Name: Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells (Video) | JoVE Journal
Uploaded: 2009-10-27T07:00:00.000+00:00
Duration: 10 min 10 s
Description: Learn platelet adhesion and aggregation assays using microfluidic flow cells under physiological shear flow. This method enables high-throughput vascular biology studies with reduced blood volumes and quantitative drug testing.

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5 minutes

Part 1: Preparation of the microfluidic channels of a BioFlux plate.
<ol>
<li>Before running the flow cell experiment, one needs to prepare the microfluidic channels with a protein coating of interest. In this case, collagen I will be used. Each channel has an inlet 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.</li>
<li> Dilute the collagen I (5mg/ml stock) coating to 200μg/ml concentration in 0.02M acetic acid. One will need 20μl for each channel used. Mix by gentle triteration with a micropipette tip.</li>
<li>Add 20 μl of coating to each respective channel to be used. One must dispense the liquid into to inner punch of the outlet well feeding the microfluidic channel of interest using a micropipette. Avoid introducing bubbles, do not push the air out of the pipette. Include one channel without collagen for a no collagen control (a negative control for adhesion and aggregation).</li>
<li>Attach the interface to the plate by first finger tightening the 4 screws and then once they are all set, use the torque driver to completely tighten. The torque driver will click when it reaches the maximum tightness.</li>
<li>Using manual mode in the BioFlux software, apply perfusion to the channels of interest at 2dyn/cm2—from the outlet well. Here one must watch for the opposite inner punch to be filled with liquid. This will take several minutes. One can either watch this occur using a low power objective on a microscope (4X) and finding the inlet well or by holding the plate and looking into the inlet wells from below. One should first see a tiny bead of liquid that slowly fills the inner punch. Once the inlet inner punch is filled, stop perfusion at once by pushing stop in the software.</li>
<li>Incubate the plate at room temperature for one hour.</li>
<li>Remove the interface and add 1 ml of PBS (plus Ca2+/Mg2+) to the outlet well. Start perfusion from the outlet well at 2dyn/cm2. Continue perfusion for 10 minutes. Stop perfusion and remove the interface.</li>
<li>Remove excess PBS from both inlet and outlet wells – never remove liquid that fills the inner punch. Block the channels using blocking solution ( 0.5% v/v BSA in PBS (plus Ca2+/Mg2+)). Add 1 ml of blocking solution to each outlet well to be used and perfuse from the outlet well at 2dyn/cm2. Continue perfusion for 10 minutes. Stop perfusion and remove the interface. Channels prepared up to this step can be used throughout the day. </li>
<li>Prior to adding blood, remove the liquid on both sides of the channel except for the inner punches.</li>
</ol>
 Part 2. Preparing the blood with GPIIb/IIIa inhibitory antibody.
<ol>
<li>While the collagen is incubating in the channels, one must prepare the whole blood. One should follow the biosafety regulations dictated by his/her institute when handling human blood.</li>
<li>Fresh human blood (from a fasting individual) drawn into sodium citrate anticoagulant should be used within 3 hours of collection. </li>
<li>Prepare blood by adding 4μM calcein AM. Prepare a 4mM stock of calcein AM in DMSO and add to blood at a dilution of 1/1000 v/v. Mix by gentle inversion.</li>
<li>Dispense 1 ml of the calcein AM labeled blood into 10 - 1.5mL microtubes. Add GPIIb/IIIa inhibitor antibody to each at the desired dilution (the molecular weight of IgG is ~150kDa). An example dilution series is from 1200nM to 9nM and includes a no antibody control and an unrelated antibody control. An excellent positive control for inhibition would be ReoPro (abciximab, Eli Lilly) at a concentration of 20ug/ml. Mix by gentle inversion. </li>
<li>Incubate at room temperature for 1 hour. Mix by gentle inversion every 10 minutes.</li>
</ol>
 Part 3: Running the flow cell experiment on the BF1000 workstation.
<ol>
<li>First, one must set up an automated protocol in the BioFlux control module. For collagen I, one should set up a protocol to run 10dyn/cm2 for 10 minutes from the outlet well. At this point, one should also set up data acquisition parameters using the BF1000 workstation, including channel positions, capture in FITC wavelength and time-lapse information. It is recommended to capture 3 fields of view using a 10x objective per channel every 30 seconds over a duration of 10 minutes total.</li>
<li>Working quickly, place 500ul of prepared blood of each condition into separate prepared channels. Place no antibody control blood into a channel that is coated with collagen and one that is just blocked. </li>
<li>Place the interface on the plate. </li>
<li>Place the plate on the BF1000 workstation microscope. Clamp on the interface.</li>
<li>Perform plate load adjustment. Also, move stage to one well containing blood. Set FTIC image capture parameters (exposure time, gain etc…). </li>
<li>In the BioFlux montage software, start acquisition to begin flow and acquisition simultaneously.</li>
<li>Remove the plate from BF1000 workstation, dispose of plate according to institutional guidelines.</li>
</ol>
Part 4: Representative Results.
Here the protocol for platelet adhesion and aggregation in microfluidic channels was presented. Treatment with a platelet aggregation inhibitor, anti-GPIIb/IIIa was also included in the protocol. Using collagen coated microfluidic channels of the BioFlux system, one should expect to see aggressive thrombus formation over time with the untreated control blood sample and no thrombus formation on the uncoated channel. In one recently performed experiment, the average size of aggregates under control conditions was 2000μm2. Figure 1. <img src="/files/ftp_upload/1644/1644fig1.jpg" alt="Static equilibrium; thermal imaging; heat distribution analysis; diagram; materials study." data-altupdatedat="1788304762000" data-alt="Figure 1">
Activated GPIIb/IIIa is potent mediator of platelet-platelet interactions and aggregation stabilization1. Adhesion to collagen activates the GPIIb/IIIa complex to elicit this response 2. After incubation with anti-GPIIb/IIIa for 1 hour prior to shear exposure, one should expect to observe a decrease in size of thrombi as well as a decrease in the frequency of thrombus formation. A dose dependent response can typically be observed at 10dyn/cm2. Figure 2. <img src="/files/ftp_upload/1644/1644fig2.jpg" alt="Dose-response curve; EC50 value, logarithmic scale, inhibition percentage, scientific analysis graph." data-altupdatedat="1788304762000" data-alt="Figure 2"> The IC50 value for this particular inhibitor was 17nM at 10 dyn/cm2. Maximum inhibition (for this donor) compared to the no antibody control was 11% at 10 dyn/cm2.
While the methods here were presented using a specific extracellular matrix protein and a specific inhibitor of platelet aggregation, the protocol is extensible to other coatings, other cell types and other inhibitors for cell adhesion assays. The key ingredients to success of such experiments are avoidance of bubbles in the microfluidic channels, correct dilution of all reagents in the correct diluents, and appropriate incubation conditions for all reagents.

<ol><li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+growing+complexity+of+platelet+aggregation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">The growing complexity of platelet aggregation.</a> Blood. 109, 5087-5095 (2007).</li>
<li>Nakamura, T., Kambayashi, J., Okuma, M., Tandon, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+the+GP+IIb-IIIa+complex+induced+by+platelet+adhesion+to+collagen+is+mediated+by+both+alpha2beta1+integrin+and+GP%5BTitle%5D)+AND+%22VI.+J+Biol+Chem%22%5BJournal%5D)">Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP.</a> VI. J Biol Chem. 274, 11897-11903 (1999).</li>
</ol>

The contributing authors are employees of Fluxion Biosciences.

While the methods here were presented using a specific extracellular matrix protein and a specific inhibitor of platelet aggregation, the protocol is extensible to other coatings, other cell types and other inhibitors for cell adhesion assays. The key ingredients to success of such experiments are avoidance of bubbles in the microfluidic channels, correct dilution of all reagents in the correct diluents, and appropriate incubation conditions for all reagents.

<table><tbody><tr><td>collagen I, Bovine</td><td>Reagent</td><td>Invitrogen</td><td>A1064401</td><td>other sources of collagen I can be used</td></tr><tr><td>PBS plus Ca/Mg</td><td>Reagent</td><td>Invitrogen</td><td>14040-117</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Reagent</td><td>EMD Millipore</td><td>EM-2930 Bottom of Form</td><td>From VWR</td></tr><tr><td>calcein AM</td><td>Reagent</td><td>Invitrogen</td><td>C1430</td><td>Calcein AM from BD can also be used</td></tr><tr><td>BioFlux 1000 System</td><td>Reagent</td><td>Fluxion Biosciences</td><td>Contact Company</td><td></td></tr><tr><td>BioFlux Plate</td><td>Reagent</td><td>Fluxion Biosciences</td><td>900013</td><td></td></tr><tr><td>antiGPIIb/IIIa</td><td>Reagent</td><td>Abcam</td><td>ab33407</td><td>an alternative # is ab15021</td></tr><tr><td>ReoPro</td><td>Reagent</td><td>Eli Lilly</td><td></td><td>by Rx only</td></tr></tbody></table>

platelet adhesion and aggregation under flow using microfluidic flow cells

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

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.

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Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

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