BY ROBERT ELDER MENTOR: DR. ADAM HIGGINS Development of a Device to Measure Cell Membrane Water Permeability

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 Conductive solvent Suspended cell flows through channel + - Ω Resistance momentarily increased

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) 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. Resistance → Volume → Permeability Hypotonic solutionIsotonic solution Electrical current

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 Dual inlet system for switching solutions quickly Heat exchanger to control temperatures Channel: 100µm deep for sensitive measurements Syringes Heat Exchanger Flow Channel Electrodes Cell Monolayer +-

Device Design Heat exchanger shell Coverslip and gasket form flow channel 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 Dye exchange results  Slower than model results  Solution exchange less than 1 second at relevant flowrates Channel Bottom Channel Top Chamber Entrance Point of Interest

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 Syringes Heat Exchanger Flowing Not flowing Initial:

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 Syringes Heat Exchanger Not Flowing Flowing Final:

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 Syringes Heat Exchanger Flowing Not flowing Goal:

Device Characterization: Heat Exchanger Short Medium Long

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 determine permeability Time (s) Intensity

Fluorescence Quenching Measurements Relative intensity changes can be used to determine permeability Time (s) Normalized Intensity 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