1. 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

2. Cryopreservation
Preservation of cells or tissues under a low temperature condition
Conventional Freezing
Vitrification
Cryopreservation is a procedure where cells or tissues are preserved under a low temperature condition.
The extremely cold temperature slows down or stop the metabolic processes.
I am interested in the cryopreservation because this procedure is helpful for storing useful tissues and cells for later use. For examples, cryopreservation is often use in preserving semen, oocytes, and ovarian tissues.
Ovarian tissue cryopreservation gives benefit for those female patients who undergo chemotherapy. Their healthy ovarian tissues can either be transplanted back after their treatment or can be used for vitro fertilization
There are two ways for the cryopreservation procedure
First one is conventional freezing- a slow freezing method
And the second is called vitrification- a supercooling procedure that rapidly makes viscous liquid into a glass state
Typically, cryoprotective agents are used in both procedures
Source: <Fertility Institute of Hawaii>
source: <The Infertility Center of Saint Louis>
source: <Nature.com>

3. Cryoprotective Agents (CPAs)
Glycerol
Dimethyl Sulfoxide (DMSO)
Protect freezing damage
Prevent ice crystal formation
Dimethyl Sufoxide, which is known as DMSO, and glycerol and two of the most common cryoprotective agents.
The addition of CPAs would benefit the cells in several ways
First is to protect the cells from freezing damage
And then prevent the extracellular and intraocular ice formation that could lead to cell death

4. 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
Although CPA plays a significant role in the cryopreservation, the addition Cryoprotective agents in vitrification procedure, further damages can introduce to the cells
Exposing cells to high concentration CPA can result in chemical toxicity damage. Multistep addition and removal of CPA is often used for minimizing the toxicity damage.
The addition and removal of CPA also causes the osmotic transport of water through cell membrane, which can lead to the volume change damage of the cell.
Last summer, I focused on determining the volume range where the cell can maintain in healthy state with the addition of CPAs.

5. Damages in Volume Change
Hypertonic
Isotonic
Hypotonic
Damage of volume changes is due to the addition of different concentration CPAs.
When cells are exposed to a hypertonic solution where there are more salts outside of a cell than inside of a cell, the water tends to flow out of the cell and causes dehydration.
On the other hand, hypotonic solution is a low concentration solution where it has less salt than inside of a cell, which causes water to run into a cell and potentially become ruptured.
Exposing cells to isotonic solution makes a cell maintain at a right volume because it creates a even osmotic transport between the cell and solution
Idealy, we want the cell to maintain osmotic transport between the cell and the solution. However, with the addition and removal of CPAs, we try to prevent too much shrinkage and swell, which lead to cell death.
As a result, determination of osmotic tolerance limits of cell volume allows for optimization of cryoprotectant addition
Determine the osmotic tolerance limits of cell volume under different temperature control.
Goal

6. Hypothesis
Temperature does affect the osmotic tolerance limits of cell volume.

7. Exposing Sodium Chloride Solution at Room Temperature
This is the type of data I am collecting this summer
The 300 osmolality is the isotonic solution
We observed that when the cells are exposed to the concentration away from the isotonic solution, the cell survival rate decreases
However, there is still a range of concentration where most cell survives,
which this range concentration can further be known be set as tolerance limits
This data was obtained by experiment conducted by Allyson Fry

8. Variables and Timeline
Effect of Different Temp.
6 degree Celsius
Room temp. (21 C)
37 degree Celsius
1 room temp.
3. 37C
To investigate the volume change, I am exposing the cell to different under different temperature control
they are tested under the room temperature, at 4 degree Celsius, and at 37 degree Celsius.
Here is a brief experimental timeline for me this summer. First I am verifying sodium chloride solution and performing at room temperature, and then at 6 degree Celsius and 37 degree Celsius respectively.
2. 6C

9. Approaches BPAEC Seeding on 96 well plates
Bovine Pulmonary Artery Endothelial Cell (BPAEC) is used
BPAEC Seeding on 96 well plates
Seeding Density: 5000 cells/ well

10. 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

11. Approaches continue. PrestoBlue Viability Assay
Higher the fluorescence units indicates greater cell survival
PrestoBlue reagent is modified by the reducing environment of the viable cell, turns red in color and becomes highly fluorescent. 
Source: <Life Technology-Invitrogen>

12. Analysis Cell Survival Cell Volume
Conservation of moles inside the Cell

13. Relative Cell Volume

14. Results: Verifying NaCl Experiment at room temperature

15. Results (Cont’d): NaCl Experiment at temperature of 6 degree Celsius

16. high temperature data resulted in high variability
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

17. Investigation of potential source of variability
Cell loss- wash steps.
Neither were the source of variability
May due to lack of practice in cell culture procedure
The experiments were repeated at the extreme conditions
Highly hypotonic
Highly hypertonic
Contamination exp.

18. Results (Cont’d): Extreme Condition
NaCl Experiment at temperature of 6 degree Celsius
NaCl Experiment at temperature of 37 degree Celsius

19. 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

20. 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.

21. Acknowledgements Howard Hughes Medical Institute Dr. Adam Higgins
Dr. Kevin Ahern
Allyson Fry
Ratih Lusianti
Cameron Glasscock
Corey Lerch