Device Development for Testing the Effects of Electric Fields upon Mammalian Cells

Device Development for Testing the Effects of Electric Fields upon Mammalian Cells

David Ede, Kevin Roehm, Sundar Madihally

Contact: david.ede@okstate.edu, Oklahoma State University

Electric fields (EF) play a key role in regeneration of the body. Tissues create electrically charged environments in response to injury. Additionally, embryos utilize EF to guide nervous system organization and growth, yet the central nervous system loses this ability to regenerate.  Use of EF is a promising tool to promote central nervous system regeneration by guiding and promoting cell growth [1,2].  Current devices for testing cell response to EF are complex and not well suited to representing bodily conditions.  Modern manufacturing techniques such as 3D printing allow for customizable compact devices, limiting the need for many pieces of equipment and permitting the creation of 3-dimensional environments.  This project developed an integrated, compact device to apply electric field to cells using 3D printing technology.  A 3-chambered tray was designed to specifications in SolidWorks and 3D Printed with a Makerbot Replicator 2. Material of construction was polylactic acid (PLA).  A central chamber housed cells and cell media while two peripheral chambers housed Ag/AgCl electrodes fixed in place by conductive agarose gels.  The design was also tested for EF propagation using the computer program COMSOL. 

Human neuroblastoma cells (brain cancer, IMR-32 line) derived from a metastatic site in the abdominal cavity, purchased from ATCC was cultured in eagle’s minimum essential medium (EMEM) with 10% fetal bovine serum (FBS).  Cells at 25,000cells/slide were seeded upon glass slides coated with 0.1% gelatin and incubated for 12-18 hours at 37°C with 5%CO2/95% Air.  Cells were subjected to direct current (DC) EF for 3 hours and 45 minutes with a voltage of 400-500mV/mm and current of 3mA. Agarose gels (0.5%) in PBS solution protected cells from electrode corrosion and conducted current into media.  After EF exposure, cells aligned parallel to the EF axis and elongated along the perpendicular axis, verifying published reports [1].  Hence, the device produced for this project effectively creates an environment to subject cells to electric fields. 

In order to better understand the developed device, it was modeled using COMSOL Multiphysics.  Current conservation was used to model the electric field using: J = σE + J_e and E= -∇V.  The constitutive equation is D = ε₀ε_rE, where σ is the electrical conductivity, ϵ is the relative permittivity, J is the flux vector, E is the electric field, V is the voltage, and Q is the volumetric current.  The conductivity of the medium is and the dielectric constant is 80, as reported by Huang et al. [2].  Based upon data reported by Oi, Shinyama, and Fujita [3], the conductivity of PLA is approximately  and its dielectric constant is approximately 3.25 at room temperature (21°C).  These results showed that the present device design maintains a higher potential in the first chamber with steep losses between each chamber. We have produced an alternative design that shows the most gradual gradient which may decrease overheating by reducing the resistance to flow, thereby making it possible to maintain the desired current with a smaller potential across the electrodes.  For future usage, we need to modify the device to allow continuous cell visualization during EF exposure.  Also, we need to refine experimental conditions such as cell density, agarose concentration, and current to a greater accuracy.

REFERENCES

[1] Patel N, Poo MM. Orientation of neurite growth by extracellular electric fields. J Neurosci 2:483–496. 1982.

[2] Huang Y, Wang XB, Becker FF, Gascoyne PR. Introducing dielectrophoresis as a new force field for field-flow fractionation. Biophysical Journal. 73:1118-29. 1997

[3] Oi T, Shinyama K, Fujita S. Electrical properties of heat-treated polylactic acid. Electrical Engineering in Japan. 180:1-8, 2012