Group 3:
Microfluidic System for
Galvanotaxis Measurements
Team Members:
Arunan Skandarajah and Devin Henson
Advisors:
Dr. Janetopoulos,
Dr. John Wikswo,
and Dr. Paul King
Physiological Presence of Electrical
Fields
• Normal development and maintenance
processes result in endogenous electric
fields1,2
– Established in wounds- ~.4-2 V/cm
– Generated across gland and organ walls
– Present during fetal organization
• Result from ion flow across barriers
Cells Respond to Electric Fields
• Endothelial cells in wounds can be
manipulated to speed or slow healing1
• Neuronal cells grow along electrically
generated paths1
• Metastasizing cells will respond in the
opposite manner of less malignant cells3
• Our model organism, Dictyostelium
discoideum, also undergoes electrotaxis at
voltages from 2-20V/cm4
Problem Statement
How can we make experimentation easier than is currently
possible?
Forrester, 2007 (2)
Issues with current technology
1. Difficult to fit bulky system on microscope stage
2. Exposed and electrified liquid dangerous to experimenters
3. Voltages as high as 500 V must be applied across device.
4. Excessive media and reagents required
Solution Process
• Utilize Microfabrication to
– Reduce size of overall device to the scale of
centimeters
• Allow for easy use on microscope stage
• Reduce the required applied voltage to ~50 V by
reducing field size
• Reduce the volume of cells and media required
– Create a closed design
• Perfuse Media through Device
– Control of ion and pH gradients
– Avoid problems with agar
• Eliminate voltage drop resulting from long bridges
Microfluidic Design
Experimental Cell Area
Resistance Distribution
Calculations
• DB Buffer = 552.49 Ω*cm
• Conductivity electrode meter from the Cliffel Lab in
SC5516
• 2% agar = 650 Ω*cm with considerable
variation
• Voltage drop due to electrolysis of water prevents
precise measurement
Current Status
•
•
•
•
Microfabrication and AutoCAD training complete
Prototypes produced
Cell successfully cultured and seeded into devices
Cell motility captured, but stated rates of movement have
not been matched
• Must establish baseline cell response to evaluate device
before proceeding further
Cells seeded in
device, imaged at 32x
using Zeiss Axiovert
microscope and
QImaging camera
Current Status
•
Two macro-scale devices for establishing cell
response
1. Coverslip method- replica from literature
- easy to seed and clean
- does not resolve any targeted problems
2. Microfluidic- “halfway”
- lower voltage, better setup
- more difficult to clean and seed
Agar bridge access holes
Agar bridge contact area
glass slide
PDMS
Mcrofluidic
channel
Coverslip roof
Preliminary Cell Motility Data
• Cells are moving at approximately 1
μm/min
• This value is 1/3 of that reported in
literature4
• Poor gas exchange?
Recent Developments
• Reached Zhao to discuss protocol for macro-scale
device
– Reduce chamber length for better gas exchange (1
cm and 5 mm lengths)
– Possible visit to UC-Davis
• Received films for fabrication of microfluidic
designs
• Need to confirm new microfluidic devices work
similarly to previous iterations for controlling pH
and ion gradients
– Find optimal flow rates and cell seeding methods
Immediate Tasks
• Try Zhao’s suggestions with shorter chamber
lengths
• Characterize flow profile and pH gradients in
new microfluidics
• Edit Kevin Seale’s code for our cell type and
phase contrast imaging
• Understand baseline response of cells in electric
field
• Select among prototypes for optimal microfluidic
channel dimensions
ImageJ Cell Tracking Software
• Requires 8-bit images
– QCapture capable of capturing in 8-bit
– Previously captured 12-bit images are
convertible to 8-bit  no lost images
• Obtained code for finding and tracking
cells (Kevin Seale, SyBBURE)
• Other macros for specific cell tracking
downloadable online
Future Direction
• Utilize optimal microfluidic design to
replicate cell response from literature
• Improvements in using our device
– Lower applied voltage necessary
– Controlled pH and ion gradients without agar
– Reduced overall device size and reagent
consumption
• Quantify data
– Program ImageJ to obtain cell displacement
– Find directedness and trajectory rates
Works Cited
1. Nuccitelli, R., A role for endogenous electric fields in wound
healing. Current Topics in Developmental Biology, 2003. 58:
p. 1-26.
2. Forrester, J.V., Lois, N., Zhao, M., McCaig, C. The spark of
life: the role of electric fields in regulating cell behaviour using
the eye as a model system, Ophthal. Res. 39:4-16, 2007.
3. Pu, J., McCaig, C.D., Cao, L., Zhao, Z., Segall, J.E., Zhao, M.
EGF receptor signaling is essential for electric-field-directed
migration of breast cancer cells, J. Cell Sci. 120:3395-3403,
2007
4. Zhao, M., Jin, T., McCaig, C.D., Forrester, J.V., Devreotes,
P.N. Genetic analysis of the role of G protein-coupled
receptor signaling in electrotaxis, J. Cell Biol. 157:921-927,
2002.
.