1. 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 – constructing prototypes based on designs provided by senior SyBBURE members, modified designs used for other purposes, and designs built explicitly on the results of our experimentation and experience. Our work has cycled through varying levels of complexities, but the most promising solution yet has been the simplest. A successful microfluidic design for this project would have four traits: Create voltage fields that are required for galvanotaxis, 7-10 V/cm, using applied potentials an order of magnitude less than current designs. Able to control pH in the experimental time frame using flow in place of agar salt bridges Be able to fit entirely on a microscope stage without any open components Over the course of the project we have accomplished the following: Reconstructed the original agar bridge model for galvanotaxis. Designed and microfabricated our 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 our own variations on a large scale system to help 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 AutoCAD was used to design the device. The design patterns were sent to Newman Printing, Inc. to create an 8 ½ x 11 sheet of film for use in fabrication. Photolithography techniques were used to create a silicon wafer master in SU-8 in VIIBRE class 100 clean rooms. Microfluidic devices were then made from PDMS poured over the negative master. The devices were cut from the PDMS mold after baking for four hours in a 60º C drying oven, and access holes were punched through the PDMS using 16 gauge needles and 2 mm biopsy punches. The PDMS mold and cover slip were placed in a plasma bonder for 20 seconds and then bonded together. Experimental Set-ups for Characterization of Cell Response Schematic for device we reconstructed to study galvanotaxis (See photo, right). [1] Galvanotaxis is the movement of cells in a particular direction in response to an electric field across the cells. This movement is significant in the physiological realm because fields have been associated with cell activity in wound healing, neural cone growth, and embryonic reorganization. The role of voltage potentials in tumor metastasis also presents a target for future treatment. Currently the setup for a galvanotaxis experiment requires large beakers of media, long agar salt bridges, dangerously high voltages to generate the necessary field across the cells, and generous cross flow to control pH and ion gradients. Such a cumbersome design makes cell study under a microscope very difficult. A microfluidic device for galvanotaxis will reduce the amount of media used, lower the flow rate necessary to control pH and ion flow, and decrease the required voltage by an order of magnitude to a value deemed safe. Additionally the device can sit entirely on the stage of a microscope with flow controlled by a nearby syringe pump. Following the development of a successful device, Dictyostelium discoideum cells were loaded into the device and studied. Once a baseline response has been established, simple modifications of our microfluidic platform will allow for quick and easy specialization of experiments and a better understanding of galvanotaxis. Experimental Set-Up From Literature 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) 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. Microfabricated PDMS Device Using Agar Bridges The completed device is attached at the inner two access holes to a Harvard Apparatus PHD 2000 Syringe Pump for flow input and to two waste containers at the outer holes. Electrodes connected to a Fisher Biotech Power Supply applied a known voltage at the two outer access holes. Another pair of electrodes attached to an Extech multimeter measured the voltage potential across the cell area. Plastic Becton-Dickinson syringes with a capacity of 10 mL and 23 G needles were placed in the pump apparatus. Tygon tubing of I.D..040” and O.D. of.080” was used to interface with the 2 mm access holes. The I.D. 020” and O.D. 060” Tygon Tubing was used to fit the 16 G holes. As shown above, flow into the center access holes reliably exits from the corresponding waste ports without causing a flow across the cells in the central area. This mechanism allowed us to eliminate the agar bridges while still isolating the cells from electrolytes or other factors like flow. Voltage, pH, and Flow Characteristics To further constrain the dimensions of the experimental area, and raise the proportion of the voltage across the cells, we developed a microfluidic channel with height ~50 µm, width ~250 µm, and length ~1 cm. The channel is accessible to the agar bridges via two 3 mm biopsy punch holes, which create a small reservoir of media for the bridges to sit in. The reservoir helps to maintain consistent fluid contact for voltage application. Before we could attempt to improve upon the difficulties associated with the current device used to study galvanotaxis, we had to reconstruct it to establish a baseline cell response and gain first-hand experience with the device’s problems. In the set up above, we utilize 30 ml glass beakers filled with DB buffering solution. The voltage is applied via two platinum electrodes connected to a Fisher Biotech power-supply. Agar bridges are constructed from glass NMR tubes bent using a Bunsen Burner. The field strength is measured across the cell area using a Extech Voltmeter. No. 1 coverslip walls are cut to shape by diamond-tip pen and sealed to glass slide using Permount adhesive. The coverslip roof provides a geometric constraint in height. 75 mm Application Electrode, Pt Measuring Electrodes, Pt 2 mm Punch Application Electrode, Pt 500 µm 5 mm 20 mm The relationship between applied and measured voltage was characterized across several flow rates to investigate the consistency, linearity, and flow independence properties of the response. Three trials were run for each of the flow speeds. No data points were recorded until the voltage across the experimental area stabilized for a given applied voltage. To assess pH and ion gradients, the designs were initially tested for proper electrical activity at the electrodes through the use of Bromothymol Blue (upper transition to blue at pH = 7.6, lower transition to yellow at pH = 6.0). Visual evaluation showed the clear pH-altering effects in the immediate vicinity of the electrodes and the potential for product diffusion. Preliminary elimination of several flow schemes and speeds was possible through the use of this pH indicator. For cell- based assays, however, we sought to demonstrate that the two outflow pH values remained within a narrow range over the time frame of the experiment with a Denver Instrument pH/mV meter. Side and Top Views of microfluidic device. Inset shows D. discoideum inside our device