(482b) Electroporation of Cells On Chip Using High Frequency Electric Fields Without Electrode-Sample Contact

Contactless Dielectrophoresis (cDEP) is a relatively new technique for investigating, isolating, and enriching mammalian cells. This technique separates cells from contact with the electrodes by using fluid electrodes which are isolated from the sample channel by thin insulating membranes. This technique has been used to isolate live mammalian cells from dead cells and beads of the same size as well as discriminate between cells with different metastatic behaviors. A significant effort has been made to expand the originally limited frequency spectrum over which these devices are effective. Current designs are capable of manipulating cells at frequencies between 1 kHz and 1 MHz in low conductivity, physiologically suitable buffers.  This wide range of operation allows investigators to manipulate mammalian cells at frequencies close to their first Clausius-Mossotti factor crossover point where the difference between similar cells is the most pronounced. This has enabled the use of cDEP devices for a number of biological applications which analyze and culture the cells off chip, post sorting. Here, we show that these devices can additionally be used to transport large molecules or genes into cells with high efficiency while maintaining a sterile environment.

The application of an external electric field causes a redistribution of charge within a cell resulting in an increase in the cell’s transmembrane potential (TMP). This, in turn, increases the free energy in the molecules in the cell membrane and the rate of transport of larger molecules into the cell. The process, known as electropermeabilization or electroporation, has been widely used in biology to transfer macromolecules, into cells while maintaining cell viability. Irreversible electroporation (IRE) results if the external field intensity increases past a certain threshold, such that the cell membrane is permanently destabilized and the cell cannot recover. Charge redistribution in presence of an electric field is not instantaneous. For a brief time, ions internal and external to the cell rearrange in response to the external field. This displacement current halts as the TMP reaches its maximum, after approximately 1 μs. If the electric field is pulsed or due to a high frequency AC voltage, a displacement current propagates through the cell giving rise to increased membrane potentials in the nucleus and organelles. In typical reversible and irreversible electroporation protocols the pulses are a single polarity with on times on the order of 100 to 1000 microseconds. In this study, we show that fields with frequency components above 10 kHz can result transient and irreversible electroporation effects.

Multi-physics models of cDEP devices were constructed in COMSOL which incorporated fluid- and electro-dynamics. The device contained constricting features in which cells are exposed briefly to an elevated electric field. By altering the magnitude of the applied voltage and the fluid velocity, the field-time dose experienced by the cells can be adjusted. Because the field varies both spatially and temporally, the simulations were used to create a time-dependent ‘pulse envelope’ that would be experienced by a cell experimentally. A three dimensional model of a single cell including internal organelles was constructed and exposed to this pulse envelope. The resulting increase in transmembrane potential was then calculated and compared literature values for cellular response and damage.

Master stamps of the two theoretical devices were created on separate silicon wafers using deep reactive ion etching. Polymer replicates were then cast in PDMS with a 10:1 ratio of polymer to curing agent. The polymer replicates were bonded to glass slides after exposure to air plasma for two minutes and stored under vacuum. Prior to experimentation, the fluid electrode channels were filled with phosphate buffered saline with a conductivity of 1.4 S/m. Cells were suspended in a sucrose solution with a conductivity of 0.01 S/m and driven through the devices using a syringe pump. A high voltage amplifier and series transformer were used to apply voltages with amplitudes up to 300 V_RMS between 10 and 800 kHz.

The amplitude of the applied voltage and the fluid velocity was adjusted to determine the experimental doses required to induce reversible and irreversible electroporation effects. This work confirms the existence of a relationship between the Clausius-Mossotti factor and the ability to induce electroporation effects. The energy required to electroporate cells was found to increase with as the frequency of the applied field was elevated. The contactless method presented here may be advantageous as it eliminates any possible electro-chemical effects or bubble formation at the fluid-electrode interface. Future work will focus on evaluating cells off chip for transfection efficiency.