1. A Novel Device for Controlling D. discoideum Movement with an Electric Field The Janetopoulos Lab, Department of Biological Sciences Arunan Skandarajah with Devin Henson Abstract Summary Cells have been shown to respond to electric fields, moving in a process known as electrotaxis. This behavior has significant implications in human physiology, but devices that allow scientists to study electrotaxis are inadequate. We are constructing a device utilizing microfluidics to address current problems and serve as an easily adaptable platform for diverse future experiments. Dictyostelium discoideum is a useful model for human cells, and we are using it to both test a new technology and to elucidate signaling mechanisms involved in electrotaxis. The current experimental setup for electrotaxis is unwieldy and potentially dangerous because of high voltages and uncovered liquids My goal is to engineer a device that makes it easier to study electrotaxis: Significantly reduce applied voltage necessary to create fields of 2-20 V/cm across cells Control pH and ion gradients using flow in place of agar salt bridges Reduce overall device size to fit easily on microscope stage Create a completely closed system for safer experimentation I am also trying to understand how cells detect and respond to electric fields Over the course of the project we have accomplished the following: Reconstructed the original agar bridge model for electrotaxis. Designed and microfabricated project-specific masters and PDMS devices. Electroplated platinum electrodes on device Built auxiliary components for a electrotaxis system: insertable electrodes and PDMS punches. Started and maintained a healthy cell culture Successfully seeded cells into devices Viewed random motility of healthy cells in devices Imaged cells with multiple fluorescent tags Observed the electrotactic response in multiple cell lines in original device Succeeded in reproducing behavior over multiple trials Utilized software to track the paths of cells I hope to continue working on this project while expanding my experience with Dictyostelium to the field of chemotaxis. By observing cells under both electrical and chemical gradients, we will be better able to elucidate the mechanisms of cell motility. I am also facing technical challenges and have several short-term goals. The electroplated design is currently an expensive and labor-intense device, though the increased precision would be worth if this device were easier to clean. I will need to explore cheaper methods of production that are still reproducible. In the near future, I hope to use ImageJ cell tracking software to quantitate cell movement and compare the behavior of cells with different mutations or at different life stages. Introduction Electrotaxis is the cell-mediated response of moving in a particular direction in an electric field Movement in electric fields is an important component of wound healing, neural cone growth, and embryonic reorganization. References Future Direction Chiang, M., Electrical fields in the vicinity of small wounds in Notophthalmus viridescens skin. The Biological Bulletin, 1989. 176(2): p. 179-183. Song, Gu, Pu, Reid, Zhao, Z., & Zhao M. Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo. Nat. Protocols 6(2) 1479-1489 Acknowledgements Thanks to our advisers on this project: Professors Janetopoulos, Wikswo, and King. A special thanks to Carrie Elzie for her help on biological issues and Ron Reiserer for his technical support and assistance with training and troubleshooting. Also, thanks to the entire VIIBRE staff for making our research possible. Results Continued Figure 1 – Electric fields can alter the wound healing process. Reproduced from Chiang, 1991 Figure 3 – Composite device of PDMS, glass, and platinum. Direction of flow as marked. Coverslip base is 24mm x 60 mm. Figure with assistance of Devin Henson = direction of fluid flow Power supply Voltmeter 16 G Punches PDMS Coverslip with deposited Pt (gray) Top View Cell Area Side View PDMS Coverslip with deposited Pt (gray) Tubing Macro-Scale System for Characterization of Cell Response Reconstructed previously used design in order to understand how cells should move Established baseline cell response using social amoeba, D. discoideum Obtained time lapse videos for cell tracking Figure 2 - Experimental Set-Up From Literature Microfluidic System for Electrotaxis Measurement Results Figure 4 – Representative sample of WF38 D. discoideum cell movement over the course of twenty minutes. Applied field strength of 13V/cm with polarity marked in white. Use of fully-contained, microfluidic system addresses four design goals Consists of a PDMS block plasma bonded to coverglass with platinum electrodes deposited Device Operation: Fisher Biotech Power Supply applies voltage at solder connections to outer electrodes – field strength measured by Extech multimeter and maintained between 2-20V/cm. A Harvard Apparatus PHD 2000 Syringe Pump moves buffer solution via Tygon tubing to flush electrode products without producing any flow in cell area Resulting device ready for characterization and use in exploring cellular response Cell movement has been reproduced with various cell lines and across field strengths of 10-15V/cm Movement has been tracked manually with data the MTrackJ plugin Qualitatively established a dependence on growing conditions or development state for migration speed.