(340f) Investigation of the Thermal and Electrical Impact of Electric Fields On Mammalian Cells Manipulated Using Contactless Dielectrophoresis

Dielectrophoresis (DEP), the motion of a particle in a non-uniform electric field, has become a robust method for analyzing nano-particles, cells, viruses, and DNA based on their physical and electrical properties. A new technique, contactless Dielectrophoresis (cDEP), 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 of the cells off chip, post sorting. To ensure that secondary assays on these cells are accurate, it is important to minimize the physiological stress experienced by the cells as they are sorted. In this study a combination of finite element modeling and experimental examination is used to investigate the effects that continuous exposure to medium and high frequency electric fields have on mammalian cells.

Multi-physics models of two cDEP devices were constructed in COMSOL which incorporated Joule heating, fluid dynamics, and heat transfer in fluids and solids. The first device contained a continuous sorting geometry with saw tooth features in which cells experience a vertically displacing DEP force. The electric field experienced by the cells cycled in magnitude between high and low as they traveled through regions with and without constrictions, respectively. The second device contained a batch sorting geometry in which cells are attracted to and trapped on insulating pillars within the sample channel. In this geometry, cells experience a constant electric field for the duration in which they are trapped. The time dependent electric field dosages were computed using previously published experimental values. A third three-dimensional model of a single cell suspended in solution was then constructed and these experimental field dosages were applied. 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. MDA-MB-231 breast cancer cells were suspended in a sucrose solution with a conductivity of 0.1 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.

Uptake of membrane impermeable dye was noticed in some experimental conditions indicating that some degree of reversible electroporation was occurring. The degree of dye uptake increased as the applied voltage was increased or as the frequency was lowered. Additionally, a small set of experimental conditions resulted in permanent destabilization of the cell membrane due to irreversible electroporation. These results are consistent with those determined numerically.  Current work is focused on evaluation of the expression of heat shock proteins after cells are driven through the devices. However, theoretical calculations predict a minimal increase in the fluid temperature, and expression of these proteins is expected to be negligible.

In this work, we showed that for the majority of experimental conditions, prolonged exposure to alternating electric fields in a cDEP device results in minimal impact on analyzed cells. However, there exists a small subset of parameters which induce temporary changes in the permeability of the cell membrane which must be accounted for when analyzing secondary assays on sorted cells.