(Invited) Rapid Formation of Single-Cell Pairs for Hybrid Cells

Abstract

Dielectrophoresis (DEP) has been used as a manipulation tool for a separation, sorting and patterning of particles and cells. We have used negative-DEP (n-DEP) which directs particles and cells to form line patterns with particles and cells rapidly toward the localized positions with relative weak electric fields. In this presentation, a microwell array electrode was used to produce vertical pairs with two different types of cells by positive-DEP (p-DEP). A microwell array (250,000 wells, 500 × 500) was fabricated on a substrate with indium tin oxide (ITO) layer by coventional photolithography. Microwells were designed in 16 μm width and 25 μm depth. Another ITO substrate was mounted on the microwell array via 30 μm thick polyester film to form a fluidic channel. Mouse myeloma cells (4.0 × 107cells/mL, 12 μm diameter) stained in blue with Hoechst 33342 were introduced into the fluidic channel. AC voltage (1.0 MHz, 10 Vpp) was immediately applied between the upper ITO and lower ITO with microwell array to guide cells in individual microwells by p-DEP. On applying the AC voltage, the dispersed cells immediately, i.e., within 1 s, stayed at positions with microwells. In the microwells trapped with two cells, cells in the top component were removed by the fluidic flow, but cells in the bottom component were retained in the microwells because the fluidic flow could not remove cells from microwells with a depth of 25 μm, i.e., twice the cell diameter or more. These results clearly indicate that the cell array could be formed on the microwell array electrode easily and rapidly by p-DEP. K562 cells (15 μm diameter) stained in green with 5-(and-6)-carboxyfluoresein diacetate succinimidyl ester (CFDA SE) were also guided into microwells occupied with myeloma cells in blue by p-DEP. Myeloma cells in blue and K562 cells in green were successively trapped into microwells by p-DEP. The presence of two cells is not particularly clear in the optical image. In contrast, both blue and green signals which are indicated the presence of myeloma cells and K562 cells are clearly observed from the individual microwells. These results strongly indicated that two different types of cells are vertically aligned in microwells to form single-cell pairs. We can obtain pairs of different cells within 1 min with the pairing efficiency of approximately 68 %. The vertical alignment of cell pairs would be a distinct advantage for producing couplets by cell fusion using electric pulse fields generated between the upper electrode and the lower microwell array electrode, which were used for the cell pairing by p-DEP. The array of two different types of cells can be easily and rapidly formed using the simple device with a p-DEP process. Figure 1