Development and Characterization of Device for Galvanotactic Response in D. discoideum The Janetopoulos Lab, Department of Biological Sciences Devin Henson and Arunan Skandarajah Abstract Microfabrication Methods Summary Cellular response to electric fields is a physiologically important research topic that has been under-explored because of deficiencies in the current devices available. In the course of this project we seek to develop a device on the microfluidic scale that will address the current problems and promise a platform for rapid production of experiment-specific devices. We have followed the development process through several iterations – reconstructing the current device to understand its flaws, implementing designs provided by senior SyBBURE members, and finally producing devices based explicitly on the results of our own experimentation and experience. Our microfluidic device aims to solve four specific design issues: Significantly reduce applied voltage necessary to create fields of 7-10 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 Over the course of the project we have accomplished the following: Reconstructed the original agar bridge model for galvanotaxis. Designed and microfabricated project-specific masters and PDMS devices. Established resistive properties of materials used in each device Used COMSOL to model flow and pressure gradients in our device. Built variations on a large scale system to establish basic cell response. Built auxiliary components for a galvanotaxis system: insertable electrodes and PDMS punches Applied concepts from macro experiments and VIIBRE generated designs to develop the current, very simple iteration of microfluidic designs. Started and maintained a healthy cell culture Successfully seeded cells into devices Viewed random motility of healthy cells in devices Set up short experiments applying varying voltages to cells to view galvanotactic response Limited success replicating cell motility results from literature; must improve device parameters in order to consistently gather useful motility data We hope to develop the device further through continuing work as members of a SyBBURE summer research and VUSRP. Our main goals throughout the summer will be to first establish the baseline cellular response and then take our microfluidic device from the design stage into the data collection stage. With a functional device, we will explore the mechanisms that allow cells to move in an electric field by using genetic variants and compare these effects with the cell’s response to other gradients, such as chemotaxis. Introduction Devices designed using AutoCAD and film-printed by Newman Printing, Inc. Photolithography techniques create a negative silicon wafer master in SU-8 in VIIBRE class 100 clean rooms Polydimethylsiloxane (PDMS) poured over the master and baked for four hours at 60° C to produce a solid mold with microfluidic channels Access holes punched into device using 2 mm biopsy punches Channels sealed against glass by plasma bonding Experimental Set-ups for Characterization of Cell Response Galvanotaxis: movement of cells in a particular direction in response to an electric field Significant in the physiological realm because fields have been associated with cell activity in wound healing, neural cone growth, and embryonic reorganization. Current experimental setup for galvanotaxis requires: 1.Large beakers of media requiring large microscope stage 2.Long agar salt bridges to control ion and pH gradients 3.Dangerously high voltages to generate necessary electric fields across cells References Future Direction [1] 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) 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. Use of fully-contained, microfluidic system addresses four design goals Consists of a PDMS block plasma bonded to coverglass with inserted platinum electrodes Device Operation: 1.Hewlett-Packard Power Supply applies voltage at outer electrodes 2.Extech multimeter measures resulting field across cells 3.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 Voltage, pH, and Flow Characteristics Reconstructed previously used design in order to understand how cells should move Established baseline cell response using social amoeba, D. discoideum Materials used to construct device: 1.30 mL glass beakers filled with DB buffer solution 2.Agar salt bridges (glass NMR tubes bent using a Bunsen burner) 3.Glass slide bottom with coverslip walls sealed using Permount adhesive and coverslip roof to constrain height of cell area 4.Fisher Biotech Power Supply for voltage application at outer platinum electrodes 5.Extech Multimeter for voltage measurement across experimental area Relationship between applied and measured voltage was characterized Chose flow speeds based on the expected diffusion of electroyte products. Established consistency, linearity, and flow independence properties of the voltage response across three trials for each of three flow speeds To demonstrate control using our chosen flow speed, effluent was tested with Denver Instrument pH/mV meter. Figure 1 - Schematic for device we reconstructed to study galvanotaxis Figure 2 – Schematic of the Microfabrication process Figure 3 - Experimental Set-Up From Literature B. Figure 4 – A. Side view of microfluidic device. Inset shows cells D. discoideum in device. Arrows indicate direction of flow. B. Top view with device dimensions. 5 mm Glass Slide PDMS Access Channels Microfluidic Channels Platinum Electrodes 500 µm 20 mm 75 mm 25 mm 2 mm punch A. 16 G punch