Group 3: Microfluidic System for Galvanotaxis Measurements

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Group 3Microfluidic System for Galvanotaxis
Measurements

• Team Members
• Arunan Skandarajah and Devin Henson
• Advisors
• Dr. Janetopoulos,
• Dr. John Wikswo,
• and Dr. Paul King

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Physiological Presence of Electrical Fields

• Normal development and maintenance processes
  result in endogenous electric fields
• Established in wounds- .4-2 V/cm
• Generated across gland and organ walls
• Present during fetal organization
• Result from ion flow across barriers

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Cells Respond to Electric Fields

• Endothelial cells in wounds can be manipulated to
  speed or slow healing
• Neuronal cells grow along electrically generated
  paths
• Metastasizing cells will respond in the opposite
  manner of less malignant cells
• Our model organism, Dictyostelium discoideum,
  also undergoes electrotaxis at voltages from
  2-20V/cm

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Problem Statement

• How can we make experimentation easier than is
  currently possible?
• Issues with current technology
• Difficult to fit bulky system on microscope
  stage
• Exposed and electrified liquid dangerous to
  experimenters
• Voltages as high as 500 V must be applied across
  device.
• Excessive media and reagents required

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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

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Resistance Distribution Calculations

• 1.81 mS/cm conductivity of DB buffer
• Conductivity electrode meter from the Cliffel Lab
  in SC5516
• Resistivity 1/1.81 552.49 O/cm
• Preliminary measurements with 2 agar
• 650 O/cm with considerable variation
• More tests necessary with different electrode
  types

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Microfluidic Design
Experimental Cell Area
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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
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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

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Preliminary Cell Motility Data

• Cells are moving at approximately 1 µm/min
• This value is 1/3 of that reported in literature

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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

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Immediate Tasks

• Work more closely with successful user of old
  device, Min Zhao
• Edit Kevin Seales code for our cell type and
  phase contrast imaging
• Microfabricate additional half-way and fully
  microfluidic systems

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Future Direction

• Quantify data
• Program ImageJ to obtain cell displacement
• Find directedness and trajectory rates
• Explore new experimental procedures
• alternating fields
• coupling with other mechanisms of motility
• knockouts, nulls, genetic modification