Effect of Temperature on Effect of Temperature on Osmotic Tolerance Limits Osmotic Tolerance Limits for Adherent Endothelial for Adherent Endothelial CellsCells

HHMI Summer 2011Department of Chemical, Biological, & Environmental EngineeringBy Kenneth HuangMentor: Dr. Adam Higgins

CryopreservationCryopreservationPreservation of cells or tissues

under a low temperature condition1. Conventional Freezing2. Vitrification

source: <The Infertility Center of Saint Louis>

source: <Nature.com>

Source: <Fertility Institute of Hawaii>

Cryoprotective Agents Cryoprotective Agents (CPAs)(CPAs)

Protect freezing damagePrevent ice crystal formation

Dimethyl Sulfoxide (DMSO) Glycerol

Additional Damages due to Additional Damages due to CPAsCPAs

• 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 Damages in Volume ChangeChange

Hypertonic Isotonic Hypotonic

GoaGoall

Determine the osmotic tolerance limits of cell volume under different temperature control.

HypothesisHypothesisTemperature does affect the

osmotic tolerance limits of cell volume.

Exposing Sodium Chloride Exposing Sodium Chloride Solution at Room Solution at Room TemperatureTemperature

This data was obtained by experiment conducted by Allyson Fry

Variables and TimelineVariables and TimelineEffect of Different

Temp.6 degree CelsiusRoom temp. (21 C)37 degree Celsius

1 room temp. 3. 37C

2. 6C

ApproachesApproaches

BPAEC Seeding on 96 well plates◦Seeding Density: 5000 cells/ well

Bovine Pulmonary Artery Endothelial Cell (BPAEC) is used

Approaches continue.Approaches continue.Expose to NaCl solution with

different concentration for 15 minutes

Positive control

Negative control

Concentration 1

Concentration 2

Concentration 3

Concentration 4

Concentration 5

Concentration 6

Approaches continue.Approaches continue.PrestoBlue Viability Assay

◦Higher the fluorescence units indicates greater cell survival

Source: <Life Technology-Invitrogen>

AnalysisAnalysisCell Survival

TreatmentIsotonicDensitySeeding

InterestTreatment

__

_

Cell Volume• Conservation of moles inside the

Cell moleionConcentratVolume

)()( interestinterest, mVmV isoIsow

Relative Cell VolumeRelative Cell Volume

interest,

interest

m

m

V

V Iso

Isow

29.470

300

Results:Results:Verifying NaCl Experiment at room temperatureVerifying NaCl Experiment at room temperature

Results (Cont’d):Results (Cont’d):

NaCl Experiment at temperature of 6 degree CelsiusNaCl Experiment at temperature of 6 degree Celsius

NaCl Experiment at temperature of 37 degree CelsiusNaCl Experiment at temperature of 37 degree Celsius

high temperature data resulted in high variability

Potential source of variability◦Cell loss during the wash of PBS++

solution◦Contamination of PBS++ solution

Results (Cont’d):Results (Cont’d):

Investigation of potential source of Investigation of potential source of variabilityvariability

Cell loss- wash steps.

Contamination exp.

• The experiments were repeated at the extreme conditions

• Highly hypotonic

• Highly hypertonic

Neither were the source of variability

• May due to lack of practice in cell culture procedure

Results (Cont’d): Extreme Results (Cont’d): Extreme ConditionConditionNaCl Experiment at temperature of 6 degree CelsiusNaCl Experiment at temperature of 6 degree Celsius

NaCl Experiment at temperature of 37 degree CelsiusNaCl Experiment at temperature of 37 degree Celsius

Experimental ConclusionExperimental 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:Future Work:Perform with a different viability

assayTo 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.

AcknowledgementsAcknowledgementsHoward Hughes Medical InstituteDr. Adam HigginsDr. Kevin AhernAllyson FryRatih LusiantiCameron GlasscockCorey Lerch