Selective Trapping and Retrieval of Single Cells Using Microwell Array Devices Combined with Dielectrophoresis

Abstract

Introduction Cell microarrays which sectionalized cells into microwells are powerful tool for elucidating exhaustively functions of cells at single cell level. However, there are some difficulties in taking advantages of the cell array. At first, the “single-cell occupancy”, which is the ratio of microwells containing one cell to total microwell, is low. Typically, the sedimentation of cells with their own weight was used to form cell array, resulting in a low “single-cell occupancy” as low as about 50%. The second difficulty is that it takes time and effort to retrieve the target cell due to the precise and careful manipulation of the micropippet at resolution of micrometer. The broad generalization of cell microarrays requires the development of techniques to efficiently fabricate cell arrays and to easily pick up cells from cell populations. Dielectrophoresis (DEP) has become attractive because it allows for easy, rapid, and mass manipulation of cells. We have previously demonstrated the dielectrophoretic trapping cells in microwells within a few seconds with an occupation efficiency over 95% using a microwell array device. The device was fabricated by placing the upper indium-tin oxide (ITO) substrate on top of the bottom ITO electrode covered with an insulating layer with a microwell pattern. Although this device has the advantage of producing cell arrays rapidly at high density, it did not selectively manipulate the cells on the microwells. In this study, we propose a simple device for flexible dielectrophoretic manipulation of cells based on the combination of positive DEP (p-DEP) and negative DEP (n-DEP). The use of the present device allows for accurate retrieval of the target cells from a microwell array with retaining the undesired on microwells, not to mention the forming of cell arrays. This device was comprised an upper substrate with microband electrodes mounted on a lower substrate with microwells on the same design of microband electrodes by 90 degree relative to the bottom substrate (Fig. A). The layout of electrodes enables to retrieve the target cell by the repulsive force of n-DEP induced by applying an AC voltage to two microband electrodes arranged above and below the microwell containing the target cell. Naturally, cell arrays can also be fabricated by the attractive force to bottom of microwell induced with p-DEP by applying an AC voltage to all microband electrodes. Experiments and Results The upper and lower ITO substrates with patterns of microband array were fabricated by a conventional photolithographic method. Width of the microband electrode and gap between the electrodes were 40 µm and 80 µm, respectively. The pattern of microwell (16 µm in diameter and 10 µm in height) on the microband electrodes on the lower substate was made of a negative photoresist SU-8. Microband array electrodes on the upper substrate were located 30 µm above the microwells on the lower substrate to form 144 intersections comprising microband electrodes containing microwells. Cell mixtures (3.0 × 106 cells/mL) of hybridoma stained in green and red were prepared in a ratio of 10:1 to demonstrate the retrieval of red cells designated as the target cell. The application of the AC signal (1 MHz, 3 Vpp) to both the upper and the lower microband electrodes with opposite phasing resulted in the formation of the cell array. A single red cell was trapped in the 1–G well and eight green cells were trapped in the others (Fig. B). Subsequently, the frequency applied to band electrode 1 on the upper substrate and the band electrode G on the lower substrate was switched from 1 MHz for p-DEP to 300 kHz for n-DEP, while the frequency applied to the other band electrodes was maintained at 1 MHz for p-DEP (Fig. C). The target red cell in 1–G well was gradually removed over a few seconds after switching the frequency, and it was then transferred downward in the image by slight fluidic flow. In contrast, the other green cells remained in the original position. The results indicated that the repulsive force of n-DEP from the strong electric field region acts on the cell in the 1–G well that comprises both band electrodes switched in the n-DEP frequency region. It is noted that p-DEP still acted on cells in wells comprising band electrodes that applied an AC signal in the p-DEP and n-DEP frequency regions, respectively. Thus, this system would make it possible to retrieve target cells selectively from the array of cells and recover them in an outlet without the microdispensers. Figure 1