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