Cell Membrane Permeability in Adherent Cells for Cryopreservative Procedures - PowerPoint PPT Presentation

Loading...

PPT – Cell Membrane Permeability in Adherent Cells for Cryopreservative Procedures PowerPoint presentation | free to download - id: 7fbee-ZDc1Z



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Cell Membrane Permeability in Adherent Cells for Cryopreservative Procedures

Description:

Improved preservation techniques for adhered cells is a step towards preservation of tissues ... Logan, Crystal, Robert, Alyson ... – PowerPoint PPT presentation

Number of Views:170
Avg rating:3.0/5.0
Slides: 24
Provided by: loweryn
Learn more at: http://oregonstate.edu
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Cell Membrane Permeability in Adherent Cells for Cryopreservative Procedures


1
Cell Membrane Permeability in Adherent Cells for
Cryopreservative Procedures
  • Nick Lowery
  • Mentor Dr. Adam Higgins
  • HHMI 2008

2
Purpose
  • Cryopreservative techniques lacking with adhered
    cells
  • Isolated cells have the best survival rate (highs
    gt90)?
  • 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

3
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

4
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

5
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

6
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

7
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
8
Methods (cont.)?
  • Resistance in the chamber is proportional to the
    volume of the cells
  • Cells expand, channel shrinks, resistance
    increases, and vice versa

Shrunken cells, small R
Swollen cells, large R
9
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

10
Experimental Setup
  • Cell membrane permeability is temperature
    dependent
  • All solutions passed through heat exchanger to
    insure constant temperature throughout system

11
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 37C
12
Old Exchanger Design
  • Problems
  • Shell too small to accommodate sufficient tubing
  • Polycarbonate not fully transparent
  • Too tall for microscopy
  • Leaks

Scale 2 in.
13
New Exchanger Design
  • Fixes problems with old design
  • Allows for versatility

Front edge length 4.5 in.
14
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

15
Resistance Measurements
16
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

17
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

18
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

19
Fluorescence Quenching (cont.)
20
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.wikip
edia.org
21
Effect of Cytochalasin D (cont.)
Normal Cells
Cells w/ Cytochalasin D
22
Future Work
  • Redesign cytochalasin D experiments so cells
    dont 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

23
Acknowledgements
  • Dr. Adam Higgins
  • HHMI Dr. Kevin Ahern
  • Pete and Rosalie Johnson, The Johnson Scholarship
    Dr. Skip Rochefort
  • Logan, Crystal, Robert, Alyson
About PowerShow.com