXlab platform.
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The Effect of Lipid Antioxidant α-Tocopherol on Cell Viability and Electrofusion Yield of B16-F1 Cells In Vitro
The Journal of Membrane Biology, 2019Co-Authors: Maša Kandušer, Mojca Kokalj Imsirovic, Marko UšajAbstract: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 1 Hz with strengths ranging from 400 to 1600 V/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 on cell 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.
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Cell Electrofusion: past and future perspectives for antibody production and cancer cell vaccines
Expert opinion on drug delivery, 2014Co-Authors: Maša Kandušer, Marko UšajAbstract:Introduction: In the past few decades, new methods for drug and gene delivery have been developed, among which electroporation and Electrofusion have gained noticeable attention. Lately, advances in the field of immunotherapy have enabled new cancer therapies based on immune response, including monoclonal antibodies and cell vaccines. Efficient cell fusion is needed for both hybridoma production and cell vaccine preparation, and Electrofusion is a promising method to achieve this goal. Areas covered: In the present review, we cover new strategies of cancer treatment related to antibody production and cell vaccines. In more detail, cell electroporation and Electrofusion are addressed. We briefly describe principles of cell electroporation and focus on Electrofusion and its influential factors, with special attention on the fusogenic state of the cell membrane, contact formation, the effect of Electrofusion media and cell viability. We end the review with an overview of the very promising field of microfluidic devices for Electrofusion. Expert opinion: In our opinion, Electrofusion can be a very efficient method for hybridoma and cell vaccine production. Advances in the development of microfluidic devices and a better understanding of the underlying (biological) mechanisms will overcome the current limitations.
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Cell Electrofusion using nanosecond electric pulses
Scientific Reports, 2013Co-Authors: Lea Rems, Matej Reberšek, Maša Kandušer, Marko Ušaj, Damijan Miklavčič, Gorazd PuciharAbstract:Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, Electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that Electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in Electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.
-
The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer
The Journal of Membrane Biology, 2012Co-Authors: Marko Ušaj, Maša KandušerAbstract: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 appli- cation of electric pulses significantly improves electrofu- sion efficiency. How hypotonic treatment improves Electrofusion is still not known in detail. Our results indi- cate that at given induced transmembrane potential elec- troporation 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.
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cell cell Electrofusion optimization of electric field amplitude and hypotonic treatment for mouse melanoma b16 f1 and chinese hamster ovary cho cells
The Journal of Membrane Biology, 2010Co-Authors: Marko Ušaj, Katja Trontelj, Damijan Miklavčič, Maša KandušerAbstract:Efficient electroporation of cells in physical contact induces cell fusion, and this process is known as Electrofusion. It has been shown that appropriate hypotonic treatment of cells before the application of electric pulses can cause a significant increase in Electrofusion efficiency. First, the amplitudes of the electric field were determined spectrofluorometrically, where sufficient permeabilization in hypotonic buffer occurred for B16-F1 and CHO cells. In further Electrofusion experiments 14 ± 4% of fused cells for B16-F1 and 6 ± 1% for CHO was achieved. These Electrofusion efficiencies, determined by double staining and fluorescence microcopy, are comparable to those of other published studies. It was also confirmed that successful electroporation does not necessarily guarantee high Electrofusion efficiency due to biological factors involved in the Electrofusion process. Furthermore, not only the extension of Electrofusion but also cell survival depends on the cell line used. Further studies are needed to improve overall cell survival after electroporation in hypotonic buffer, which was significantly reduced, especially for B16-F1 cells. Another contribution of this report is the description of a simple modification of the adherence method for formation of spontaneous cell contact, while cells preserve their spherical shape.
Xiaolin Zheng - One of the best experts on this subject based on the ideXlab platform.
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a high throughput dielectrophoresis based cell Electrofusion microfluidic device
Electrophoresis, 2011Co-Authors: Jun Yang, Zhengqin Yin, Irina Svir, Shizhi Qian, Bin Xia, Jiawen Yan, Wensheng Hou, Xiaolin ZhengAbstract: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.
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a cell Electrofusion microfluidic device integrated with 3d thin film microelectrode arrays
Biomicrofluidics, 2011Co-Authors: Jun Yang, Sang W. Joo, Shizhi Qian, Xiaolin ZhengAbstract:A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell Electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays to induce electroporation and Electrofusion. Compared to the design with thin film microelectrode arrays deposited at the bottom of the side walls, the 3D thin film microelectrode array could induce electroporation and Electrofusion under a lower voltage. The staggered electrode arrays on opposing side walls induce inhomogeneous electric field distribution, which could avoid multi-cell fusion. The alignment and pairing efficiencies of K562 cells in this device were 99% and 70.7%, respectively. The electric pulse of low voltage (∼9 V) could induce Electrofusion of these cells, and the fusion efficiency was about 43.1% of total cells loaded into the device, which is much higher than that of the convectional and most existing microfluidics-based Electrofusion devices.
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A high‐throughput dielectrophoresis‐based cell Electrofusion microfluidic device
Electrophoresis, 2011Co-Authors: Jun Yang, Zhengqin Yin, Irina Svir, Shizhi Qian, Bin Xia, Jiawen Yan, Wensheng Hou, Xiaolin ZhengAbstract: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 (
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Chip‐Based Cell Electrofusion
Advanced Engineering Materials, 2010Co-Authors: Jun Yang, Li-ping Zhao, Zhengqin Yin, Jie Chen, Irina Svir, Xiaolin ZhengAbstract:Cell 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.
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Polyimide Membrane Based Cell-Electrofusion Chip
Chinese Journal of Analytical Chemistry, 2009Co-Authors: Jun Yang, Zhengqin Yin, Xiaolin Zheng, Jing Yang, Yi Cao, Xing-guo Zhang, Bin XiaAbstract:Abstract A novel flexible cell-Electrofusion chip was fabricated using flexible printed circuits board (FPCB) technique, by which microelectrode array was fabricated on the copper foil laminated on the surface of the polyimide membrane. On this chip, cell Electrofusion could be carried out at low voltage (
Zhengqin Yin - One of the best experts on this subject based on the ideXlab platform.
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A Cell Electrofusion Chip for Somatic Cells Reprogramming.
PloS one, 2015Co-Authors: Yuxiao Zeng, Jun Yang, Zhengqin YinAbstract:Cell fusion is a potent approach to explore the mechanisms of somatic cells reprogramming. However, previous fusion methods, such as polyethylene glycol (PEG) mediated cell fusion, are often limited by poor fusion yields. In this study, we developed a simplified cell Electrofusion chip, which was based on a micro-cavity/ discrete microelectrode structure to improve the fusion efficiency and to reduce multi-cell Electrofusion. Using this chip, we could efficiently fuse NIH3T3 cells and mouse embryonic stem cells (mESCs) to induce somatic cells reprogramming. We also found that fused cells demethylated gradually and 5-hydroxymethylcytosine (5hmC) was involved in the demethylation during the reprogramming. Thus, the cell Electrofusion chip would facilitate reprogramming mechanisms research by improving efficiency of cell fusion and reducing workloads.
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The structure and electric field distribution of cell Electrofusion chip.
2015Co-Authors: Yuxiao Zeng, Jun Yang, Zhengqin YinAbstract:(A) Cell trapping and pairing under positive-DEP force. (B) The electric field distribution in the cell Electrofusion chip. (C) A 3D schematic of the cell Electrofusion chip based on micro-cavity/ discrete microelectrode structure.
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a high throughput dielectrophoresis based cell Electrofusion microfluidic device
Electrophoresis, 2011Co-Authors: Jun Yang, Zhengqin Yin, Irina Svir, Shizhi Qian, Bin Xia, Jiawen Yan, Wensheng Hou, Xiaolin ZhengAbstract: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.
-
A high‐throughput dielectrophoresis‐based cell Electrofusion microfluidic device
Electrophoresis, 2011Co-Authors: Jun Yang, Zhengqin Yin, Irina Svir, Shizhi Qian, Bin Xia, Jiawen Yan, Wensheng Hou, Xiaolin ZhengAbstract: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 (
-
Chip‐Based Cell Electrofusion
Advanced Engineering Materials, 2010Co-Authors: Jun Yang, Li-ping Zhao, Zhengqin Yin, Jie Chen, Irina Svir, Xiaolin ZhengAbstract:Cell 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.