Development of a Device to
Measure Cell Membrane
Water Permeability
BY ROBERT ELDER
MENTOR: DR. ADAM HIGGINS
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

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
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
Measuring Permeability
 The Coulter principle

Particles flowing through a channel change the electrical
resistance in proportion to their size
Resistance
momentarily increased
-
+
Ω
Conductive
solvent
Suspended cell flows
through channel
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
Measuring Permeability
 The rate of the volume change is related to the
membrane permeability (P)
d
V

P
Z
C
C


i
n
t
e
r
n
a
l
e
x
t
e
r
n
a
l
d
t
 P is permeability
 C is total solute concentration
 Z is a collection of other constants
Measuring Permeability Overview
 Goal: build a device to measure permeability by
using the Coulter principle to determine volume
changes.
Isotonic solution
Hypotonic solution
Electrical current
 Resistance → Volume → Permeability
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
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
Device Overview
+
Syringes
Heat Exchanger
Electrodes
-
Flow Channel
Cell Monolayer
 Dual inlet system for switching solutions quickly
 Heat exchanger to control temperatures
 Channel: 100µm deep for sensitive measurements
Device Design
Coverslip and gasket
form flow channel
Heat exchanger shell
Clear plastic allows
microscopy
Device Design
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

Device Characterization: Solution Exchange
 Model results

Solution exchange is much faster than volume changes
Channel Top
Chamber
Entrance
Point of
Interest
Channel Bottom
 Dye exchange results
Slower than model results
 Solution exchange less than 1 second at relevant flowrates

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

Initial:
Flowing
Syringes
Not flowing
Heat Exchanger
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

Final:
Not Flowing
Syringes
Flowing
Heat Exchanger
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
Goal:
Flowing
Syringes
Not flowing
Heat Exchanger
Device Characterization: Heat Exchanger
Different Tubing Lengths
Very long & metal tubing
1.02
1.20
Metal
1.00
0.80
Accomplished Temperature Change
Accomplished Temperature Change
1.00
Long
0.60
Medium
Short
0.40
0.20
0.98
Very long
0.96
0.94
0.92
0.90
0.88
0.86
0.00
0
50
100
150
200
250
Flow rate (ml/hr)
300
350
0
50
100
150
200
250
Flow rate (ml/hr)
300
350
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
Fluorescence Quenching Measurements
 Relative intensity changes can be used to
Intensity
determine permeability
Time (s)
Fluorescence Quenching Measurements
 Relative intensity changes can be used to
Normalized Intensity
determine permeability
Time (s)
Time (s)
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
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
Acknowledgements
 Howard Hughes Medical Institute
 University Honors College
 Dr. Adam Higgins
 Dr. Kevin Ahern
 Nick Lowery, Crystal Gupta, Logan Williams
 Andy Brickman, Manfred Dittrich