A Novel Device for Controlling D. discoideum Movement
with an Electric Field
Arunan Skandarajah with Devin Henson
Abstract
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.
The Janetopoulos Lab, Department of Biological Sciences
Tubing
16 G
Punches
PDMS
PDMS
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.
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.
Results Continued
Microfluidic System for Electrotaxis
Measurement
Voltmeter
Power supply
Top View
Coverslip
with
deposited Pt
(gray)
Cell Area
Side View
Coverslip
with
deposited Pt
(gray)
= direction of fluid
flow
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
Figure 1 – Electric fields can alter the wound
healing process. Reproduced from Chiang, 1991
• 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
• 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
Results
Summary
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
Future Direction
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.
Macro-Scale System for
Characterization of Cell Response
References
Chiang, M., Electrical fields in the vicinity of small wounds in Notophthalmus
viridescens skin. The Biological Bulletin, 1989. 176(2): p. 179-183.
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.
Figure 2 - Experimental Set-Up From Literature
•
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
• 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.
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.