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

The overall goal of the following experiment is to measure the mechanical properties of the endothelial glycocalyx using an atomic force microscope or a FM.This is achieved by first culturing, a monolayer of endothelial cells under a physiological flow in a bioreactor in order to build up the glycocalyx layer as a second step. Micron sized beads are attached to a FM cantilevers and used as a probe to measure the resistive force of the cell body and the glycocalyx during indentation. Next, the indentation curves are fit with a two layer model in order to determine the glycocalyx modulus and thickness results are obtained that show that the endothelial glycocalyx has a modulus of 0.7 kilo pascals and a thickness of 380 nanometers based on the two layer analysis.

Hi, I'm Rick Wa.I'm chair of biomedical Engineering at the University of Rochester. There's been a lot of discussion in recent years about the possible role of the endothelial glycocalyx in inflammation, in particular as to whether it might act as a protective barrier to prevent unwanted cell adhesion during inflammation. One of the keys to resolving the questions that surround this issue is the ability to measure the physical properties, namely the thickness and the stiffness of this layer, both in normal conditions and during the inflammatory response.

I'm Graham Marsh. I'm a graduate student at the University of Rochester Department of Biomedical Engineering. The big trouble in characterizing the endothelial glycocalyx is it's incredibly soft.

The approach that we came up with is to affix a large bead to the tip of A an A FM cantilever so that the forces measured will be resolvable with the A FM.To begin this procedure, construct a flow chamber like the one shown here, capable of growing cells under a shear stress of 1.0 Pascal. Prepare the flow chamber for the experiment by first cleaning the glass slides in piranha solution for 15 minutes. Rinsing them three times with distilled water and drying them at 70 degrees Celsius for 30 minutes.

Next, coat the slides with amino propyl trixi, cy lane, or apte by vapor deposition. Add one milliliter of apte to a flask that is attached to the vacuum chamber with the sample. Then cap the flask and pull a vacuum of 0.7 bar for one hour.

During this time, refresh the vacuum three times. Next, cut a new flow chamber gasket from a 0.4 millimeter thick sheet of silicone using a silhouette SD cutting tool. The channel measures 6.4 millimeters wide by 19 millimeters long, and the outer diameter is cut to 35 millimeters in order for it to fit into the asylum culture dish.

Using these dimensions, calculate the flow rate necessary to generate a sheer stress of one pascal with the equation shown here. Q represents the flow rate. Tau is the sheer stress.

Mu is the viscosity of the medium. H is the channel height, and W is the width of the flow chamber. The next step is to align the top piece of the flow chamber with the gasket in the cell culture dish and secure it with a magnetic ring.

Connect the flow ports in the cell culture dish to three wave valves with lines to the 30 milliliter syringes. Then sterilize the entire assembly by flushing with isopropyl alcohol for five minutes. Then remove the alcohol and allow it to air dry in the tissue.

Culture hood Prewarm 4%fetal calf serum in McCoy's medium to 37 degrees Celsius. After being left to dry for five to 10 minutes, flush out whatever alcohol remains with 30 milliliters of the prewarm fetal calf serum. Then fill the syringes with 20 milliliters of veek MCDB 1 31 growth medium.

Next, cap the syringes to prevent contamination. When moving the system out of the sterile hood, fit the cap for the catch reservoir with the needle that reaches into the fluid to draw medium back to the upstream reservoir. Finally, attach 0.2 micron sterile filters to the air inlets on the caps next harvest human umbilical vein.

Endothelial cells grown to confluence in a T 25 flask. Re suspend the pelleted cells at 100, 000 cells per milliliter in medium and draw the solution up into a syringe. Then inject the suspension into the flow chamber through the three-way valve.

Allow the cells to settle and adhere to the glass substrate for two hours. Before beginning flow, move the bioreactor and cells into an incubator where the cells are allowed to settle and adhere for two hours. Tubing connecting the reservoirs to a peristaltic pump passes through the back of the incubator Once the cells have adhered, set the peristaltic pump to the desired flow rate.

Media moves between the reservoirs, creating a pressure head and a flow through the bioreactor that produces a wall shear stress of one pascal. Allow the cells to incubate with continuous flow at 37 degrees Celsius for one to five days until the chamber is confluent. To begin cantilever preparation, clean a tip plus a FM cantilever in nitric acid for five minutes, followed by a rinse with distilled water.

Once dry, functionalize the cantilever with apdi in a vapor deposition chamber as previously shown for the glass slides. Then prepare a solution of five milligrams per milliliter by weight NHS Sulf O lc, biotin in Hank's buffered salt solution. Submerge the cantilevers in the solution for 15 minutes.

This reaction conjugates the CY and end hydroxy oxide and presents biotin molecules on the surface of the cantilevers. On the day before, start preparing a solution of biotin free medium by combining 20 milliliters of vexcel culture medium containing 20%serum with 200 microliters of strept ENC coated beads, and incubate for 12 hours while rotating. On the day of the experiment.

Pull the magnetic beads to the bottom of the tube with a magnet for two minutes and transfer the supernatant containing biotin free medium to a new tube. Sterilize the media by passing it through a 0.22 micrometer filter and put it into a new tube. The next step is to remove the flow chamber from the cell culture dish and then wash the cells three times with two milliliters of 37 degree Celsius, biotin free medium.

Then add one microgram of 2.4 micrometer diameter, stripped Aden coated beads. Add a concentration of 10 micrograms per milliliter into the cell culture dish and gently mix it. Next, calibrate the A FM tib by attaching it to a strippin bead and pressing it into a plain glass surface.

In order to accomplish this land the tip on the glass surface next to a bead, retract the tip, then position the apex of the cantilever over the bead and press down for several seconds. Finally, use the calibrated cantilever to indent the samples. The 2.4 micrometer beads offer a larger contact area with the cell surface so that the mechanical properties of the soft glycocalyx layer can be more easily detected.

To accomplish this position, the cantilever over a cell near the cell nucleus, use a soft approach of the tip onto the cell to set the cantilever height approximately three micrometers above the cell surface. Then indent the surface 20 times at a rate of one micrometer per second to a maximum force of seven. Nano newtons allow for six seconds of time to elapse between successive contacts.

Calculate the thickness of the glycocalyx layer using equations related to the hertz theory. The force of indentation F is described by this equation where delta is the indentation depth. R is the sphere radius, and estar is the combined modulus of the materials under stress.

In order to separate the combined elastic modulus estar into cellular and glycocalyx components, treat the glycocalyx layer as a uniform, thin soft film on the surface of the cell body and use the two layer model of Clifford and C.In this equation, PQ and N are constant empirically determined from polymer fits. M is a constant from the polymer model, and Z is related to the thickness of the thin film or glycocalyx layer by the equation shown here. The force measured by the A FM measured in nano Newtons begins to increase after contact with the surface and is initially dominated by the glycocalyx layer component.

Once the glycocalyx layer is compressed, the cell body becomes the major component of deformation. The blue two layer fit line was calculated by fitting four free parameters to get it to match the indentation data shown in red from the equation used the cell modulus, glycocalyx, layer modulus, and glycocalyx layer thickness can all be determined. The properties from 25 cells were compiled into the histogram shown here.

The average glycocalyx layer modulus was determined to be 0.7 plus or minus 0.5 kipa cals, and the glycocalyx layer thickness was determined to be 380 plus or minus 50 nanometers. This approach gives us the tool we needed to answer fundamental questions about the role of the glycocalyx in regulating cell adhesion during inflammation. We're now able to address questions about how the properties of the glycocalyx are altered during inflammation.

By what means those changes are accomplished. This approach can be extended in a number of ways. For example, one could use it to test novel, more sophisticated theoretical descriptions of the properties of the glycocalyx.

Or on a more practical level, one might be able to use this to test bio-inspired surface coatings, designed to protect implantable devices from immune response in the host. Thanks very much for your interest in our work.