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Paper37-SFB 2003 Reno - Cell Mapping

Paper37-SFB 2003 Reno - Cell Mapping - SFB 2003 Reno NV AFM...

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Unformatted text preview: SFB 2003 Reno, NV AFM Quantification of Receptor-Ligand Interactions on the Surface of Living Cells on Adam W. Feinberg1 and Anthony B. Brennan1,2 Department of Biomedical Engineering 2 Department of Materials Science & Engineering University of Florida Gainesville, FL 1 Department of Biomedical Engineering Outline • • • • • • • • • Goals and objectives Introduction to AFM technique Model cell system Materials and methods Cell topography Biochemical Force Probe Cell surface receptor mapping Specific Interaction Force and Distance Acknowledgements 2 Department of Biomedical Engineering Research Goals • To improve the interaction between cells and To manmade materials for use in medical devices and tissue engineering constructs and • To analyze the biointerface on the micro and To nano scale by quantifying the physical, mechanical and chemical properties of the material and cells material 3 Department of Biomedical Engineering Specific Objectives • To probe the cellular reaction to a surface by To probing specific interactions probing • To maintain the following conditions – – – Cell must be living Cell must be minimally perturb Experiment must be conducted on a relevant time Experiment scale scale • Approximate a physiologic process in vitro Approximate in – Leukocyte rolling on endothelial cells 4 Department of Biomedical Engineering 5 AFM Schematic LED Based Laser AFM Control Head with Z Stepper Motor and Piezoelectric Scanner 4 Quadrant Photo Detector Laser Beam X-Y Piezoelectric Scanner Video Camera for Tip Alignment and Sample Viewing 0.06 N/m Z Piezoelectric Scanner 0.32 N/m AFM tip 0.12 N/m 0.68 N/m X-Y Translation Stage– Software Controlled Stepper Motors Sample Department of Biomedical Engineering Adhesion Theory • Biointerface – – – Pristine Surface Biopolymer adsorption Surface Surface rearrangement rearrangement – Cellular Adhesion – Remodeling • We will examine the We exposed cell surface exposed 6 Department of Biomedical Engineering Model Cell System • • • Porcine vascular endothelial cells Standard 35mm polystyrene culture dish P-selectin receptors expressed on free P-selectin membrane surface membrane • Sialyl Lewis X (sLeX) iis functional component of s the leukocyte ligand P-selectin glycoprotein ligand 1 (PSGL-1) ligand • Experiments conducted at simulated physiologic Experiments conditions in Hanks balanced salt solution conditions 7 Department of Biomedical Engineering Materials & Methods • Substrates – Polystyrene culture dish • Model Cell System – Porcine vascular endothelial cells (PVECs) • Analysis – Cell Topography – Receptor mapping on cell surface – Receptor-ligand binding force 8 Department of Biomedical Engineering Endothelial Cell Culture • Porcine vascular endothelial cells (PVECs), Porcine harvested from the pulmonary artery harvested • Obtained from Dr. Edward Block, Veterans Obtained Hospital, Gainesville, FL Hospital, • Cultured at 37°C, 5% CO2 for 2 to 5 days • Grown to confluence in polystyrene petri dish (3 Grown to 5 days) to • Rinsed to remove media, recovered with HBSS Rinsed and transferred immediately to the AFM for imaging imaging 9 Department of Biomedical Engineering 10 AFM Force Curve Measurements a) d) Laser Photo Detector Cantilev er Used to measure adhesion between Used the AFM tip and sample the Laser Photo Detector Cantilev er Sample b) Sample Laser Photo Detector e) Cantilev er Photo Detector c Laser Photo Detector Cantilev er Sample Sample c) Laser f) Cantilev er Laser Photo Detector a b Cantilev er f d e Sample Sample Department of Biomedical Engineering Force Volume Imaging http://www.di.com/AppNotes/ForceVol/FV.array.html • 2-dimensional array of 2-dimensional first curves first • The topographic image The is reconstructed from the force curves the • Each pixel represents a Each force curve that may be analyzed individually to quantify the tip/sample interaction interaction 11 Department of Biomedical Engineering AFM Imaging 12 Department of Biomedical Engineering 13 AFM Simulates Leukocyte Rolling • Leukocytes are recruited from flowing blood at sites of Leukocytes inflammation by endothelial cell, surface adhesion molecules molecules • The AFM is used to simulate this rolling mechanism in The vitro and measure the receptor-ligand binding vitro Leukocyte AFM Probe Ligand Selectin Endothelial Cell Substrate Department of Biomedical Engineering Affect of Contact Force High contact force allows High cytoskeleton to be imaged cytoskeleton Low contact force allows Low membrane to be imaged membrane 14 Department of Biomedical Engineering Topography Data (Living Cells) Average PVEC size on Polystyrene 40.0 ± 9.3 µ m long 40.0 long 16.3 ± 3.3 µ m wide wide length width • • • Cell density Cell morphology Imaging of membrane and Imaging sub-membrane structures sub-membrane • Cell volume by Cell approximation as a half ellipsoid ellipsoid • Inter-cell bridging: Inter-cell desmosomes, tight junctions, etc… junctions, • Time response 15 Department of Biomedical Engineering 16 Topographic Image Time Series Cells remain viable up to 6 hours after removal from incubator 2 hours hours 45 minutes 45 2 hours hours 10µm 3 hours hours 30 minutes 30 4 hours hours 15 minutes 15 10µm 10µm Department of Biomedical Engineering 17 AFM Tip Functionalization 3-aminopropyl triethoxysilane Silica Silica Microsphere Sialyl Lewis X linked to Bovine Serum Albumin Glutaraldehyde HO O H2N H3C H + O Si O + H H H H H H O H3C H3C H3C + HO OH Ac NH O HO OH O HO O H OH O NH Ac O O O OH O OH HO HO O Functionalization of microsphere verified using immunoflourscent staining of the sLeX OH Department of Biomedical Engineering Interaction Between Microsphere and Cell Controls • Functionalized microsphere Functionalized at each of the chemical modification steps imaged on petri dish and petri dish with PVECs with • Specific interactions seen Specific only with sLeX modified tip only on PVECs on Ligand forms concentric Ligand rings on the microsphere microsphere Stressed receptor-ligand bond Endothelial cell membrane surface Retracting AFM cantilever Silica microsphere SLeX (ligand) Selectin (receptor) 18 Department of Biomedical Engineering Interaction Geometry Cantilever Functionalized Microsphere Cell Substrate We acknowledge the above geometrical interactions may We occur, but the effects should average out over hundreds of force curves across the cell body force 19 Department of Biomedical Engineering Selectin Mapping • • • 2D and 3D force volume images of PVECs cultured on polystyrene, 2D specific interactions are indicated by the green dots specific Specific interactions are located in clusters near the periphery of cells Only 1 force curve out of 100 occurred on the nucleus 20 Department of Biomedical Engineering 21 Selectin Mapping - 10,000 µ m Selectin 2 Green dots indicate location of specific adhesion events Large scan area shows multiple cells Department of Biomedical Engineering 22 Force Curve Adhesions Tip Deflection [nm] Receptor-Ligand is Compressed Extend Retract Molecules Unravel C-C-C Backbone is Stretched Bond Rupture Interaction Force Interaction Distance 0 Single Interaction Adhesion Event Z Displacement [nm] 3000 Department of Biomedical Engineering 23 Force Curves on Live PVECs Specific Interactions Specific SleX AFM Probe 10% occurrence) (less thanon PVECs With Specific Adhesions (less Nonspecific Interactions 4 typical curves 4 typical curves 0 Force [nN] -5 -10 -15 -20 -25 0 500 1000 1500 2000 Z-Separation [nm] • • Controls were performed with the microsphere coated with 3aminopropyltriethoxysilane (APTES) or with APTES + glutaraldehyde Controls showed only nonspecific interactions identical to the curves Controls shown above shown 2500 Department of Biomedical Engineering Data Analysis Each Force Curve is Opened in WSxM, Converted to Force vs. Distance and Adhesion Peaks are Recorded Adhesion Peak Values Are Grouped Together and Used to Form a Histogram Each Force Curve is Individually Exported as a Deflection vs. Distance Curve Histogram is Smoothed With a Binomial Filter Non-Specific Specific Force Volume Image from AFM An Autocorrelation Function is Applied to the Filtered Histogram Periodic Interaction (if present) is Read from the Autocorrelation Function Plot 24 Department of Biomedical Engineering Binomial Filter • An envelope filter used to smooth the histogram data An (Excel 2002) (Excel • Filter coefficients based on Pascal’s Triangle Pascal’s Triangle 2nd Order Filter for Distance Interaction Histogram 6th Order Filter for Force Interaction Histogram 25 Department of Biomedical Engineering Autocorrelation Function Measure of the dependence of force series values at one time on the values at another time (SPSS 11). Given the data series x(n), n=1, 2, ...N, the autocorrelation function at lag k is defined as: 1 RXX (k ) = N −k N −k ∑ x ( n) x ( n + k ) n =1 http://www.cbi.polimi.it/glossary/Autocorrelation.html Bendat-JS, Persol-AG (1986) Random Data - Analysis and Measurement Procedures, 2nd Edition, John Wiley & Sons, NY 26 Department of Biomedical Engineering 27 Specific and Non-Specific Interactions Histogram of Non-Specific Interaction Force (20 pN Bins) a) Unfiltered 6th Order Binomial Filtered 6 Histogram of Specific Interaction Force (20 pN Bins) b) 20 Unfiltered 6th Order Binomial Filtered 5 4 Frequency Frequency 15 3 10 2 5 1 0 0 0 1 2 0 3 1 2 3 Force [nN] Force [nN] Autocorrelation of Specific Interactions d) 0.2 Autocorrelation c) 0.4 0.0 -0.2 -0.4 0 1 2 Force [nN] 3 Department of Biomedical Engineering Interaction Distance 28 Department of Biomedical Engineering Receptor-Ligand Interaction Mean Interaction Force from 6th Order Binomial Filtered Histogram (20 pN bins) Binomial FsLe(x)-selectin = 183 ± 40 pN Interaction Distance (12 nm bins) F sLe(x)-selectin = 73 ± 41 nm sLe(x)-selectin 29 Department of Biomedical Engineering Pulling Rate vs. Rupture Force • Rupture force is dependent on Rupture pulling velocity of the AFM tip pulling • P-selectin was grafted to glass P-selectin substrate and PSGL-1 was grafted to the AFM tip grafted • Extrapolating to the Extrapolating experimental rate of 12 µ m/s m/s indicates a rupture force of 175 to 200 pN for this system to • Similar values of ~200 pN seen Similar for high-speed video microscopy of leukocyte and Fritz, J., Katopodis, A.G., Kolbinger, F., et al., Proc. EC interactions EC Natl. Acad. Sci., 1998. 95: p.12283-12288. • This agrees well with the This measured force of 183 ± 40 pN measured 30 Department of Biomedical Engineering Conclusions • Demonstrated the simultaneous quantification of Demonstrated receptor-ligand binding force, interaction distance and receptor location on LIVING cells receptor • The AFM can be used to determine topography and The mechanical properties of biomaterial and cell surfaces on the nano to micro scale and combined on • Cellular response to materials can now be evaluated Cellular based on the specific chemistry expressed on the cell membrane membrane • Cell structure can be compared between in vivo and in Cell in vitro and used to optimize the biointerface vitro 31 Department of Biomedical Engineering Future Work • Verify that selectins are being probed using a Verify complimentary technique – antibody staining complimentary • Extend this methodology to other cell/material and Extend receptor-ligand systems receptor-ligand • Use this technique to evaluate expression of cell Use surface receptors as a function of substrate topography and/or chemistry topography • Improved model incorporating ligand density, contact Improved area, and deformability area, • Force measurements at different pH, addition of Force cytokines cytokines • Force measurements in flow 32 Department of Biomedical Engineering Acknowledgements • Financial support from the Financial Office of Naval Research Office • Major Analytical Major Instrumentation Center at UF UF • Dr. Edward Block • Dr. C. Keith Ozaki • Dr. Charles Seegert • Mr. Lee Zhao • Mr. Thomas Estes • Mr. Zaher Abrouhamze • • • • • • • • • Mr. Wade Wilkerson Mr. Clayton Bohn Mrs. Leslie Wilson Ms. Amy Gibson Mr. Kenneth Williams Mr. Kiran Karve Ms. Michelle Carman Mr. Thomas Estes Mr. Brian Hatcher 33 ...
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