team electrode mentor: dr. john p. fisher students: sagah ahmed, natalie anzures, zach bosley,...
TRANSCRIPT
Team ELECTRODEMentor: Dr. John P. Fisher
Students: Sagah Ahmed, Natalie Anzures, Zach Bosley, Brendan Bui, Ariana Feizi, Sudi Jawahery, Courtney Koenig, Katie Lakomy, Megan Lin, Poorna Natarajan, Eisha Nathan, Hiba Sayed, Eduardo Solano
Evaluating Linear-Radial Electrode Conformations for Tissue Repair and Organizing a Device for Experimentation
Diabetes
Prevalence of Diabetes In 2010, 1.9 million Americans over the
age of 20 were diagnosed with diabetes
Formation of Diabetic UlcersNerve damage, poor circulation, lack of
tissue repair
Issues with Current TreatmentsRequires constant application by the
patient, doesn’t directly target the wound, can be very expensive
http://www.executivehm.com/news/diabetes-in-the-us/
EFFECTS of VEGF & bFGF in wound healing
Body’s natural bioelectric healing system
Angiogenesis and Inflammation
Wound healing and electrical stimulation
Epithelial Cells
Hypothesis
Inflammation of damaged tissue is the major factor suppressing angiogenesis in diabetic ulcers
The application of an electrical stimulus will alleviate the effects of the inflammatory tissue response in wounds
Project Purpose: Create an electrical stimulation device that will improve wound healing in diabetic ulcers.
We are examining the effects of…
In Vitro ExperimentsCell Culture
Freezing/ Thawing Cells are frozen throughout the experiment so a
constant source of cells is available
Nourishment Cells are grown in flasks containing Rat Aortic
Endothelial Cell Medium changed every 48 hours
Passaging Cells are moved to bigger and more flasks as
needed
Plating Before experimentation, cells are plated on 6-well
plate containers
Device Design
ϟUse best parameters determined from experimental trials
ϟCompare effectiveness of a linear setup vs. a radial setup
ϟCheap and small prototype for a more marketable device
Electrical Circuit
Battery
Flow
Flow
+
Device:Linear and Radial Apparatuses
Experimental set-up of a linearly applied electric field. The anode and cathode are 1.5-cm apart.
Experimental set-up of a radially applied electric field. The radius is 1.5 cm.
In Vitro Experiments:Parameters
ϟTest voltages:
ϟ0.01 V
ϟ0.1 V
ϟ1.0 V
ϟ50 Hz
ϟApplied for one 30-minute period
ϟ6 wells: 3 control, 3 experimental
Pulsed Monophasic Current
Time
Volt
ag
e
DEVICE IN ACTION! (electrical stimulation of cells at 0.1 volts, 30 min per well for 3 exp wells)
Negative electrode
Positive electrode
Control(no stimulation)
Experimental
Cell ANALYSIS
Measuring Cell Proliferation
Fluorescently stain live (green) and dead cells (red) in our sample
Live/Dead samples are examined under a fluorescent microscope and cells counted using Computer Software Image J
No significant cell death has been observed after successful cell culture has been achieved
Live/Dead Assay Picture 10x, taken on 10/10/12
100 um
Cellular Proliferation
Control
Experimental
Measuring VEGF/bFGF Expression Levels
Extraction of mRNA and samples are frozen for further testing (ELISA, qRT-PCR)
mRNA levels are tested using the Nanodrop
We are looking for levels of mRNA detection that are high enough to provide us with accurate results in further testing
Cell Migration
Purpose: To observe migration of cell population 3 and 6 hours after electrical stimulation
2 well plates, each with 6 wells.
3 wells control, 3 wells experimental
Cells scraped into 2 cm wide line
Timeline
0 hrs: Stimulation and Plate 1 Imaging
3 hrs: Plate 1 Imaging
6 hrs: Plate 2 Imaging
2 cm cells
No cells
No cells
Migration Imaging/Analysis
Take 3 images along each side of the line of cells (6 pictures per well)
Measure distance from starting position to edge of cell population
Take measurements at several points along the line of cells
Average the distances measured in each image
Image J program
Migration data, 10/9/12
Left side view, scraped line of cells at edge
Current and Future Objectives
FALL 2012:
Experimental testing for Specific Aim 1 (linear device)
Data Analysis for Specific Aim 1
SPRING 2013:
Experimental testing for Specific Aim 2 (radial device)
Protein Cell analysis (ELISA with VEGF and bFGF)
http://www.bmes.org/
Advice for Future Gemstone Teams!
TEAM DYNAMICS
4 Subgroups: Electrical Device, Cell Culture, Cell Migration, Cell Analysis
CURRENT/FUTURE CHALLENGES:
Communication between all teammates/subgroups
Even distribution of workload amongst team members
Coordination having resources prepared and experimental testing times
Keeping up with compiling data and analysis of results
MAKE FRIENDS AND HAVE FUN!!!
GEMSTONE TIPS FOR SUCCESS:
Pick a mentor who is involved in research related to your topic and has RESOURCES
Be flexible with your schedule because plans may change unexpectedly
Apply for grants!
Concluding remarks & THANKS
We would like to thank:
Dr. John P. Fisher, Team ELECTRODE Mentor
Dr. Mario Dagenais, Electrical Device Professor
Mr. Jim Miller, Librarian
Lab Graduate Students:
Kim Ferlin, Bao Nguyen, Martha Wang, and Tiecheng Zhu
Gemstone directors and staff: Dr. Frank Coale, Dr. Kristan Skendall, Mrs. Heather Creek, Dr. James Wallace, Dr. Rebecca Thomas
QUESTIONS?
Please visit our website at: http://teams.gemstone.umd.edu/classof2014/electrode/index.html