Microorifice-Based High-Yield Cell Fusion on Microfluidic Chip: Electrofusion of Selected Pairs and Fusant Viability

Abstract

Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. The aim of this paper is to verify feasibility of high-yield cell fusion on a microfluidic chip. This paper also examines viability of the fusant created on the chip. We fabricated a microfluidic chip to fuse selected cell pairs and to study postfusion behavior. We used a self-forming meniscus-based fabrication process to create microorifice with a diameter of 2-10 microm on the vertical walls in a microfluidic channel. When 1 MHz was applied to electrodes located on both sides of the microorifice, dielectrophoretic force attracted the cells toward microorifice to form a cell pair. Once the cells get into contact, fusion pulse was applied. Real time imaging of cells during fusion and cytoplasmic dye transfer between cells indicated success of cell fusion. We found that when high frequency voltage for dielectrophoresis was swept from 1 MHz to 10 kHz in 100 micros, cell fusion was initiated. The effective electric field strength was 0.1-0.2 kV/cm. We analyzed viability by imaging fusant going into cell division phase after 48 h of incubation. We conclude that fabricated microfluidic chip is suitable for high-yield one-to-one fusion and creation of viable fusants. This technology should be a useful tool to study fusion phenomena and viability of fusants, as it allows imaging of the cells during and after the fusion.

A microfluidic chip for highly efficient cell capturing and pairing

“…Recently, micro-orifice structure was reported to assure one-to-one cell pairs and follow-up fusion procedure. 23,24 A meniscus-based fabrication approach was adopted to create microorifice with a diameter of 2-10 lm on the vertical walls in a microfluidic channel. Only seven cells from each side of the wall were attracted to the microorifice to form a cell pair (95-100%) under a 1 MHz alternating current.…”

A cell electrofusion microfluidic device integrated with 3D thin-film microelectrode arrays

“…13 Recently, microfluidics-based cell electrofusion is obtaining more and more attention due to its low voltage, low Joule-heating effect, and precise controllability. 11,[13][14][15][16][17][18][19][20][21][22][23][24][25][26] Due to the short distance between the patterned microelectrodes, a low voltage is sufficient to generate high electric field required for electrofusion. Therefore, it eliminates the requirement for the expensive high-voltage power generator.…”

“…The existing studies on microfluidics-based cell electrofusion mainly focused on improving cell pairing and fusion efficiencies. Typically, positive DEP under AC electric field is used to control cell pairing, [14][15][16][17][18][19][20][21] and DC pulses are used for reversible electroporation and electrofusion in the microfluidic devices. Compared with the conventional electrofusion methods, the microfluidic devices have advantages of low voltage and high efficiency in cell pairing.…”

“…13 However, this method requires modification of cells. Gel et al 18,19 recently developed a novel micro-orifice based cell electrofusion microfluidic device without using 3D microelectrodes. The micro-orifice, whose diameter is smaller than that of the cells, creates field constriction resulting in membrane breakdown occurring nowhere but in the micro-orifice.…”

“…However, it still uses parallel plate electrodes with a large distance to induce reversible electroporation and fusion and thus has inherent disadvantages of the convectional electrofusion method. In the existing studies, both planar thin-film microelectrode [18][19][20][21] and bulk microelectrode 11,13,14,16 have been widely used. The planar thin film microelectrodes are deposited at the bottom edge of the side walls of the microchannel and do not cover the entire side walls.…”