Determining cryoprotectant toxicity
with adherent endothelial cells
http://www.pages.drexel.edu/~nb93/images/heart.gif
Source: http://www.2n2u.com/wpcontent/uploads/2011/02/Vascular.jpg
Source: http://www.pages.drexel.edu/~nb93/images/heart.gif
Cameron Glasscock
Oregon State University
Chemical, Biological, and Environmental Engineering
Mentor: Dr. Adam Higgins
HHMI Summer 2011
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
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
Source: http://www.benbest.com/cryonics/DMSO.jpg
Source: http://www.bmrb.wisc.edu/metabolomics/standards/glycerol/lit/3416.png
Procedures
1.
Endothelial cells seeded onto well plates
Source http://www.porvair-sciences.com/acatalog/205003_1.jpg
2.
Exposure to cryoprotectant solutions
Source: http://us.123rf.com/400wm/400/400/phakimata/phakimata0806/phakimata080600061/3131934-bluemulti-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/trans
port%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
PrestoBlue
reagent
for apoptosis
1) Add reagent
to cells
2) Incubate
3) Read fluorescence
Experimental Variables
Cryoprotectant Type
DMSO
Ethylene
Glycol
Glycerol
Concentration
1m
3m
5m
Exposure Time
60 min
40 min
20 min
Temperature
4C
21C
Propylene
Glycol
7m
37C
10 min
5 min
0 min
Data Analysis
37C Glycerol
(treated sample fluorescence


Represented on cell
survival versus time
plot
Fit to exponential
regression of the form:
N=e
 kt
1
1M
3M
5M
7M
0.8
Cell Survival
Cell survival =
)
seeding density fluorescence
control sample fluorescence
0.6
0.4
0.2
0
0
10
20
30
40
Exposure Time (Min)
50
60
70
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
0.08
0.07
toxicity rate k (min^-1)
N=e
 kt
0.06
y = 0.0108x - 0.0049
R² = 0.9871
0.05
0.04
0.03
0.02
0.01
0
0
1
2
3
4
5
Concentration (molal)
6
7
8
Experimental Results
Initial Experiments


Results were highly
variable
Possible Sources of
Variability
Uneven seeding
distribution
 Cell loss during wash steps

y = e-0.007x
R² = -1.32
1
Cell Survival
1,3-molal Glycerol at 21C
 Used 96-Well Plates

1-molal Glycerol 21C
1.2
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
1 molal CPA Exposure Time (Min)
60
3-molal Gycerol 21C
1.2
y = e-0.018x
R² = 0.0957
1
Cell Survival

0.8
0.6
0.4
0.2
0
0
10
20
30
40
3 molal CPA Exposure Time (Min)
50
60
Investigating Seeding Distribution
placing well
plates with freshly seeded
cells at room temperature
for 1 hour before placing
in 37C incubator
Pre-Incubated 1,3-Glycerol Toxicity Data
1.2
1-molal
1
Cell Survival

Uneven seeding
distribution caused by
thermal gradients
Pre-incubation to reduce
variability
y = e-0.014x
R² = -1.515
0.8
0.6
0.4
0.2
0
 Involves
0
10
1.20
20
30
40
50
1 molal CPA Exposure Time (Min)
60
3-molal
1.00
Cell Survival

y = e-0.023x
R² = -5.331
0.80
0.60
0.40
0.20
0.00
0
10
20
30
40
50
3 molal CPA Exposure Time (Min)
60
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
Effect of Wash Steps
1.2
Normalized Fluorescence

1
0.8
0.6
0.4
0.2
0
No Wash
5 Washes
Revised Experiments

24-well plates
 Avoid
cell loss during wash steps
 Increased well size helps to reduce variability
Effect of Wash Steps
1-molal Glycerol 21C
1.2
1.2
y = e-0.006x
R² = -3.083
1
1
Cell Survival
Normalized Fluorescence
1.4
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0
0
No Wash
5 Washes
0
10
20
30
40
Exposure Time (min)
50
60
70
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