1. What causes a change in fluorescence?
Measurement of Cell Membrane Permeability in Cultured Endothelial Cells
Background on Cryopreservation
Tissues are cooled to subzero temperatures stopping all biological activity.
Allows for storage of mammalian cells while maintaining cell viability.
Ice formation inside the cell causes cell death.
Cryoprotectant molecules (CPAs) inhibit ice formation in cells during freezing.
Optimization of CPA addition requires modeling of membrane permeability.
Exposing cells to osmotic pressure gradients causes swelling or shrinking.
Note: This schematic shows blood cells not neurons. Endothelial cells have a different shape
Permeability Modeling
Lucy Keenlyside and Melissa Robertson
OSU School of Chemical, Biological, and Environmental Engineering
Modeling equations describing water crossing the cell membrane
Experimental
Volume flux of water across membrane
Extracellular (e)/intracellular (i) osmotic pressure
Water permeability parameter (hydraulic permeability)
Heat Exchanger Shell
Cell Exposure
Flow chamber allows for control over the exposure,
osmotic pressure
and temperature of solution to which cells are exposed.
High-speed microscopic observation of endothelial cell fluorescence change.
Heat exchanger was validated to maintain constant temperature within 0.5 ̊C
Modeling equations describing solute (CPA) crossing the cell membrane
Extracellular (e)/ intracellular (i) osmolality
CPA permeability parameter
Molar flux of CPA
Syringe Pump
Highspeed Microscope
Cells are exposed to isotonic and anisotonic solutions with use of a syringe pump.
Endothelial cell images and membrane permeability modeling are done using MATLAB and Image Pro.
CPA molar volume
Fluorescence intensity over time. Sharp jumps and drops in the curve represent cell reaction to solution change. Above: Raw intensity data. Right: Normalized intensity data.
Relative Intensity (I) I
Time (t) t
Relating cell volume to fluorescence intensity
Intensity can be measured
Isotonic – solution with an osmotic pressure equal to that in the cell
Hypotonic – solution with an osmotic pressure greater than that in the cell
Hypertonic – solution with an osmotic pressure less than that in the cell
Endothelial cells exposed to an isotonic solution.
Measured relative fluorescence intensity
Permeability
Average
Standard Deviation
Number of Replicates
water
18.6
[GPa-sec]-1
7.76 12
DMSO
[sec]-1
0.056 8
ppg
[sec]-1
0.077 4
Endothelial cells exposed to a hypertonic solution.
Objective
Use fluorescence quenching to measure relative cell volume changes in response to osmotic pressure gradients.
Quantify membrane permeability of cultured endothelial cell.
Future Work
Heat exchanger prototype can be rebuilt as an improved heat exchanger unit.
Fluorescence quenching experiments utilizing several anisotonic solutions to determine the permeability of water and other molecules across the cell membrane.
Permeability data will be taken for different types of cells.
What causes a change in fluorescence?
Inside each cell are small fluorescent and quenching molecules. When the cell shrinks, the molecules get closer together increasing quenching and fluorescence appears dimmer (see left). When the cell expands, the molecules get further apart and fluorescence is brighter.
Acknowledgments
The project team would like to thank Dr. Adam Higgins for sponsoring and Dr. Philip Harding for managing this project, as well as Andy Brickman and Manfred Dittrich for contributing to design and construction. Allyson Fry is acknowledged for her work culturing and treating the endothelial cells.