(482f) Dielectrophoretic Separation of Electroporated Cells

An exposure of a cell to a sufficiently high external electric field results in electroporation, which alters electrical properties of cells and their dielectrophoretic response. We designed and fabricated a dielectrophoretic field-flow fractionation (DEP-FFF) device which allows for fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells.

The chamber was constructed from a single Pyrex wafer with interdigitated Au electrodes, a spacer, and a top cover glass, making the events in the chamber observable under most optical microscopes. The dimensions were optimized based on numerical computations of the electric field, its gradient and the fluid-flow velocity profile. The electrodes were fabricated using photolithography. A double-sided self-adhesive tape of 100 µm thickness was used as a spacer, with an opening cut in its middle to form a channel, and with water-resistant acrylic glue of the tape holding the glass plates together and providing a tight seal. The glue loses its adhesive properties above 70°C, allowing for easy disassembly of the chamber in hot water and its thorough cleaning.

We first optimized the flow rate and electric field parameters for efficient DEP-FFF separation of moderately heat-treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, and we tested their fractionation in the chamber. DEP-FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the groups, implying undesired transition from negative into positive DEP, where the cells are pulled towards the electrodes at the bottom of the chamber. We tested DEP-FFF chamber with another cell line B16F1 and verified that our system can be used for different cell types. Throughput of this chamber is high, 10⁵ cells can be separated in one batch in 6 min. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency.