The scope of this research is to assess platelet function under physiologic conditions. We specifically study platelet function using microfluidic devices and biological surfaces. And using this approach, we were able to answer questions related to platelet dysfunction and hemostatic resuscitation for applications in transfusion medicine.
Current platelet function assays often test the ability of platelets to aggregate in response to a specific agonist of choice. Detection methods might include light transmission or electrical impedance, and these assays may utilize process samples, and are tested stagnantly. By using this microfluidic approach to test platelet function, we incorporate fluid dynamics to add physiologic relevance.
Fluid dynamics play a critical role in biological mechanisms involved in platelet function during hemostasis. In bleeding patients, we are particularly concerned about platelet function, and therapeutic interventions that may enhance that function for better outcomes. Using this platelet function testing approach, we are able to track the contributions of different platelet populations in an experiment and unlock a way to assess therapeutic options for trauma and other bleeding conditions.
To begin, pour the silicone elastomer base into a weighing dish. Add the silicone curing agent at a ratio of ten-to-one and stir the mixture thoroughly to ensure proper cross-linking of the silicone polymer chains. Place the master mold into a Petri dish and pour the uncured PDMS onto the mold evenly.
Then, place the Petri dish inside a vacuum desiccator for 30 minutes to remove bubbles from the mixture. To finish curing the PDMS, place the mold into an oven set to 70 degrees Celsius for 90 minutes. After complete PDMS curing, use a razor blade or scalpel to cut out the microfluidic cast.
Punch holes at the edges of the channels. Now, place the glass slide and the microfluidic cast with the etched side upward into a plasma cleaner. Start the vacuum pump and seal the chamber.
Then, turn the plasma cleaner to the High setting and leave the setup in the cleaner for 30 seconds. After that, turn off the plasma cleaner and release the vacuum. To bind the plasma-cleaned cast and glass slide, gently press the sides that were facing up in the plasma cleaner together.
Place the microfluidic device in an oven or on a hot plate at 70 degrees Celsius for 10 minutes to finalize the binding process. Now, coat the microfluidic chamber with type-one equine fibrillar collagen reagent through a designated outlet to the designated inlet. Store the device in a warm, humid, closed container to prevent the coated collagen from evaporating within the channel.
After one hour, rinse the device with PBS in the opposite direction of the coating to flush out the collagen solution. To begin, incubate citrated whole blood with a fluorescence conjugated CD41 antibody, using a fluorophore of choice. Stain the blood for 30 minutes on a nutating rocker.
Next, turn on the microscope and associated software. Set the withdrawal syringe pump level with the microscope stage and adjust the syringe pump settings according to the experimental requirements. Then, place the microfluidic device on the microscope stage and tape its edges to the stage to prevent movement.
Now, connect the one-sixteenth of an inch internal diameter tubing to an elbow connector, and then to a 10 milliliter syringe with a one-sixteenth of an inch internal diameter connector. Fill the 10 milliliter syringe with sterile PBS and connect it to the syringe pump. Then, insert the elbow connector into the outlet of the microfluidic device.
Prepare inlet lines approximately 10 centimeters long with an elbow connector on one end and an angled cut on the other end. Now, connect the inlet elbow connector to the device inlet and position the inlet line into a waste microcentrifuge tube placed on an angled holder. Use the 10-times objective lens to focus on the microfluidic device channel edges.
Next, prime the lines with PBS. Manually advance the syringe pump to clear the channel of any PDMS or debris. Check near the channel inlet and outlet for any obstructions.
Now, open the saved image capture settings or create a new image capture procedure for time series images captured every one to two seconds with a bright field channel and fluorescent channels corresponding to the CD41 antibodies in the blood sample. Obtain the filtered citrated blood sample and mix it by pipetting up and down just before starting the experiment. Place the sample on the angled microcentrifuge holder.
Then, position the inlet line into the blood sample and start recording the image capture. Slowly withdraw the syringe to fill the dead volume in the tubing. Once the blood reaches the channel, immediately press Play on the syringe pump to resume flow at the desired sheer rate.
Run the experiment until the platelets have fully occluded the stenotic region of the microfluidic device, or until the experimental endpoint, which is typically 10 minutes. Once the experiment is complete, stop the image capture, and stop the syringe pump. Finally, save the image capture to the appropriate storage.
