A cell electrofusion microfluidic chip with micro-cavity microelectrode array

A new cell electrofusion microfluidic chip with 19,000 pairs of micro-cavity structures patterned on vertical sidewalls of a serpentine-shaped microchannel has been designed and fabricated. In each micro-cavity structure, the two sidewalls perpendicular to the microchannel are made of SiO2 insulator, and that parallel to the microchannel is made of silicon as the microelectrode. One purpose of the design with micro-cavity microelectrode array is to obtain high membrane voltage occurring at the contact point of two paired cells, where cell fusion takes place. The device was tested to electrofuse NIH3T3 and myoblast cells under a relatively low voltage (~9 V). Under an AC electric field applied between the pair of microelectrodes positioned in the opposite micro-cavities, about 85–90 % micro-cavities captured cells, and about 60 % micro-cavities are effectively capable of trapping the desired two-cell pairs. DC electric pulses of low voltage (~9 V) were subsequently applied between the micro-cavity microelectrode arrays to induce electrofusion. Due to the concentration of the local electric field near the micro-cavity structure, fusion efficiency reaches about 50 % of total cells loaded into the device. Multi-cell electrofusion and membrane rupture at the end of cell chains are eliminated through the present novel design.

[1]  Shizhi Qian,et al.  A cell electrofusion microfluidic device integrated with 3D thin-film microelectrode arrays. , 2011, Biomicrofluidics.

[2]  Keunchang Cho,et al.  A novel electroporation method using a capillary and wire-type electrode. , 2008, Biosensors & bioelectronics.

[3]  Shizhi Qian,et al.  A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device , 2011, Electrophoresis.

[4]  C. Milstein,et al.  Continuous cultures of fused cells secreting antibody of predefined specificity , 1975, Nature.

[5]  Xiaolin Zheng,et al.  Somatic and stem cell pairing and fusion using a microfluidic array device , 2011 .

[6]  Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown. , 1981, Biochimica et biophysica acta.

[7]  Joel Voldman,et al.  An active bubble trap and debubbler for microfluidic systems. , 2008, Lab on a chip.

[8]  I. Wilmut,et al.  "Viable Offspring Derived from Fetal and Adult Mammalian Cells" (1997), by Ian Wilmut et al. , 2014 .

[9]  Frank H. Ruddle,et al.  Pluripotent teratocarcinoma-thymus somatic cell hybrids , 1976, Cell.

[10]  Ning Hu,et al.  Polyimide Membrane Based Cell-electrofusion Chip , 2009 .

[11]  Guillaume Tresset,et al.  A Microfluidic Device for Electrofusion of Biological Vesicles , 2004, 17th IEEE International Conference on Micro Electro Mechanical Systems. Maastricht MEMS 2004 Technical Digest.

[12]  O. Baysal,et al.  On-demand particle enrichment in a microfluidic channel by a locally controlled floating electrode , 2011 .

[13]  Leandro Lorenzelli,et al.  A dielectrophoresis-based microdevice coated with nanostructured TiO2 for separation of particles and cells , 2011 .

[14]  R. Jaenisch,et al.  Microfluidic Control of Cell Pairing and Fusion , 2009, Nature Methods.

[15]  Elizabeth H. Chen,et al.  Unveiling the Mechanisms of Cell-Cell Fusion , 2005, Science.

[16]  Kang-Ming Chang,et al.  A circuit design of a low-cost, portable and programmable electroporation device for biomedical applications , 2012 .

[17]  L. Lilge,et al.  Controlled electroporation of the plasma membrane in microfluidic devices for single cell analysis. , 2012, Biomicrofluidics.

[18]  D. Chiu,et al.  A microfluidics platform for cell fusion. , 2001, Current opinion in chemical biology.

[19]  Chang Lu,et al.  Microfluidic cell fusion under continuous direct current voltage , 2006 .

[20]  Sang Hoon Lee,et al.  An electrofusion chip with a cell delivery system driven by surface tension , 2008 .

[21]  O Orwar,et al.  Microfluidic device for combinatorial fusion of liposomes and cells. , 2001, Analytical chemistry.

[22]  D. Miklavčič,et al.  Cell–Cell Electrofusion: Optimization of Electric Field Amplitude and Hypotonic Treatment for Mouse Melanoma (B16-F1) and Chinese Hamster Ovary (CHO) Cells , 2010, The Journal of Membrane Biology.

[23]  Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells , 2007 .

[24]  Z. Wen,et al.  Characterization of a capacitance-coupled contactless conductivity detection system with sidewall electrodes on a low-voltage-driven electrophoresis microchip , 2010, Analytical and bioanalytical chemistry.

[25]  Y. Huang,et al.  Introducing dielectrophoresis as a new force field for field-flow fractionation. , 1997, Biophysical journal.

[26]  Liqun Wu,et al.  Dielectrophoretic capture voltage spectrum for measurement of dielectric properties and separation of cancer cells. , 2012, Biomicrofluidics.

[27]  Hidehiro Oana,et al.  Dielectrophoretic cell trapping and parallel one-to-one fusion based on field constriction created by a micro-orifice array. , 2010, Biomicrofluidics.

[28]  Frances S. House,et al.  An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies. , 2008, Journal of immunological methods.

[29]  Ning Hu,et al.  Chip‐Based Cell Electrofusion , 2010 .

[30]  M Washizu,et al.  High-yield electrofusion of biological cells based on field tailoring by microfabricated structures. , 2008, IET nanobiotechnology.

[31]  Albert van den Berg,et al.  On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation , 2011, Electrophoresis.

[32]  Hou Wen SOI-based Cell Electrofusion Chip , 2009 .

[33]  Hywel Morgan,et al.  Trapping single human osteoblast-like cells from a heterogeneous population using a dielectrophoretic microfluidic device. , 2010, Biomicrofluidics.

[34]  Ashutosh Sharma,et al.  Electrokinetic particle translocation through a nanopore containing a floating electrode , 2011, Electrophoresis.

[35]  Masayasu Suzuki,et al.  Cell Leading into Microwell Array by Using Negative Dielectrophoresis , 2012 .

[36]  M. Washizu,et al.  Microorifice-Based High-Yield Cell Fusion on Microfluidic Chip: Electrofusion of Selected Pairs and Fusant Viability , 2009, IEEE Transactions on NanoBioscience.

[37]  H. Kaji,et al.  A porous membrane-based culture substrate for localized in situ electroporation of adherent mammalian cells , 2007 .

[38]  Ning Hu,et al.  Study of high-throughput cell electrofusion in a microelectrode-array chip , 2008 .

[39]  Z. Wen,et al.  A microsystem of low-voltage-driven electrophoresis on microchip with array electrode pairs for the separation of amino acids , 2009, Analytical and bioanalytical chemistry.

[40]  Hidehiro Oana,et al.  Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli , 2006 .

[41]  Ronald Pethig,et al.  Interaction between cells in dielectrophoresis and electrorotation experiments. , 2010, Biomicrofluidics.

[42]  Kevin Eggan,et al.  Nuclear Reprogramming of Somatic Cells After Fusion with Human Embryonic Stem Cells , 2005, Science.

[43]  M. Washizu,et al.  Cell membrane voltage during electrical cell fusion calculated by re-expansion method , 2007 .

[44]  U. Zimmermann,et al.  Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA. , 1981, Plant physiology.

[45]  Yu Sun,et al.  Electrodeformation for single cell mechanical characterization , 2011 .

[46]  Fang Yang,et al.  Dielectrophoretic separation of colorectal cancer cells. , 2010, Biomicrofluidics.

[47]  Norio Nakatsuji,et al.  Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells , 2001, Current Biology.

[48]  O. Baysal,et al.  Manipulating particles in microfluidics by floating electrodes , 2010, Electrophoresis.

[49]  Shizhi Qian,et al.  DC dielectrophoretic particle-particle interactions and their relative motions. , 2010, Journal of colloid and interface science.