Oregon State University Chemical, Biological, and Environmental Engineering Mentor: Dr. Adam Higgins HHMI Summer 2011 Cameron Glasscock http://www.pages.drexel.edu/~nb93/images/heart.gif Determining cryoprotectant toxicity with adherent endothelial cells Source: http://www.2n2u.com/wp-content/uploads/2011/02/Vas cular.jpg Source: http://www.pages.drexel.edu/~nb93/images/heart.gif
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Oregon State UniversityChemical, Biological, and Environmental EngineeringMentor: Dr. Adam HigginsHHMI Summer 2011
The toxicity rate k is then plotted against concentration
Regression gives toxicity as a function of concentration
Mathematical representation of toxicity
Next step: Create a 3D regression to represent toxicity as a function of both concentration and temperature
N = e kt
0 1 2 3 4 5 6 7 80
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
f(x) = 0.0107605 x − 0.00485950000000001R² = 0.987118231556779
Concentration (molal)
toxi
city
rate
k (m
in-̂1
)
Experimental Results
Initial Experiments 1,3-molal Glycerol at 21C Used 96-Well Plates
Results were highly variable
Possible Sources of Variability Uneven seeding
distribution Cell loss during wash steps
0 10 20 30 40 50 600
0.10.20.30.40.50.60.70.80.9
1f(x) = exp( − 0.00729400776742069 x )R² = 0.612594885081736
1 molal CPA Exposure Time (Min)
Cell
Surv
ival
1-molal Glycerol 21C
0 10 20 30 40 50 600
0.10.20.30.40.50.60.70.80.9
1f(x) = exp( − 0.0182753697676657 x )R² = 0.688002966392353
3 molal CPA Exposure Time (Min)
Cell
Surv
ival
3-molal Gycerol 21C
Investigating Seeding Distribution
Uneven seeding distribution caused by thermal gradients
Pre-incubation to reduce variability Involves placing well
plates with freshly seeded cells at room temperature for 1 hour before placing in 37C incubator
0 10 20 30 40 50 600
0.10.20.30.40.50.60.70.80.9
f(x) = exp( − 0.0144499724211674 x )R² = 0.366479033924925
1 molal CPA Exposure Time (Min)
Cell
Surv
ival
1-molal
0 10 20 30 40 50 600.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70f(x) = exp( − 0.0228374914463214 x )R² = 0.613941727098219
3 molal CPA Exposure Time (Min)
Cell
Surv
ival
3-molal
Pre-Incubated 1,3-Glycerol Toxicity Data
Investigating Cell Loss During Wash Steps
Experiment Cells seeded onto 96-well plate Wells were washed with a PBS buffer solution PrestoBlue measurement taken after wash steps
0
0.2
0.4
0.6
0.8
1
1.2
Effect of Wash Steps
No Wash 5 Washes
Nor
mal
ized
Fluo
resc
ence
Revised Experiments
24-well plates Avoid cell loss during wash steps Increased well size helps to reduce variability
0 10 20 30 40 50 60 700
0.10.20.30.40.50.60.70.80.9
1f(x) = exp( − 0.00569213844112215 x )R² = 0.507030559558598
1-molal Glycerol 21C
Exposure Time (min)
Cell
Surv
ival
0
0.2
0.4
0.6
0.8
1
1.2
Effect of Wash Steps
No Wash 5 Washes
Nor
mal
ized
Fluo
resc
ence
Experimental Conclusion
Initial experiments using 96-well plates yielded inconclusive data
Attempts to isolate cause of data variability Seeding distribution Cell loss due to wash steps
Experiment revised with some improvement using 24-well plates
Future Work
Improve experimental method Try different cell viability assays
Optimization of cryoprotectant addition/removal for vitrification using: Mathematical function for toxicity Osmotic tolerance limits Cell permeability data
Acknowledgements
HHMI Kevin Ahern Mentor: Dr. Adam Higgins Allyson Fry Ratih Lusianti Kenneth Huang Corey Lerch