Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

The bio flux system is an automated instrument and microfluidic device for running live cell assays under controlled sheer flow. This system uses well plate microfluidic technology to integrate micron scale flow cell devices into SBS standard Well plates. BioFlex can be used to conduct platelet adhesion and aggregation studies with high throughput and reduced blood volumes.

Whole blood labeled with fluorescent dye is added to the wells and then flowed through the channels. Under controlled sheer flow, high resolution microscopy data is produced, which quantifies platelet adhesion and aggregation in the presence of drug compounds and other varying parameters. Hi, I'm Mike Schwartz from the RD lab at Flexion Biosciences.

Today we will show you a procedure for the use of microfluidic flow cells in platelet adhesion assays. We use this protocol in our lab for a variety of vascular biology assays. So let's get started.

Beef, before running the flow cell experiment, the microfluidic channels of the bio flux plate must be prepared with a protein coating of interest collagen one will be used in this experiment. Each of the 24 experimental channels of the bio flux plate connects to an inlet well and an outlet well for this plate. The inlet well is the left well feeding the channel and the outlet well is on the right, dilute the collagen one stock to a concentration of 200 micrograms per milliliter.

In 0.02 molar acetic acid, 20 microliters will be needed for each channel used. Since this experiment requires 11 channels, make 200 microliters of collagen coating. One channel remains uncoated mixed by gentle iteration with a micro pipette tip.

Add 20 microliters of coating to each respective channel by using a micro pipette to dispense the liquid into the inner punch of the well. Feeding the microfluidic channel of interest as shown here with the yellow dye include one channel without collagen as a negative control for platelet adhesion and aggregation. After the collagen coating has been added to all the respective channels, attach the interface to the plate.

If you are using the bio flux 1000 interface, you can clamp down both latches onto the stage. If you are using the Bio flux 200 interface first finger, tighten the four screws. And then once they are all set, use the torque driver to tighten completely.

The torque driver will click when it reaches the maximum. Using the manual mode in the bio flux software, apply perfusion to the channels of interest at 2D per centimeter squared from the outlet. Well use a low power objective on a microscope to find the inlet well and observe the opposite inner punch being filled with liquid.

You should first see a tiny be of liquid, slowly filling the inner punch after several minutes and when the inlet inner punch is filled, stop profusion on all channels by pushing stop in the software. Incubate the plate at room temperature for one hour. During this incubation period, you can begin preparing the whole blood at the end of the one hour incubation.

Once the blood has been prepared, remove the interface and add one milliliter of PBS plus calcium and magnesium ions to the outlet. Well start profusion from the outlet well at two dines per centimeter squared and continue profusion for 10 minutes. After 10 minutes, stop the profusion After removing the interface, remove excess PBS from both the inlet and outlet wells.

Never remove liquid that fills the inner punch. To block the channels, add one milliliter of blocking solution to each outlet. Well to be used perfuse from the outlet well at two dine per centimeter squared and continue perfusion for 10 minutes.

Stop profusion after 10 minutes and remove the interface. The channels are now ready and can be used throughout the day while the plate is being coated with collagen. During a one hour incubation, fresh human blood from a fasting individual should be prepared for imaging and running into the plate.

Blood should be drawn into sodium citrate, anticoagulant and used within three hours of collection. First, add a four millimolar stock of calcium M in DMSO to the blood at a dilution of one to 1000 volume per volume. To obtain four micromolar calcium am mixed by gentle inversion.

Next, dispense one milliliter of the calcium am labeled blood into ten one point five milliliter micro tubes. Add GP two B three a inhibitor antibody to each tube at the desired dilution. Be sure to include a no antibody negative control and an unrelated antibody positive control mixed by gentle inversion.

Incubate the tubes at room temperature for one hour mixing by gentle inversion every 10 minutes prior to running the flow cell experiment. An automated protocol should be set up in the bio flux control module for collagen.One. Set up a protocol to run the blood at ten nine centimeters squared for 10 minutes from the outlet.

Well, using the BioFlex 1000 workstation data acquisition parameters should be set up, which include the desired stage positions acquisition in the fitz wavelength and time-lapse setting. A typical image acquisition plan for this protocol would comprise one field of view per channel to be captured using a 10 x objective every 30 seconds over a total duration of 10 minutes. Additional fields of view and alternate TimeLapse settings are also possible.

Once the automated protocol and data acquisition parameters have been set up, return to the BioFlex plate and remove the liquid on both sides of the channel except for the inner punches. Taking care to work quickly, place 500 microliters of blood of each condition into the output wells of each channel. Note that the output wells are being used to deliver the blood because it is the shortest path to the viewing zone and will provide a more immediate reaction of the blood to the collagen coating.

In addition, place the control blood with no antibody into a channel that is coated with collagen and one that is just blocked. Place the plate on the BioFlex 1000 workstation microscope and clamp on the interface. Perform the stage list calibration by locating the first calibration point which is defined as the origin.

Then locate and mark the second calibration point. Apply this calibration to the appropriate stage list. This ensures that all of the viewing locations on the plate can be found automatically.

Move the stage to one of the channels containing blood set fits e image capture parameters such as exposure time and gain in the BioFlex montage software. Launch the appropriate workflow by clicking on the desired button. This will start the flow protocol and image acquisition simultaneously.

At the end of the experiment, remove the plate from the BIOFLU 1000 workstation and dispose of it according to institutional guidelines. Using collagen coated microfluidic channels of the bio flux system. Aggressive thrombus formation is observed over time with the untreated control blood sample.

In contrast, no thrombus formation is observed with the uncoated channel. In one recently performed experiment, the average size of aggregates under control conditions was 2000 micrometer squared. GP two B three A is a potent mediator of platelet platelet interactions and aggregation stabilization when activated by adhesion to collagen as shown here after incubation with anti GB two B three a antibody for one hour prior to sheer exposure, a decrease in size of thrombi as well as a decrease in the frequency of thrombus formation is observed.

A dose dependent response can typically be observed at ten nine centimeter squared and the IC 50 value for this particular inhibitor was 17 nanomolar. Maximum inhibition for this donor compared to the no antibody control was 11%at 10 dine centimeter squared. A 48 well high shear plate is also available for running whole blood experiments up to 200 D per centimeter squared or 5, 000 inverse seconds.

We've just shown you how to use microfluidic flow channels to conduct physiologically relevant vascular biology experiments. When doing this protocol, it's important to remember to dilute the collagen in the correct buffer. Always use correct pipetting technique to avoid introducing air bubbles and always follow your lab's biosafety regulations.

So that's it. Thanks for watching and good luck with your experiments.