Download - Cameron Glasscock
Oregon State UniversityChemical, Biological, and Environmental EngineeringMentor: Dr. Adam HigginsHHMI 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/Vascular.jpgSource: http://www.pages.drexel.edu/~nb93/images/heart.gif
Cryopreservation
Storage of biological materials Tissue engineering,
transplantation medicine, and other cell-based therapies
The problem: Ice crystal formation causes damage
Source: http://en.wikipedia.org/wiki/File:Iceman_(Bobby_Drake).png
Cryoprotectant chemicals
Reduces damage caused by ice crystal formation Vitrification
Addition and removal causes two types of damage Osmotic damage Toxicity damage
Source: http://blog.bioethics.net/cryopreservation.jpg
Source: http://www.benbest.com/cryonics/DMSO.jpg Source: http://www.bmrb.wisc.edu/metabolomics/standards/glycerol/lit/3416.png
Project
Goal: Determine toxicity of cryoprotectant chemicals with adherent endothelial cells.
Hypothesis: Cryoprotectant type, concentration, temperature, and exposure time have an effect on cryoprotectant toxicity
Glycerol
Procedures
1. Endothelial cells seeded onto well plates
2. Exposure to cryoprotectant solutions Source http://www.porvair-sciences.com/acatalog/205003_1.jpg
Source: http://us.123rf.com/400wm/400/400/phakimata/phakimata0806/phakimata080600061/3131934-blue-multi-channel-pipet-used-for-pipetting-a-96-well-plate-with-pink-solution-on-white.jpg
Procedures (Continued…)
Toxicity damage needs to be isolated from osmotic damage Multi-step addition/removal
during cryoprotectant exposure
Predict procedures with permeability and osmotic tolerance limits data
Source: http://www.ccs.k12.in.us/chsteachers/amayhew/Biology%20Notes/transport%20notes.htm
Procedures (Continued…)
3. Toxicity measured using fluorescent cell viability assay PrestoBlue.
High fluorescence indicates more living cells
Source: http://www.invitrogen.com/etc/medialib/en/images/ics_organized/applications/cell_tissue_analysis/popups.Par.16964.Image.-1.-1.1.gif
Procedures (Continued…)
PrestoBlue measurements taken twice Directly before solution exposure to give initial
seeding density fluorescence 24 hours after solution exposure to give fluorescence
after treatment Accounts for apoptosis
PrestoBlue reagent
1) Add reagent to cells
2) Incubate 3) Read fluorescence
1m
Experimental Variables
DMSO
60 min 40 min 20 min 10 min 5 min 0 min
4C 37C21C
GlycerolEthylene
GlycolPropylene
GlycolCryoprotectant Type
Concentration
Exposure Time
Temperature
3m 5m 7m
Data Analysis
Represented on cell survival versus time plot
Fit to exponential regression of the form:
treated sample fluorescence( )seeding density fluorescenceCell survival = control sample fluorescence
N = e kt
0 10 20 30 40 50 60 700
0.2
0.4
0.6
0.8
137C Glycerol1M Exponential (1M)
3M Exponential (3M)5M Exponential (5M)7M Exponential (7M)
Exposure Time (Min)
Cell
Surv
ival
Accounting for Multi-Step Add/Rem
Toxicity accumulated from lower concentrations
Accounted for with derived correction factor:
2-Step Add/Rem Procedure
Toxicity Function
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
