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Bioengineering

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Published: February 21st, 2013

DOI:

10.3791/50163

1Department of Biomedical Engineering, University of Rochester

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

1. Methods

1.1 Cell Flow Chamber

A flow chamber, shown in Figure 1, was constructed so that cells could be grown under a shear of 1.0 Pa (10 dyn/cm2) and then transferred directly to an Asylum MFP3D AFM (Santa Barbara, CA).

  1. The flow chamber was prepared for the experiment by first cleaning the glass slides in piranha solution (3:1 H2SO4:H2O2) for 15 min and then washing them with di.......

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In a typical experiment, 20 force-vs-distance curves were obtained from a given region of the cell, typically in the perinuclear region, near, but not on, the nucleus (within ~2 μm). The curves were aligned to account for any sample drift over the duration of the measurement and then averaged to remove cantilever noise, as shown in Figure 4. The curves were analyzed and fit with the two layer model that was developed for determining the modulus and thickness of thin polymer films 1. From fi.......

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We used values calculated from the two-layer model and Hertz theory to model the interaction of a leukocyte circulating in the blood with the endothelial wall. We have calculated that a microvillus on the leukocyte with a diameter of 50 nm under a 10 pN load would indent approximately 150 nm into the glycocalyx, only a fraction of the total thickness. This indicates that the glycocalyx, with properties as measured in this experiment, is a significant barrier to cell-cell interaction and can be a large steric hindrance wh.......

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The authors would like to thank Elena Lomakina, Richard Bauserman, Margaret Youngman, Shay Vaknin, Jessica Snyder, Chris Striemer, Nakul Nataraj, Hung Li Chung, Tejas Khire, and Eric Lam for their assistance with this project. This project was funded by NIH #PO1 HL 018208.

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Comments
McCoy's Medium Gibco 16600-082
Fetal Calf Serum Hyclone SH30070
Endothelial Cell Growth Medium Vec Technologies MCDB-131
Pooled Human Umbilical Vein Endothelial Cells Vec Technologies PHUVEC/T-25
Sulfuric Acid JT Baker 9681-02
Hydrogen Peroxide VWR BDH3742-1
(3-aminopropyl)triethoxysilane Aldrich 440140-100ML
Isopropyl Alcohol VWR BDH8999-4
Trypsin Cellgro 25-054-C1
Hank's Buffered Salt Solution Gibco 14175-095
sulfo-NHS-LC-Biotin Thermo Scientific 21335
Streptavadin beads Dynabeads 112.06D
MFP-3D AFM Asylum Research
Tipless Cantilevers Nanoworld ARROW-TL1-50
Silhouette SD Quickutz Silhouette-SD
Silicone Rubber Stockwell Elastomerics SE50-RS
30 ml Syringes Benton Dickinson 309650
18 gauge needles Benton Dickinson 305196
Extension Sets Hospira 4429-48
4 way valves Teleflex W21372
Male/Female Port Caps Smith's Medical MX491B
Peristaltic Pump Watson-Marlow 401U/D
Peristaltic Tubing Watson-Marlow 903.0016.016
sterile filters Pall Life Science 4652

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