Straight Channel Microfluidic Chips for the Study of Platelet Adhesion under Flow

Microfluidic devices have become an integral method of cardiovascular research as they enable the study of shear force in biological processes, such as platelet function and thrombus formation. Furthermore, microfluidic chips offer the benefits of ex vivo testing of platelet adhesion using small amounts of blood or purified platelets. Microfluidic chips comprise flow channels of varying dimensions and geometries which are connected to a syringe pump. The pump draws blood or platelet suspensions through the channel(s) allowing for imaging of platelet adhesion and thrombus formation by fluorescence microscopy. The chips can be fabricated from various blood-compatible materials. The current protocol uses commercial plastic or in-house polydimethylsiloxane (PDMS) chips. Commercial biochips offer the advantage of standardization whereas in-house chips offer the advantage of decreased cost and flexibility in design. Microfluidic devices are a powerful tool to study the biorheology of platelets and other cell types with the potential of a diagnostic and monitoring tool for cardiovascular diseases.

In contrast to the conventional flow chamber that usually requires milli-liter sample volumes, the microfluidic biochip only requires micro-liter samples, making it a perfect analytical tool for small volumes (e.g., pediatric or rare samples and mouse studies). In addition, the biochips described in this protocol are reusable, making them an economic and versatile choice for microfluidic studies.

A. Fabrication of PDMS biochips
The microfluidic biochips can be fabricated with PDMS (Sylgard 184 kit) casted from a master mould on a silicon wafer by photolithography (Qin et al., 2010). Most universities and institutes have photolithography and clean room facilities for the fabrication of the master mould. In this protocol, the photolithography is conducted at the Research and Prototype Foundry at University of Sydney Nano Institute. It is possible to perform photolithography without a foundry. The equipment required would include: 1. Air plasma system, 2. Oven for PDMS curing, 3. Desiccator for PDMS degassing, 4. Programmable spin coater, 5. UV lamp-LED exposure, 6. Programmable hot plate. We refer interested readers to: Microfluidic device design, fabrication, and testing protocols or Elveflow. 5. Crosslink the film pattern by baking on a hotplate and ramping the temperature at 5 °C/min, starting at 23 °C and holding at 90 °C to dry out the solvents. The ramping profile is achieved by proximity, plus vacuum contact bake steps, for a total duration of 805 s. 6. Allow the film to cool on the hotplate to room temperature.
Note: Keep the film on the hotplate to avoid thermal stress.  b. Once the curing agent is added to the PDMS base, the PDMS will slowly begin to cure and harden at room temperature. This will be noticeable after 4 h. After 16 h, the PDMS will become too viscous to practically work with.
13. Pour the PDMS mixture over the pre-made mold, creating a 4-5 mm thick film.
14. Place the PDMS and mold in a Nalgene ® vacuum desiccator and degas for 30 min.
Note: The amount of time required to degas the mixture is dependent on the volume and surface area of PDMS. The mixture must be degassed completely, otherwise bubbles will form during the curing process which will change the morphology of the device. For 200 g of PDMS placed in a 100 mm diameter Petri dish, 2 h is sufficient to completely degas the mixture. 15. Bake (cure) the PDMS mixture and mold in the oven at 80 °C for 4 h. 16. Cut out the cured PDMS chips from the mold gently and carefully.   1. Place the biochip in 400 ml of 10% extran and sonicate for 15 min.

Blow dry the biochip completely using compressed air.
Note: Make sure the biochip is completely dry before proceeding. Remnants from small droplets can compromise the contact between the biochip and the coverslip leading to leakage during a microfluidics experiment.
Note: The PDMS will swell slightly and show indentations after the Butan-1-ol treatment. This is normal and the PDMS will return to its original shape and size once sonicated in dH2O. 4. Blow dry the biochip completely using compressed air. Place the biochip in 10% extran and sonicate for 10 min.

Remove the fibrinogen in the inlet well, replace with 100 μl of Tyrode's buffer or PBS and repeat
Step D2 to wash the channel.

5.
Remove the Tyrode's buffer or PBS from the inlet well and replace with 100 μl of blocking buffer.

Repeat
Step D2 to block the channel with blocking buffer. Incubate for 30 min at 25 °C. 6. Remove the blocking buffer from the inlet well, replace with 100 μl of Tyrode's buffer or PBS and repeat Step D2 to wash the channel.
Note: Once washed, keep the Tyrode's buffer or PBS in the channel until image acquisition.
This will prevent the formation of air bubbles in the channel while flowing cells through the biochip.

Human platelets
The procedure of drawing human blood by venipuncture is determined by the Institution's Ethics and Protocols for human blood sampling.
1. Draw 8 ml venous blood by venipuncture into an ACD tube.
2. Centrifuge at 200 x g for 20 min, no brake, and separate the platelet-rich plasma (supernatant).
3. Allow platelet-rich plasma to rest in the water bath at 37 °C for 30 min. 4. Add PGE1 to the platelet rich plasma (1 µM final concentration) immediately prior to centrifugation.
5. Centrifuge at 800 x g for 20 min, no brake, and discard the platelet-poor plasma (supernatant). 6. Resuspend the platelet pellet in HEPES-Tyrodes' buffer with glucose. Perform a platelet count on the hematology analyzer and adjust the platelet concentration to 3 x 10 5 /µl (Nesbitt et al., 2009). Five minutes before perfusing through the microfluidic channel, add calcein to a final concentration of 1 µg/ml and keep the platelets in the dark until use. Use the labeled platelets within an hour as the calcein is effluxed out of the cell and cell fluorescence will be lost.

Mouse platelets
The procedure of drawing mouse blood by venipuncture is determined by the Institution's Animal          Figure 9A). After the video camera preview has been initiated the channel will be displayed on the screen ( Figure 9B).   5. To start the assay, enter the desired parameters for flow by adjusting the shear units, the acquisition time and capture delay ( Figure 11A). After all the steps have been performed, click on "start assay" to initiate the pump and data acquisition. To stop the assay, click on "stop assay".

Video 2. Assembly of the in-house PDMS biochip onto a microscope stage
To acquire an image, click on "acquire image to file" on the "Setup VenaFlux page". This will 19 www.bio-protocol.org/e3195 save the image at the pre-specified location as a bitmap image ( Figure 11B). To acquire a video, click on "acquire video to file". The analysis of the images and image stacks of the videos can be performed by ImageJ as described below or using the ImagePro Premier 64-bit software.
We prefer ImageJ for our analysis as it is flexible and can include adjustable macros.

A. PDMS biochips
Two methods of manual analysis are described for the analysis of platelet adhesion over time: platelet counting and analysis by fluorescence intensity. This protocol describes the use of Fiji/ImageJ on PC for analysis, however the process is the same for Mac users.

Platelet adhesion analysis by counting
The steps involved in platelet count and platelet sum fluorescence data analysis (Figures 13-14) can be found in Video 3.    5. Move to the next frame on the image stack, click on additional platelets that have adhered and de-select platelets no longer present as described in step 3. Record the platelet counts ( Figure   13) and repeat for each frame in the image stack.

Platelet adhesion analysis by fluorescence intensity analysis
The steps involved in platelet sum fluorescence data analysis (Figures 15-22) can be found in Video 4. Figure 15. Accessing analysis parameters. The parameters that will be displayed on a measurement readout can be selected from the set measurements menu. To access the menu, select the "analyze" tab (highlighted in blue), and click "Set measurements…" (circled in red).

Figure 17. Region of Interest (ROI) manager on ImageJ.
The ROI manager can be accessed by selecting "Analyze" and then "Tools" (highlighted in blue) and clicking on "ROI manager…" (circled in red). 5. Click on the "Freehand selections" tool on the tool bar ( Figure 18). 6. For each frame on the time stack, on the 488 nm channel, circle a platelet or platelet aggregate and click "Add" on the ROI manager window (Figure 19).

Figure 19. Storing regions of interest on the ROI manager.
A region of interest, drawn using the freehand selections tool, can be stored on the ROI manager either by pressing "t" on the keyboard or clicking "Add" on the ROI manager (circled in red).