Effect of Temperature on Osmotic
Tolerance Limits for Adherent
Endothelial Cells
HHMI Summer 2011
Department of Chemical, Biological, & Environmental Engineering
By Kenneth Huang
Mentor: Dr. Adam Higgins
Cryopreservation

Preservation of cells or tissues under a
low temperature condition
1. Conventional Freezing
2. Vitrification
Source: <Fertility Institute of Hawaii>
source: <The Infertility Center of Saint Louis>
source: <Nature.com>
Cryoprotective Agents (CPAs)
Dimethyl Sulfoxide (DMSO)
Glycerol
Protect freezing damage
 Prevent ice crystal formation

Additional Damages due to CPAs
• Chemical Toxicity damage while the cells sit
under the CPAs.
• Damage of volume change due to osmotic
transport of water during the addition/
removal procedure
Damages in Volume Change
Hypertonic
Goal
Isotonic
Hypotonic
Determine the osmotic tolerance limits
of cell volume under different
temperature control.
Hypothesis

Temperature does affect the osmotic
tolerance limits of cell volume.
1.2
1.2
1
1
Cell Survival
Cell Survival
Exposing Sodium Chloride Solution
at Room Temperature
0.8
0.6
Logistic Fit
0.4
PrestoBlue
Hypotonic
0.2
0.8
0.6
0.4
PrestoBlue
Hypertonic
0.2
Logistic Fit
0
0
0
100
200
Treatment (mmol/kg)
300
300
1300
2300
3300
4300
Treatment (mmol/kg)
This data was obtained by experiment conducted by Allyson Fry
Variables and Timeline
Effect of Different Temp.
 6 degree Celsius
 Room temp. (21 C)
 37 degree Celsius
3. 37C
1 room temp.
2. 6C
Approaches
Bovine Pulmonary
Artery Endothelial Cell
(BPAEC) is used

BPAEC Seeding on 96 well plates
◦ Seeding Density: 5000 cells/ well
Approaches continue.

Expose to NaCl solution with different
concentration for 15 minutes
Positive control
Concentration 1
Concentration 2
Concentration 3
Concentration 4
Concentration 5
Concentration 6
Negative control
Approaches continue.

PrestoBlue Viability Assay
◦ Higher the fluorescence units indicates
greater cell survival
Source: <Life Technology-Invitrogen>
Analysis

Cell Survival
Treatm ent_ Interest
Seeding_ Density
Isotonic_ Treatm ent

Cell Volume
• Conservation of moles inside the Cell
Volume  Concentrat ion  mole
Vw,Iso (miso )  Vinterest (minterest )
Relative Cell Volume
Vinterest
mIso
300


 4.29
Vw, Iso
minterest
70
Results:
Verifying NaCl Experiment at room temperature
1.2
1
1
Cell Survival
1.2
Cell Survival
0.8
0.6
Logistic Fit
0.4
PrestoBlue
Hypotonic
0.2
0.8
0.6
0.4
PrestoBlue
Hypertonic
0.2
Logistic Fit
0
0
0
100
200
300
300
1.2
0.8
0.6
Hypotonic Data
Logistic Fit
0
Cell Survival
1
0.2
3300
4300
Hypertonic Data
1.4
0.4
2300
Treatment (mmol/kg)
Treatment (mmol/kg)
Cell Survival
1300
1.2
Logistic Fit
1
0.8
0.6
0.4
0.2
0
0
100
200
300
Treatment (mmol/Kg)
300
2300
4300
Treatment (mmol/Kg)
Results (Cont’d):
NaCl Experiment at temperature of 6 degree Celsius
1.200
1.200
1.000
Cell Survival
0.800
0.600
0.400
Hypotonic Data
0.200
Logistic Fit
0
100
0.800
0.600
0.400
Hypertonic Data
0.200
0.000
200
300
Logistic Fit
0.000
300
Treatment (mmol/Kg)
1300
2300
3300
4300
Treatment (mmol/Kg)
NaCl Experiment at temperature of 37 degree Celsius
1.6
1.8
1.4
1.6
1.2
1.4
Cell survival
Cell survival
Cell Survival
1.000
1
0.8
0.6
1.2
1
0.8
0.6
0.4
0.4
0.2
0.2
0
0
0
100
200
treatment (mmol)
300
400
0
1000
2000
3000
treatment (mmol)
4000
5000
Results (Cont’d):
high temperature data resulted in high
variability
 Potential source of variability

◦ Cell loss during the wash of PBS++ solution
◦ Contamination of PBS++ solution
Investigation of potential source of variability
Viability/ Seeding Density


Cell loss- wash steps.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Neither were the
source of variability
•
0
1
2
3
# of washes
4
5
Contamination exp.
1.2
Cell survival
1
0.8
0.6
0.4
0.2
0
0hr old PBS
0hr new PBS
24hr old PBS
24hr new PBS
•
May due to lack of practice in
cell culture procedure
The experiments
were repeated at the
extreme conditions
• Highly hypotonic
• Highly hypertonic
Results (Cont’d): Extreme Condition
NaCl Experiment at temperature of 6 degree Celsius
1.4
1.2
Cell survival
1
0.8
0.6
0.4
0.2
0
control
5
25
50
300
4000
Concentration (mOsm)
4500
EtOH
NaCl Experiment
at temperature of 37 degree Celsius
1.6
1.4
Cell survival
1.2
1
0.8
0.6
0.4
0.2
0
Control
5
25
50
300
4000
Concentration (mOsm)
4500
EtOH
Experimental Conclusion
◦ Temperature does effect the osmotic tolerance limit
◦ Initial high temperature data resulted in high
variability
◦ Neither wash steps or suspected contamination were
the source of variability
◦ Both low and high temperature controlled data
showed differences in cell survival in extreme
condition experiment
 Low temperature data: relatively high cell survival
 High temperature data: relatively low cell survival
◦ However, the accurate tolerance limits were not
determined for specific temperature controlled
experiment
Future Work:
Perform with a different viability assay
 To investigate the actual relative cell
volume tolerance limits for a wide range
of temperature conditions
 Optimization of addition or removal of
CPAs for cryopreservation along with
toxicity data and permeability data.

Acknowledgements
Howard Hughes Medical Institute
 Dr. Adam Higgins
 Dr. Kevin Ahern
 Allyson Fry
 Ratih Lusianti
 Cameron Glasscock
 Corey Lerch
