Purpose

Cryopreservative techniques lacking with
adhered cells



Isolated cells have the best survival rate (highs
>90%)
Improved preservation techniques for adhered cells
is a step towards preservation of tissues
Move towards freezing of organs in the future,
virtually eliminating waiting lists
Research Goal

Develop a device to quickly and accurately
measure hydraulic cell membrane permeability
in adherent cells



Adherent cells respond differently to standard
cryopreservation procedures
Most methods focus on suspended cells
Current method used for adherent cells
(fluorescence quenching) inconvenient
Cryopreservation



Main problem with cryopreservative procedures
is cells dying in the process
Virtually all cell deaths occur during cooling and
warming phases
2 main mechanisms of cell injury / death
Cryopreservation (cont.)

Intracellular Ice


Ice crystals form
during cooling, can
damage organelles
and membranes
within the cell
Upon warming,
crystals melt, causing
damage from osmotic
effects

Extracellular Ice


As extracellular
solution freezes,
remaining unfrozen
solution concentrates
Osmotic pressure
draws water out of the
cell, dehydrating the
cell, or damaging it
from excessive
shrinkage
Cryopreservation (cont.)



Slow cooling rates cause extracellular ice
formation
Fast rates form intracellular ice
For best survival rates, median optimal cooling
rate needs to be determined


Different rates for different types of cells
Membrane permeability determines this rate
Methods

Cell membranes resist electric current

Adhere cells to wall of flow chamber, flow
electrolyte solution through, current will pass
through the solution, over the cells
Solution
Current
Cells
Methods (cont.)

Resistance in the chamber is proportional to the
volume of the cells

Cells expand, channel shrinks, resistance
increases, and vice versa
Swollen cells, large R
Shrunken cells, small R
Methods (cont.)



Cell swelling/shrinking induced by flowing
isotonic soln. over cells, then quickly switching
to anisotonic soln.
Measure change in voltage across chamber,
calculate change in resistance, which is
proportional to change in cell volume
Measure elapsed time for change in cell
volume, calculate cell membrane permeability
Experimental Setup

Cell membrane permeability is temperature
dependent

All solutions passed through heat exchanger to
insure constant temperature throughout system
Heat Exchanger
“Shell”
Inlets - solutions
of varying tonicities
Tubing
coils
Outlet
Flow chamber: 19 x 3 x 0.1 mm,
on underside of heat exchanger
Water
bath at 37°C
Old Exchanger Design

Problems




Shell too small to
accommodate
sufficient tubing
Polycarbonate not
fully transparent
Too tall for
microscopy
Leaks
Scale = ~2 in.
New Exchanger Design


Fixes problems with
old design
Allows for versatility
Front edge length = 4.5 in.
Electrical Experiments

Testing resistance of various solutions and
dimensions of the flow chamber

Most work has been explaining various anomalies
in the data


Electrical noise from various sources, air bubbles, other
aspects
Goal: to establish baseline to compare tests of
cells to

Know what to expect (and avoid) during cell testing
Resistance Measurements
Electrode Polarization

Occurs at current
carrying electrodes in
an electrolyte solution



Charged electrodes
attract ions of opposite
charge from solution
Ions form “wall” around
electrode, making it
harder for current to
push through
More ions accumulate
over time, so
resistance will also
gradually increase
over time
Electrode Polarization (cont.)

Four electrode setup

Two electrodes carry current, two measure
resistance




Ions build up, but measurement electrodes unaffected
Can keep current DC circuit (mostly) intact
Need to accommodate extra electrodes
AC circuit

Electrodes alternate sign at a given frequency




Ions are alternately attracted and repelled, so no buildup
Will have to create new circuit
Resistance measurement changes
Will only need two electrodes
Fluorescence Quenching



Comparison to resistance tests
Cells loaded with calcein AM, a fluorescent
molecule
Time lapse taken of cells as iso/hyper/isotonic
solutions flowed over cells


Computer software used to measure intensity
Hypertonic solutions cause cells to shrink,
decreasing fluorescence intensity

Increased solute concentrations allow for other
molecules to interact with calcein AM and “steal”
the energy otherwise used to fluoresce
Fluorescence Quenching (cont.)
Effects of Cytochalasin D

Cytochalasin D disrupts
the cellular cytoskeleton
by interfering with actin
polymerization


Two F-actin (polymerized
actin) strands form a helix
called a microfilament,
which provides the
cytoskeletal structure
Testing via fluorescence
quenching to see if
cytochalasin D affects
membrane permeability
Cytoskeleton of mouse embryo fibroblasts
en.wikipedia.org
Effect of Cytochalasin D (cont.)
Normal Cells
Cells w/ Cytochalasin D
Future Work



Redesign cytochalasin D experiments so cells
don’t shear off, analyze data to see if there is
any effect on permeability
After sufficient fluorescence quenching data
has been taken, integrate electrodes
Proceed to taking electrical resistance data,
hope that it matches the fluorescence data
Acknowledgements
Dr. Adam Higgins
HHMI & Dr. Kevin Ahern
Pete and Rosalie Johnson, The Johnson
Scholarship & Dr. Skip Rochefort
Logan, Crystal, Robert, Alyson