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 http://en.wikipedia.org/wiki/Osmotic_pressure 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 0.111 [sec]-1 0.056 8 ppg 0.165 [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.