Department of Mechanical Engineering, University of Tokyo, Tokyo 113-8656, Japan.
IEEE Trans Nanobioscience. 2009 Dec;8(4):300-5. doi: 10.1109/TNB.2009.2035252.
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.
微孔融合利用电场收缩来确保选定的细胞对进行高产的一对一融合。本文旨在验证在微流控芯片上实现高产细胞融合的可行性。本文还研究了芯片上融合产物的生存能力。我们制造了一个微流控芯片来融合选定的细胞对，并研究融合后的行为。我们使用自形成的弯月面制造工艺在微流控通道的垂直壁上制造了直径为 2-10 微米的微孔。当在微孔两侧的电极上施加 1 MHz 的频率时，介电泳力将细胞吸引到微孔处形成细胞对。一旦细胞接触，就施加融合脉冲。融合过程中细胞的实时成像和细胞间细胞质染料转移表明细胞融合成功。我们发现，当用于介电泳的高频电压从 1 MHz 扫到 10 kHz 时，细胞融合开始。有效电场强度为 0.1-0.2 kV/cm。我们通过在孵育 48 小时后观察融合产物进入细胞分裂阶段来分析其生存能力。我们得出结论，制造的微流控芯片适合高产的一对一融合和有活力的融合产物的产生。这项技术应该是研究融合现象和融合产物生存能力的有用工具，因为它允许在融合过程中和融合后对细胞进行成像。
