The overall goal of the following experiment is to obtain a sheer adhesion map of cell particle adhesion from a single experiment using synthetic microvascular networks. This is achieved by first coating the microfluidic device with the protein of interest in this case avadon. As a second step, biotinylated particles are injected into the device at physiological shear rates, which bind to the avadon coated device with differential patterns depending on the local shear rates.
Next, the number of bound particles is counted in order to generate a sheer adhesion map of the particles using a computationally created database of sheer rate values in the network. Results show that a sheer adhesion map can be obtained from a single experiment resulting in significant savings of time and reagents as compared to the conventional parallel plate flow chambers. The primary advantage of this technology over existing methods, such as linear flow channels, is that we can generate shared a maps from a single experiment resulting in a significant saving in cost and time.
This method can help answer key questions in the field of drug delivery, such as the ability of the drug delivery vehicles to bind with the desired targets. We first had the idea for this method when we were using paddle plate flow chambers for understanding cell particular lesion. Under fluid conditions, Though, the focus of this assay is to provide basic insights into s particle and cell adhesion.
The syn vivo platform can be applied to a variety of other systems such as cancer and CNS delivery, infection and inflammation thrombosis and microcirculation dysfunction. Visual demonstration of this method is critical as the micro fluidic device steps are difficult to learn, getting accustomed to small sample volumes, and the best practices to avoid bubble formation are better communicated visually. Demonstrating the procedure will be Ashley Smith, a research scientist from our lab.
The SynVivo SMN microfluidic device is comprised of two sets of parallel ports, one for flowing in surface coating, moieties and or cells for seating, and the other for running the assay. To begin completely submerge the device in a Petri dish containing sterile deionized water and place the dish into a vacuum. Desiccate allow the desiccate to run until all of the air is removed from the channels of the device.
This should take approximately 15 minutes before removing the device from the water. Use fine point forceps to place tigon tubing primed with water into each port of the device. The tubing should be approximately one inch in length.
The device can now be removed from the water using a pipette or syringe. Place a drop of water around the base of one inlet port tubing. Carefully remove the tubing.
The drop of water will prevent air from entering the device. Next, prepare a one milliliter syringe loaded with avadon at a concentration of 20 micrograms per milliliter. Connect the syringe to a 24 gauge stainless steel needle and tubing Clamp the inlet port not being utilized with a jaw clamp.
Insert the tubing into the inlet port of the device. Inject avadon at a flow rate of one microliter per minute for 10 minutes to allow complete perfusion of the device. At the end of the flow time.
Clamp all the tubing with jaw clamps and place the device at four degrees overnight. Allow the device to come to room temperature. Then place the device on an inverted fluorescence microscope equipped with a motorized stage and a high performance camera.
Prepare a solution of two micron biotinylated particles at a concentration of 5 million particles per milliliter in phosphate buffer saline or PBS. Load the particles into a one milliliter syringe. Prepare a second one milliliter syringe of PBS.
Only Load each syringe onto a syringe pump, and then connect them to needle and tubing using a pipetter syringe. Place a drop of water around the base of the inlet port tubing and carefully remove the tubing used to coat the device. Carefully insert the tubing for biotinylated particles and for PBS into each of the inlet port.
Then start injecting biotinylated particles at a flow rate of 2.5 microliters per minute. Monitor the inlet port on the microscope at the first sign of particles. Begin the timer and continue flow for three minutes.
At the end of the three minutes, stop the flow of biotinylated particles while simultaneously starting the flow of PBS at a flow rate of 2.5 microliters per minute. Allow PBS to flow in the device for three minutes to wash off unbound particles. To begin acquisition, use the scan large image function in the imaging software to acquire the image of the entire device, then sequentially, number the bifurcations in the device and create a circular area of interest with twice the diameter of the channels.
In this case, set the area of interest diameter to 200 microns since the channel diameter is 100 microns. Use the automated count function in the imaging software to export the number of particles in each area of interest to a spreadsheet. Likewise, use the automated account feature to export the number of particles in the entire device.
CFD simulations are run and the results are stored in a database for further analysis. The information included in the results contain wall shear rates, velocity particle flux, and adherence to the device. The information here is used to determine the number of particles that adhere at each area of interest given a defined input of a particle concentration.
To generate a shear adhesion map, calculate percent of adhesion as detailed in the text protocol as a final step. Plot the sheer adhesion map using the sheer rate at each bifurcation of the networks and the percent adhesion values. This image shows a typical avidan coated syn vivo SMN device following binding of two micron biotinylated particles.
Note that particles preferentially adhere near the bifurcations in the network. The numbered bifurcations in the syn vivo SMN network can be seen here. This image is the sample wall shear map generated by the CFD model of the device.
The shear rate varies in the device ranging from 250 inverse seconds to 15 inverse seconds as observed in the micro vasculature in vivo. Note that these varying shear rates cannot be obtained simultaneously in linear microfluidic channels as they provide a constant shear rate for each flow rate. The plot shown here of the values of the shear rate at each of the bifurcations of the network reveals that the shear rate patterns are complex and cannot be obtained with a simple analytical relationship unlike linear flow channels.
Furthermore, multiple bifurcations are present in the network that fall in the same sheer bin. Adding to the statistical confidence of the data shown. Here are sample results of the CFD analysis for particle fluxes in the network, which are used to compute the particle fluxes in the branches and bifurcations of the experimental network.
The sheer adhesion map computed from a single syn vivo SMN experiment follows the inverse relationship as observed from sheer adhesion maps obtained from linear channel experiments. However, using a SynVivo assay, a single experiment allows generation of this sheer map, whereas multiple experimental runs are acquired from the linear channels. After watching this video, you should have a good understanding of how to use SynVivo SMN devices for generating the shared adhesion from a single experiment.
When performing this experiment, it's important to remember not to introduce bubbles to the SynVivo SMN microfluidic device After its development. This technique paved the way for researchers in the field of inflammation to study leukocyte endothelium interaction at different sheer rates in a single experiment. In the next generation of devices, researchers can study leukocyte rolling adhesion and migration in a single assay, something which is not currently possible.
Following this procedure, other methods investigating adhesion of drugs, drug delivery vehicles, and cells can be performed to investigate questions such as the physiological effect of flow on cell cell, cell drug and drug vehicle adhesion.
