BY ROBERT ELDER

1
Development of a Device to Measure Cell
MembraneWater Permeability

• BY ROBERT ELDER
• MENTOR DR. ADAM HIGGINS

2
Permeability

• Permeability is a property of the cell membrane
• Measures how easily a substance can cross the
  membrane
• Different for each substance and cell type
• The cell membrane is relatively permeable to
  water
• Significance of water permeability
• Cryopreservation
• Biosensing

3
Significance for Cryopreservation

• Cryopreservation techniques that can keep
  biological matter intact for years
• Water permeability affects the amount of water in
  a cell, which affects viability
• Too much water ? intracellular ice ? physical
  damage
• Too little water ? high concentration ? chemical
  damage
• Cryoprotectants that alter permeability are a
  possibility
• Current cryogenic techniques are successful only
  with cell suspensions, where isolated cells float
  in fluid

4
Significance for Biosensors

• Certain toxins form membrane pores that increase
  permeability
• Examples
• Plague bacterium
• Staphylococcus aureus
• A device to detect permeability changes could be
  used to detect these toxins

5
Measuring Permeability

• The Coulter principle
• Particles flowing through a channel change the
  electrical resistance in proportion to their size

Conductive solvent
Suspended cell flows through channel
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Measuring Permeability

• Concentration differences across membranes cause
  water to flow, which causes cells to shrink or
  expand
• Isotonic same concentration inside and outside
  cell results in no net water flow

7
Measuring Permeability

• The rate of the volume change is related to the
  membrane permeability (P)
• P is permeability
• C is total solute concentration
• Z is a collection of other constants

8
Measuring Permeability Overview

• Goal build a device to measure permeability by
  using the Coulter principle to determine volume
  changes.
• Resistance ? Volume ? Permeability

9
Existing Measurement Methods

• Examples
• Fluorescence quenching
• Concentration change of marker molecules due to
  cell uptake
• Problems
• Complex modifications to sample
• Specialized equipment
• Not portable
• Time consuming
• A more convenient method should be developed to
  accelerate research efforts

10
Project Goals

• Complete device construction and characterization
• Develop a model to relate resistance changes to
  permeability
• Compare our measurements to those from
  established techniques (fluorescence quenching)
• Test the effect of different substances on
  permeability

11
Device Overview

• Dual inlet system for switching solutions quickly
• Heat exchanger to control temperatures
• Channel 100µm deep for sensitive measurements

12
Device Design
13
Device Design
14
Device Characterization Solution Exchange

• How long does it take to switch solutions?
• Relevance
• Cells respond to concentration changes in seconds
• Switching concentrations must be much faster
• Method
• Use dye solutions to visualize solution exchange
• Compare to mathematical model of diffusion and
  fluid flow

15
Device Characterization Solution Exchange

• Model results
• Solution exchange is much faster than volume
  changes
• Dye exchange results
• Slower than model results
• Solution exchange less than 1 second at relevant
  flowrates

Channel Top
Chamber Entrance
Point of Interest
Channel Bottom
16
Device Characterization Heat Exchanger

• Dual inlet system can result in rapid temperature
  changes in channel
• Initially, isotonic solution pumped temperature
  depends on heat exchanger
• Then, anisotonic solution pumped temperature
  equal to shell temperature

17
Device Characterization Heat Exchanger

• Dual inlet system can result in rapid temperature
  changes in channel
• Initially, isotonic solution pumped temperature
  depends on heat exchanger
• Then, anisotonic solution pumped temperature
  equal to shell temperature

18
Device Characterization Heat Exchanger

• Goal minimize the initial temperature change
  when switching solutions (i.e. get isotonic
  temperature equal to anisotonic)
• Method increase tube length, investigate tube
  material

19
Device Characterization Heat Exchanger
Long
Medium
Short
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Fluorescence Quenching Measurements

• Purpose obtain permeability measurements for
  comparison
• Fluorescence intensity is directly related to
  cell volume
• Fluorescence is quenched (decreased) when
  hypertonic solution shrinks cells

21
Fluorescence Quenching Measurements

• Relative intensity changes can be used to
  determine permeability

Intensity
Time (s)
22
Fluorescence Quenching Measurements

• Relative intensity changes can be used to
  determine permeability

Normalized Intensity
Time (s)
Time (s)
23
Effect of Cytochalasin D on Permeability

• A cell-permeable mycotoxin
• Potent inhibitor of actin polymerization
• Changes cell morphology and possibly permeability
• Possible cryoprotectant but tests inconclusive

24
Results

• Response of resistance measurements was too slow
  to measure volume changes accurately
• Further work may be pursued to decrease response
  time
• Fluorescence quenching experiments were
  successful and gave results of the predicted
  order of magnitude

25
Acknowledgements

• Howard Hughes Medical Institute
• University Honors College
• Dr. Adam Higgins
• Dr. Kevin Ahern
• Nick Lowery, Crystal Gupta, Logan Williams
• Andy Brickman, Manfred Dittrich