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Jun 2018
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Straight Channel Microfluidic Chips for the Study of Platelet Adhesion under Flow
直通道微流控芯片用于研究流动中的血小板黏附    

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Abstract

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.

Keywords: Microfluidics, Biochip, Platelet, Thrombosis, Biorheology

Background

Platelets primary function is to maintain blood in circulation by sealing off any breach of vessel integrity which would otherwise lead to exsanguination. For this function, platelets have evolved specialized cell surface receptors to allow adhesion to the endothelium and subendothelium at varying shear rates, ranging from 200 s-1 in venous circulation to > 1 x 104 s-1 in stenotic arteries (Chatzizisis et al., 2008). Shear stress is implicated in the development of atherosclerotic lesions (Chatzizisis et al., 2008). Shear rate and stress impact protein expression (Morigi et al., 1995) and function (Yago et al., 2004; Ju et al., 2013 and 2015). Hence, the effect of flow on cell function must be included in vascular research.

Flow chambers, introduced in 1973 by Baumgartner (Baumgartner, 1973), incorporate the parameters of shear rate, which is the velocity gradient throughout a moving fluid, and shear stress, which is the force experienced by the wall of a conductance vessel due to the friction force of a moving fluid. The application of microfluidics has enabled significant discoveries in platelet biology. For example, by perfusing blood under controlled shear rates over adhesive proteins, it was found that platelets utilize their receptor glycoprotein Ib alpha (GPIbα) to adhere to von Willebrand factor (vWF), whereas, they use their receptor integrin alpha IIb beta 3 (αIIbβ3) to adhere to fibrinogen (Ruggeri, 2009; Ju et al., 2018; Passam et al., 2018). Under low-intermediate shear rates (< 1 x 103 s-1), typically found in veins and large arteries, platelet adhesion is predominately mediated by integrin αIIbβ3. Under high shear rates (1 x 103-1 x 104 s-1), typically found in arterial microcirculation and in arterial stenosis, platelet adhesion is predominantly vWF-dependent (Reininger et al., 2006; Ruggeri et al., 2006; Jackson, 2007). Because of the effect of fluid dynamics on cell function and protein expression, microfluidics is a powerful tool to study cells in circulation.

Commercial microfluidic devices have been developed. The rheology of biological fluids passing through these devices has been well characterized (Lane et al., 2012). However, these devices are limited by high cost and inflexible geometry designs. Recent studies in the field have utilized polydimethylsiloxane (PDMS) elastomers to fabricate microfluidic flow chambers, or ‘biochips’, with typical channel geometries of 10-1,000 μm in x and y-axes and 10-200 μm in the z-axis. PDMS is a transparent and elastic material which has been widely used to create microfluidic channels with various geometries such as stenosis (Nesbitt et al., 2009; Tovar-Lopez et al., 2013), bifurcation (Tsai et al., 2012), aneurysm (Mannino et al., 2015), and spiral channels for two-dimensional cell sorting (Hou et al., 2016). 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.

Using microfluidics, we have recently shown that a member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5) regulates platelet adhesion to fibrinogen in a shear-dependent manner (Passam et al., 2018). The current protocol describes the fabrication and assembly of an in-house straight-channel biochip. The protocol also describes the application of straight-channel commercial and in-house chips for the study of platelet adhesion under controlled shear conditions.

Materials and Reagents

  1. 2 ml glass Luer lock syringe (Tömopal, catalog number: 140-1502)
  2. 24 mm x 50 mm x 0.17 mm #1 borosilicate rectangular coverslips (Thermo Fisher, catalog number: MENCS24501GP)
  3. 150 mm x 20 mm Petri dish (SARSTEDT, catalog number: 82.1184.500)
  4. Medical grade Tygon tubing, 0.8 mm ID and 1.6 mm OD (Watson-Marlow)
  5. 20 ml Syringe Luer Lok Tip (BD, catalog number: 302830)
  6. Silicon wafer, P-type boron dope, 4 inches (Research and Prototype Foundry, University of Sydney Nano Institute)
  7. 365 nm UV lamp (Research and Prototype Foundry, University of Sydney Nano Institute)
  8. 1.5 ml transfer pipettes (Thermo Fisher, catalog number: 282TS)
  9. ConnectaTM 3-way stopcock, 2 female 1 rotating male Luer lock connector (BD, catalog number: 394995)
  10. Venous blood collection set (BD, Vacutainer Safety-Lok Blood Collection Set with Pre-Attached Holder, 21 G, catalog number: 368654) 
  11. Scotch tape
  12. Whole Blood Tube w/ Acid Citrate Dextrose (ACD) Sol A (BD, catalog number: 364606)
  13. Acid Citrate Dextrose (store at 4 °C, shelf-life: 6 months) (Sigma-Aldrich, catalog number: C3821-50ML)
  14. Bovine serum albumin (store dessicated albumin at 4 °C, shelf-life: 6 months) (Sigma-Aldrich, catalog number: A8531-1VL)
  15. Butan-1-ol (Ajax Finechem, catalog number: AJA107-2.5LGL)
  16. Calcein AM (store at -20 °C, shelf-life: 6 months) (Thermo Fisher, catalog number: C1430)
  17. Rat anti-mouse glycoprotein Ib antibody conjugated to Dylight fluor 488 (Emfret Analytics, catalog number: X488)
  18. DMSO, store in the dark at room temperature (Life Technologies, catalog number: D12345)
  19. Extran® MA 02, store at room temperature (Merck, catalog number: 1075532500)
  20. Fibrinogen, 4 mg/ml (store at -80 °C, shelf-life: 12 months) (Haematologic Technologies, catalog number: HCI-0150R)
  21. Von Willebrand Factor (vWF), 0.3 mg/ml (store at -80 °C, shelf-life: 12 months) (Haematologic Technologies, catalog number: HCVWF-0190)
  22. Photoresist SU-8 2000 (MicroChem, catalog number: SU-8 2050)
  23. Photoresist Developer Microposit Thinner Type P (Rohm and Haas)
  24. Isopropanol (IPA) (Sigma-Aldrich, catalog number: W292907)
  25. Edge bead remover (MicroChem, EBR PG)
  26. ddH2O
  27. Sylgard(R) 184 Silicone Elastomer, store at room temperature (polydimethyl siloxane; PDMS) (Dow Corning, catalog number: 1317318)
  28. Prostaglandin E1 (PGE1), 5 mg/ml (14 mM) in 100% ethanol (store at -80 °C, shelf-life: 6 months) (Sigma-Aldrich, catalog number: P5515-1MG)
  29. HEPES (Sigma-Aldrich, catalog number: H3375-100G)
  30. NaCl (Sigma-Aldrich, catalog number: S7653-250G)
  31. KCl (Sigma-Aldrich, catalog number: P933-500G)
  32. Na2HPO4 (Sigma-Aldrich, catalog number: RES20908-A702X)
  33. KH2PO4 (Sigma-Aldrich, catalog number: P0662-500G)
  34. NaHCO3 (Sigma-Aldrich, catalog number: S5761-500G)
  35. MgCl2 (Sigma-Aldrich, catalog number: M8266-100G)
  36. CaCl2 (Sigma-Aldrich, catalog number: C5670-100G)
  37. D-glucose (Sigma-Aldrich, catalog number: G8270-100G)
  38. 1x PBS (Phosphate Buffered Saline) (store at room temperature, shelf-life: 6 months) (see Recipes)
  39. 10% Extran® (store at room temperature, shelf-life: 1 year) (see Recipes)
  40. Blocking buffer (store at 4 °C, shelf-life: 2 weeks) (see Recipes)
  41. HEPES-Tyrode’s buffer with glucose, store HEPES-Tyrode’s without glucose at room temperature for 6 months, add glucose fresh each time (see Recipes)

Equipment

  1. For in-house chips
    1. Centrifuge (Eppendorf, catalog number: 5810000017)
    2. Heidelberg tabletop maskless aligner (Heidelberg Instruments, catalog number: MLA100)
    3. Pipettes (Eppendorf, catalog number: 3120000917)
    4. 1 and 6 mm Harris Uni-Core Biopsy punch (World Precision Instruments, catalog numbers: 501907, 501910)
    5. Water bath (Ratek instruments, catalog number: WB20)
    6. Nalgene® vacuum desiccator (Sigma-Aldrich, catalog number: D2797-1EA)
    7. Heratherm Gravity convection oven (Thermo Fisher, catalog number: 51028112)
    8. Hotplate (SAWATEC, catalog number: HP-150)
      Note: All hot plates used for this process are an integral part of our Ritetrack Equipment, which also contains the coater/developer modules used for this project.
    9. PHD ULTRATM Programmable Infuse/Withdraw syringe pump (Harvard Apparatus, catalog number: 70-3007)
    10. Sonicator (Thermoline, catalog number: UB-405)
    11. Tabletop centrifuge (Thermo Fisher, catalog number: 75002415)
    12. Olympus fluorescent microscope IX81 60x oil immersion objective NA 1.35

  2. For commercial chips
    1. Inlet and Outlet pins (Cellix, catalog number: SS-P-B1IC-B1OC-PACK200)
    2. Tygon tubing for BiochipConnect (Cellix, catalog number: TUBING-TYGON-BIC-B1OCROLL100FT)
    3. Vena8 Fluoro+TM biochips (Cellix, catalog number: V8CF-400-100-02P10)
    4. Mirus Evo Nanopump (Cellix)
    5. AxioObserver A1 Inverted Epi-Fluorescence microscope (Zeiss, Germany)
    6. ExiBlu CCD camera (Q imaging, Canada)

  1. For platelet separation
    1. Sysmex KX21 Hematology Analyzer (Sysmex America, Inc. Lincolnshire, Illinois, USA)

Software

  1. For in-house chips
    1. Fiji/ImageJ 1.52h (https://fiji.sc/)
    2. GraphPad Prism (GraphPad software, https://www.graphpad.com/scientific-software/prism/)

  2. For commercial chips
    1. VenaFlux 2.3 imaging software
    2. ImagePro Premier 64-bit software image analysis 
    3. GraphPad Prism (GraphPad software, https://www.graphpad.com/scientific-software/prism/)
    Note: The VenaFlux 2.3 imaging software and ImagePro Premier 64-bit image analysis software are pre-installed in the PC of the Venaflux platform (Cellix Ltd., Unit 1, Longmile Business Park, Longmile Road, Dublin 12, Ireland, info@wearecellix.com).

Procedure

  1. 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.

    1. Dehydrate a 4 in. silicon wafer at 200 °C for 20 min, then apply an adhesion promoter for 30 s at 120 °C.
    2. Spin coat the wafer with SU-8 2050 (high contrast, epoxy-based) photoresist using a spread cycle of 70 and 22 x g for 30 s and a development cycle of 1,000 and 0.5 x g for 30 s in order to achieve a desired film thickness in the z-axis (e.g., 50 μm in this protocol).
    3. Conduct a cycle of edge bead removal for 30 s using edge bead removal solvent.
    4. Directly write the pattern to the SU-8 film using a dose of 365 nm UV light at 100 mJ/cm2.
    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.
    7. Develop unexposed SU-8 photoresist using fresh developer solution for 3.5 min in a rocker.
    8. Rinse the wafer with IPA. 
    9. Dry the wafer using pressurized nitrogen. 
    10. Hard-bake the wafer at 150 °C for 30 min.
      Note: Alternatively, follow the Permanent Epoxy Negative Photoresist Processing Guidline from MicroChem.

    Note: The following Steps (A11-A17) can be performed in the research lab (these steps do not require a clean room). (Figure 1)
    1. Transfer the silicon wafer into a 150 mm Petri dish, SU-8 side facing upwards, and secure with scotch tape.
    2. Mix the Sylgard 184 kit PDMS base with the kit curing agent at a ratio of 10:1 by weight. For example, to prepare 198 g of PDMS, mix 180 g of the PDMS base with 18 g of the curing agent.
      Notes:
      1. Due to the viscosity of the base and curing agent, it is easier to prepare the kit by weight. It is important that the base and curing agent are mixed completely to prevent inconsistencies in the curing of the final product. Mix thoroughly until no visible streaks can be seen. This process will create a large amount of small bubbles that need to be removed by degassing.
      2. 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.
    3. Pour the PDMS mixture over the pre-made mold, creating a 4-5 mm thick film.
    4. 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.
    5. Bake (cure) the PDMS mixture and mold in the oven at 80 °C for 4 h.
    6. Cut out the cured PDMS chips from the mold gently and carefully.
      Notes:
      1. For the design used in this protocol, the biochip can be cut from the mold and the remaining PDMS can be left on the mold to reduce the amount of PDMS required for fabrication in the future.
      2. The typical PDMS cut-out block has a length of 4 cm, width of 2 cm and height of 5 mm. Avoid larger PDMS cut-outs as these can bend the coverslip and obscure the plane of focus during image acquisition.
      3. Take care not to damage or crack the mold whilst cutting out the biochip. Gradually cut out the PDMS by making consecutive, linear cuts into the PDMS and gently lift the PDMS off from the silicon wafer.
    7. With the channel side facing upward, use a 6 mm diameter biopsy punch to cut out a hole at one end of the channel. Punch another hole at the opposite end of the channel using a 1 mm diameter biopsy punch.
      Note: The 6 mm hole is used as a well to hold the sample for the microfluidics experiment once the biochip is placed on a coverslip. The 1 mm hole is used to connect the channel to the syringe pump.


    Figure 1. Fabrication of PDMS biochips. The PDMS biochip produced in this protocol features a 6 mm diameter inlet (well) which can hold up to 100 μl of sample, and a 1 mm diameter outlet which connects the channel to the pump. The channel has a length of 12 mm, width of 0.4 mm and a height of 0.05 mm (A, B, and C). The manufactured silicon wafer mask contains indentations which form the shape of the channel (D).

  2. Cleaning the biochip
    Dust and other impurities can affect the coating of coverslips with fibrinogen and obstruct the field of view under the microscope. Dust may also interfere with the adhesion of PDMS to the coverslip and the adhesion of platelets to the coated coverslip. The following is a gentle cleaning procedure to remove dust and impurities from the surface of PDMS and coverslips.
    1. Place the biochip in 400 ml of 10% extran and sonicate for 15 min.
    2. 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.
    3. Place the biochip in 100% Butan-1-ol and sonicate for 30 min.
      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.
    5. Blow dry the device using compressed air. Place the biochip in dH2O and sonicate for 10 min.
    6. Blow dry the device using compressed air.
    7. With the channel side facing down, place the biochip on a #1 coverslip. Press the PDMS down on the coverslip with the palm of your hand and smooth out any bubbles with your finger.
      Notes:
      1. A good seal needs to be made between the PDMS and the coverslip to prevent leakage of cells from the sides of the channel.
      2. The coverslip should be cleaned to remove any dust and smudges that may interfere with contact between the PDMS and the coverslip. The coverslip can be cleaned using Steps B1 and B5.
      3. The PDMS can be reused after the microfluidics experiment. Clean the PDMS again by following Procedure B. Store the PDMS in a sealed container away from light. The PDMS can be reused until it is permanently bent and does not stick properly to a coverslip. In our experience, the PDMS can be reused at least 10 times.
      4. The size of the coverslip used depends on the size of the biochip. Choose a coverslip size that is closest to the area of the biochip.

  3. Coating the channel of the biochip
    The steps involved in the coating of the biochip (Section C) can be found in Video 1.
    1. Place 100 μl of fibrinogen (40 μg/ml final fibrinogen concentration in PBS, pH 7.4) in the 6 mm inlet of the biochip.
    2. Using a 1.5 ml transfer pipette, draw the fibrinogen through the channel and into the pipette.
      Notes:
      1. A good seal between the transfer pipette and the 1 mm outlet must be made, otherwise the fibrinogen will not transverse the channel properly. Bubbles in the pipette indicate that good suction was not achieved.
      2. Do not drain the inlet or the channel completely. Draw up enough of the fibrinogen solution until you are certain that the channel has been filled with the fibrinogen solution. To remove the pipette from the outlet, apply positive pressure on the transfer pipette until the fluid stops drawing into the pipette, then remove the pipette.
      3. To check that the channel has been filled with the coating substrate (fibrinogen), rotate the biochip back and forth under light. Due to the refractive index mismatch between air and PDMS, an empty channel will be slightly less transparent than a channel filled with fluid, leading to a shimmering of the channel under light.
    3. Incubate the biochip for 2 h at 25 °C.
      Note: The incubation period and temperature will depend on the coating substrate used to coat the channel. However, the longer the channel is left incubating, the greater the risk of the channel leaking.
    4. 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.

      Video 1. Coating of the in-house PDMS biochip

  4. Isolation of platelets from whole blood
    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 105/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 Ethics and local protocols.
    1. Draw 1 ml of blood by cardiac puncture after CO2 euthanization or by venipuncture of the inferior vena cava under anesthesia.
    2. Centrifuge the blood on a tabletop centrifuge at 400 x g for 5 min.
    3. Collect the platelet-rich plasma (supernatant) and 1/8 of the upper red blood cell layer.
    4. Add 200 µl of HEPES-Tyrodes’ buffer with glucose to the red blood cell layer and gently mix. Centrifuge the red blood cell layer at 400 x g for 5 min.
    5. Collect the supernatant and 1/8 of the upper red blood cell layer and pool with the platelet-rich plasma from Step 3.
    6. Centrifuge the pooled platelet-rich plasma at 200 x g for 6 min.
    7. Collect the platelet-rich plasma (supernatant) without disturbing the red blood cell layer.
    8. Add 200 µl of HEPES-Tyrodes’ buffer with glucose to the red blood cell layer and mix. Centrifuge the red blood cell layer at 200 x g for 6 min.
    9. Collect the supernatant without disturbing the red blood cell layer and pool with the platelet-rich plasma from Step 7.
    10. Allow the platelet-rich plasma to rest for 10 min at room temperature. At the end of the incubation, add PGE1 (0.5 µM final concentration) and centrifuge the plasma at 500 x g for 10 min.
    11. Discard the platelet-poor plasma (supernatant) and resuspend the pellet in HEPES-Tyrode’s buffer with glucose. Perform a platelet count on the hematology analyzer and adjust the platelet concentration to 300 x 103/µl.
    12. Prior to performing a microfluidics assay, incubate the platelets with anti-GPIb 488 antibody (3 µg/ml final concentration) for 10 min at room temperature, in the dark.
    Notes:
    For both human and mouse platelets:
    1. All centrifugation steps are at room temperature.
    2. Platelet suspensions cannot be stored at 4 °C at any step. 
    3. Platelets should be used within 4 h of collection by venipuncture.
    4. Perfusion assays are performed in the dark.

  5. Image acquisition
    1. Assemble the syringe pump as shown below (Figure 2). Connect the 2 ml Luer lock glass syringe to the horizontal facing female Luer lock on the 3-way connector. Slip the silicone tubing onto the male Luer lock and screw the rotating lock over the tubing. Connect a water-filled 20 ml plastic Luer lock syringe to the final female Luer lock and place the glass syringe on the PHD syringe pump. Secure the glass syringe in place.


      Figure 2. Setup of the syringe pump. A. Assembly of the 2 ml glass syringe, plastic 20 ml syringe filled with water (to flush the tubing after each experiment) and tubing (with a 1 mm diameter metal adapter) on a 3-way stopcock connector. B. Assembly of glass syringe on the pump. C. Rotation of the stopcock to enable flow from tubing to glass syringe. D. Schematic of the microfluidic chip set-up. Dashed arrows indicate the direction of flow. The yellow square in the chip represents the platelet suspension.

    2. Bend a 30 mm length piece of 1 mm diameter steel tubing in half at a 90° angle and insert the other end into medical grade Tygon tubing. Insert the metal tubing into the 1 mm hole outlet of the biochip (Figure 3). Secure the biochip onto the stage of the microscope and bring the plane of the coverslip into focus (Figure 4).
      Note: The steps involved in the assembly of the biochip to the microscope (Figures 2-3) can be found in Video 2.

      Video 2. Assembly of the in-house PDMS biochip onto a microscope stage


      Figure 3. Final assembly of the microfluidics system. A. The 1 mm diameter metal tubing is connected to the 1 mm hole of the PDMS biochip. B. The system is then assembled to an inverted microscope.


      Figure 4. Planes of focus. Imaging of the biochip placed under an Olympus fluorescent microscope using a 60x objective. The plane of focus is displayed below the coverslip (A), above the coverslip (B) and on the coverslip (C). When the coverslip is in focus, the jagged edge of the PDMS (an artifact of the fabrication process) can be seen within the channel.

    3. Empty the inlet well and start the acquisition. Acquire a time-stack for both the DIC channel and fluorescent channel (excitation: 488 nm, emission: 520 nm) for ve+ stained platelets (Figure 5), setting the acquisition parameters to take images at regular time intervals.
    4. Place 100 μl of 3 x 105μl washed platelets in the inlet well of the biochip.
    5. Start the syringe pump and draw fluid through the channel at a rate of 5 μl/min (500 s-1) for 5 min. Alternatively, to expose the platelets to other shear rate and stress levels, use the simplified formulas (1, 2) below to calculate the flow rate needed. A table has been provided as a quick reference to calculate shear rates from flow rates for the chip used in this protocol (Table 1).



      where,
      Q = flow rate (μl/min)
      W = width of the channel (cm)
      h = height of the channel (cm)
      γ ̇ = desired shear rate (s-1)



      where,
      W = width of the channel (cm)
      h = height of the channel (cm)
      μ = viscosity (Pa∙s)
      τ = Wall shear stress (Pa)

      At 37 °C
      μ (Washed platelets in HEPES-Tyrode’s buffer with glucose) = 0.71 x 10-3 Pa∙s
      μ (Whole blood) = 3.8 x 10-3 Pa∙s = 0.038 poise
      μ (Water) = 0.695 x 10-3 Pa∙s

      1 centipoise = 1 mPa∙s = 0.001 Pa∙s 1 Pa = 10 dyn/cm2 = 1 Newton/m2



      Table 1. The flow rate of the platelet suspension required to achieve the desired shear rate and shear stress in the straight channel PDMS chip



      Figure 5. Adhesion of mouse platelets on fibrinogen under flow. Perfusion of platelets, labeled with anti-GPIb-488 antibody, on PDMS channels coated with fibrinogen (40 μg/ml) at a shear rate of 500 s-1. Time course of adherent platelets as captured by epi-fluorescence (top row) and DIC (bottom row) microscopy at 0, 6, and 20 min. Scale bars = 20 µm.

  6. Platelet adhesion using Vena8 Fluoro+ biochips
    Vena8 Fluoro+ biochips are commercial biochips manufactured by CellixTM. They are 8 channel biochips with a coverslip of 0.17 mm in width. The dimension of each channel is 0.1 mm in height, 0.4 mm in width and 25 mm length. Each channel on the biochip has a capacity of 8 microliters.
    1. Aspirate 10 μl fibrinogen (40 μg/ml in PBS pH 7.4) or vWF (100 μg/ml in PBS, pH 7.4) using a 20 μl pipette.
    2. Place the tip of the pipette into one opening of the channel of a Vena8 Fluoro+ biochip (Figure 6). Depress the plunger of the pipette to fill the channel with the coating substrate.


      Figure 6. Coating of Vena8 Fluoro+ channels. A. A 10 μl pipette is placed into the opening of a channel and the channel was filled with the coating substrate. Once filled, the channel will be transparent. B. The first 4 channels of a biochip (marked green) are filled with coating substrate. The other 4 channels are empty, appearing grey.

    3. Place the biochip in a container with a tissue moistened with dH2O and close the container. Incubate the biochip overnight at 4 °C.
    4. Remove the coating substrate from the reservoir.
    5. Aspirate 10 μl of blocking buffer with a 10 μl pipette and fill the channel with the blocking buffer. Incubate the channel for 1 h at room temperature.
    6. Aspirate 10 μl of PBS with a 10 μl pipette and fill the channel to wash. Repeat Step F6 once.
    7. Connect the tubing to the biochip using the outlet pins.
    8. Submerge the tubing of one end of the channel in an Eppendorf tube filled with the washed platelets (Figure 7). Connect the tubing of the other end of the channel to the Mirus Evo Nanopump. Initiate the pump to draw the platelet suspension through the channel of the biochips. A table for the flow rates required to achieve the desired shear rate can be found at Cellix website under the technical specification. Alternatively, use the dimensions of the channel and the Formulas 1 and 2 in Procedure E to calculate the flow rate.


      Figure 7. Setup of the Vena8 Fluoro+ biochip. An Eppendorf tube is used as a reservoir to hold the washed platelets. The other end of the channel is connected to tubing, which is attached to the Mirus Evo Nanopump.

  7. Image acquisition
    1. Assemble the syringe pump as shown below (Figure 8A): connect the inlet pin (1) (which is submerged in the Eppendorf containing platelet solution or whole blood) to the entry on one of the 8 channels on the chip. Connect the outlet pin (2) to the outlet of the channel and to the tubing (which is connected to the pump).

        
      Figure 8. Assembly of the Cellix microfluidics system. A. Insertion of the Vena8 Fluoro+ biochip into the temperature-controlled stage holder. The inlet pin (1) is connected to the entry of one of the 8 channels on the chip. The outlet pin (2) is connected to the outlet of the channel and to the tubing leading to the pump. B. Setup of the Cellix microfluidics system. (1) Hard drive; (2) Lamp; (3) Motor control; (4) Screen display; (5) Stage control; (6) ExiBlu CCD camera; (7) Epifluorescence microsocope; (8) Temperature control; (9) Mirus Evo Nanopump.

    2. Switch the “Power on” button of the PC Hardrive (1), lamp (2), motor control (3), screen display (4), stage control (5), ExiBlu CCD camera (6), Epifluorescence microsocope (7), temperature control (8), Mirus Evo Nanopump (9) (Figure 8B). Initiate the VenaFlux Assayx64 software installed on the PC. This will bring up the Venoflux interface. 
    3. Click on “new protocol’ or “open protocol” (if you have already set your parameters). Both options will bring up the same screen page (Figure 9A) with a list of commands. Each command is activated by right click of the mouse, which should be followed in the order displayed. The sequence of commands is: 1. VenFlux Setup, 2. Initialize VenFlux platform, 3. Start Video camera preview, 4. Geometry set up, 5. Update geometry, 6. Washout pump, 7. Washout cable, 8. Wash/connect biochip, 9. Washout chip, 10. Cell assay (Figure 9A). After the video camera preview has been initiated the channel will be displayed on the screen (Figure 9B).
      Notes:
      1. For steps “washout pump” and “washout cable”, the outlet pin is removed. The pump flows the connected pump solution (water or PBS) into the cable whose free end should be placed into a waste collector.
      2. For step “wash/connect biochip”, the outlet pin is connected to the cable but not to the outlet channel of the biochip. The needle of the outlet pin is held above the outlet position of the channel. When you right-click “wash/connect biochip”, the pump will deliver a squirt of 100 μl pump solution into the outlet well to avoid air entering into the channel when the needle is inserted into the outlet. This step can be omitted if you have already included some fluid in the well by pipetting.
      3. For the step “washout chip”, the outlet pin has to be inserted into the outlet of the channel. The default setting is to deliver 40 μl of pump fluid at 1 μl/sec to wash out the channel.
      4. For the step “cell assay”, enter the parameters for flow by adjusting the shear units, the acquisition time and capture delay (if required). After all steps have been entered, right click on “cell assay” to initiate the pump and acquire data. To stop the assay, click on “stop assay”.
      5. To view the channel on the screen, switch the microscope connection to the camera. Switch to eye piece for fine focus of the channel.
      6. The steps involved in data acquisition (Figures 9-11) can be found in video webinars by Cellix, Ltd: Cellix Webinar: VenaFlux Platform Technical Presentation;
        Cellix Webinar: Cellix Biochips with standard Syringe Pumps for Perfusion Assays.


      Figure 9. Initiating the VenaFlux Protocol. A. On the “Start with VenaFlux page” each command is activated by right click which should be followed in the order displayed. B. After the video camera preview has been initiated the channel will be displayed on the screen. The 2 horizontal black lines are the borders of the channel (20x magnification).

    4. The Vena8 Fluoro+ biochip has embedded markings to assist with the mapping of the biochip to the VenaFlux assay x 64 software. Each channel 1-8 has marked positions above channels 2, 3…6 which can be visualized under the microscope with 2, 3…6 dots respectively. On the dialogue screen of the VenaFlux assay x 64, at the top of the screen, click the option “Set up Venaflux”. This will bring another dialogue box displaying the x, y coordinates named “Set Vena8/VenaEC origin and channel/position spacing”. Click on Vena8 for biochip type. Using the joystick move the stage to focus on channel 1, position 2 (Figure 10A). Click “update position” on the left-top position. Using the joystick move the stage to channel 8, position 6 (Figure 10B). Click “update position” on the right-bottom position. Then press set. Coming back to the “Start with VenaFlux” page, you can click on the map of the biochip displayed at the right upper corner of the screen and this will automatically move the stage to the position.
      Note: There are no dots on the biochip for positions 1 and 8. The mapping of the Vena8 chip is based on positions 2 and 6, therefore the range of acquisition is smaller than the biochip’s dimensions.


      Figure 10. Mapping the Vena8 biochip. A. On the “Setup VenaFlux” page, move the stage to focus on channel 1, position 2 and click “update position” on the left-top position. B. Move the stage to channel 8, position 6. Click “update position” on the right-bottom position. Then press set.

    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”. To acquire an image, click on “acquire image to file” on the “Setup VenaFlux page”. This will 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.


      Figure 11. Initiating the VenaFlux Assay. A. Under “Cell assay” (circled in yellow), right click on the “variable flow rate dispensing” option and adjust the flow parameters on the “step properties view” icon (circled in brown). B. Click on “acquire image” (circled in red in panel A) to capture the image as bitmap file. Displayed is an example of an image captured of human platelets labeled with calcein 1 μg/ml (white dots) and perfused on a Vena8 channel coated with vWF 100 μg/ml. 

Data analysis

  1. 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.

    Video 3. ImageJ analysis of platelet adhesion by counting

    1. Open Fiji/ImageJ version 1.52h. 
    2. Click on the “Multi-point” tool on the tool bar. Double click on the “Multi-point” tool and on the “Point tool” window, check the “Label points” and “Show on all slices” checkbox (Figure 12).


      Figure 12. ImageJ multi-point tool. A. The multi-point tool is located on the toolbar of the ImageJ window (circled in red). B. Double-clicking the multi-point tool will open the point tool window. This window can be used to count placed markers and to adjust the display options of markers placed on the image.

    3. Click on every platelet in the field of view to count the number of adhered platelets (Figure 13). Verify the cell type by ensuring that the platelets are ve+ stained on the 488 nm channel.


      Figure 13. Counting platelets using the ImageJ multi-point tool. A. Platelets are counted on the DIC channel. B. The platelets are verified using the fluorescence channel to check for anti-GPIb-488 staining. Clicking on an image, with the multi-point tool selected, will leave a marker with a number on the image. Holding “alt” and then clicking on a marker will remove the marker. Each subsequent click or removal will increase or decrease the number on the marker by one respectively. Use this feature to count the number of platelets on the image. Alternatively, after clicking on each platelet on the image, use the counter on the point tool window (Figure 12) to determine the number of platelets counted.

    4. Enter the platelet count onto GraphPad prism or Microsoft Excel.
    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.
    6. Plot the platelet count against time (Figure 14).


      Figure 14. Number of platelets adhered to a fibrinogen channel over time. Wild type C57BL/6 mouse platelets were washed and flowed across PDMS biochips coated with fibrinogen 40 μg/ml at a shear rate of 500 s-1. The platelet count in the field of view was determined every 3 min. The total number of adherent platelets was plotted against time using GraphPad prism. Dots represent the mean platelet count at the specific time point and the error bars represent the standard error of mean from n = 3 experiments.

    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.

    Video 4. ImageJ analysis of platelet adhesion by fluorescence intensity

    1. Open Fiji/ImageJ.
    2. Click the “Analyze” tab and select “Set Measurements” (Figure 15).


      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).

    3. Check the “Area”, “Mean gray value”, “Integrated density”, and “Median” checkboxes (Figure 16).
      Note: Each of these measurements provides a description of the platelets. For single platelets, “Area” describes the area taken up by the region of interest drawn around platelets. “Mean gray value” and “Median” are both methods of quantifying the mean fluorescence intensity of a single platelet or platelet aggregate. “Integrated density” quantifies the total intensity for a selected area.


      Figure 16. Analysis parameters used for measuring platelet fluorescence intensity. Various parameters can be selected for measuring fluorescence in a region of interest. For the analysis of platelet fluorescence intensity in this protocol, we have selected “Area”, “Standard deviation”, “Mean gray value”, and “Integrated density”.

    4. Click the “Analyze” tab, click “Tools”, and select “ROI manager…” (Figure 17).


      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).

        
      Figure 18. Freehand selections tool on ImageJ. The freehand selection tool (circled in red) can be used to trace a region of interest around a platelet or platelet aggregate. The region of interest can then be stored on the ROI manager, and the parameters, set in Step B3, can be measured.

    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).

    7. Click and drag the region of interest to an area without platelets near the original position (Figure 20) and click “Add” on the ROI manager window. This measures the background fluorescence.


      Figure 20. “Background” region of interest. The region of interest drawn in Step B6 can be used for selecting a new region of interest to subtract background fluorescence intensity measurements. Click and drag the region of interest away from the platelet/platelet aggregate and add the new area to the ROI manager.

    8. Select both regions on the ROI manager window and click “Measure” (Figure 21).


      Figure 21. Measurement of regions of interests. After the desired regions of interest are selected (highlighted in blue), and “Measure” is clicked (circled in red), a new window will open, showing the measurements of the parameters selected in Step B3.

    9. Subtract the Mean/Median/RawIntDen of the background fluorescence measurement from the corresponding platelet measurement. Enter the data onto GraphPad prism or Microsoft Excel.
    10. Repeat steps 5-9 for each platelet and platelet aggregate in the frame, sum the fluorescence, and record the value. Repeat for all frames in the image stack.
    11. Plot the fluorescence intensity over time on GraphPad prism or Microsoft Excel (Figure 22).


      Figure 22. Fluorescence of platelets adhered to a fibrinogen channel over time. Wild type C57BL/6 mouse platelets were washed and flowed across PDMS biochips coated with fibrinogen 40 μg/ml at a shear rate of 500 s-1. The sum of platelet fluorescence in the field of view was determined every 3 min. The sum of platelet fluorescence was plotted against time using GraphPad prism. Dots represent the mean sum of fluorescence at the specific time point and the error bars represent the standard error of mean from n = 3 experiments.

    Statistical analysis of the change in platelet adhesion over time
    1. Open GraphPad Prism.
    2. Select the “grouped” tab.
    3. Check the “enter _____ replicate values in side-by-side columns”, entering the maximum number of replicates performed in the experiment.
    4. In the column titles, enter the time points that are to be analyzed. Make sure that the time between each point is equal.
    5. Enter the experimental data under the corresponding time points on the first row.
    6. Click “Analyze” and under the “Column analyses” drop down menu, select “One-way ANOVA (and non-parametric)”.
    7. Choose analysis parameters based on the desired analysis:
      1. For data that have a Gaussian distribution, under the “Assume Gaussian distribution?” section, click “Yes, use ANOVA” option (Notes 1 and 2).
      2. For data that do not have a Gaussian distribution, e.g., data that are skewed in distribution, under the “Assume Gaussian distribution?” section, click “No, use nonparametric test” (Notes 1 and 2).
    8. Select “Ok”, and under the “Results” drop-down menu to the left of the page, select the analysis for the data and check the P-value. A P-value < 0.05 indicates statistical significance.

    Statistical analysis of treatment effect on platelet adhesion at a specific time point
    1. Open GraphPad Prism.
    2. Select the “grouped” tab.
    3. Check the “enter _____ replicate values in side-by-side columns”, entering the maximum number of replicates performed in the experiment.
    4. In the column titles, enter the titles of the treatments that are to be analyzed.
    5. Enter the data of the treatments at a specific time point in the first row.
    6. Click “Analyze” and under the “Column analyses” drop-down menu, select “t-test (and nonparametric tests)”.
    7. Choose the analysis parameters based on the desired analysis:
      1. For data that have a Gaussian distribution, under the “Assume Gaussian distribution?” section, click “Yes, use parametric test” option (Notes 1 and 2).
      2. For data that do not have a Gaussian distribution, e.g., data that are skewed in distribution, under the “Assume Gaussian distribution?” section, click “No, use nonparametric test” (Notes 1 and 2).

  2. Commercial biochips
    Images are captured using the accompanying VenaFlux 2.3 imaging software and videos are captured at a rate of 1 frame per 5 s (30 frames). Images are analyzed at positions 2, 4 and 6 (located at 6, 14 and 22 mm from the entry site of the platelet suspension) of the channels. These positions are representative of flow nearest, mid-way and furthest from the entry of the platelet suspension into the channel. The platelet count and sum of platelet fluorescence are measured at 3-minute intervals using Image J or the ImagePro Premier 64-bit software http://www.mediacy.com/imagepro. Data are exported into Excel or GraphPad Prism for statistical analysis.

Recipes

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    pH 7.4
    Note: Prepare in advance and store at room temperature for up to 1 year.
  2. 10% Extran
    40 ml Extran® MA 02
    360 ml ddH2O
    pH 7.0
    Note: Prepare fresh extran 10% dilution before use and discard after use.
  3. Blocking buffer
    2% bovine serum albumin
    1x phosphate buffered saline
    pH 7.4
    Note: Prepare albumin 2% solution in advance and store in aliquots at -20 °C until use for up to 1 year. Avoid freeze-thawing aliquots more than 2 times.
  4. HEPES-Tyrode’s buffer with glucose
    20 mM HEPES
    134 mM NaCl
    0.34 mM Na2HPO4
    2.9 mM KCl
    12 mM NaHCO3
    1 mM MgCl2
    1 mM CaCl2
    5 mM D-glucose, added prior to washing platelets
    pH 7.4
    Note: Prepare the HEPES-Tyrode’s buffer in advance (without the D-glucose) and store at room temperature for up to 1 year. Add the D-Glucose to an aliquot of the HEPES-Tyrode’s buffer and use within 24 h.

Acknowledgments

We thank Ethel Ilagan, Fangyuan Zhou, Shaun P. Jackson and his lab for helpful discussion. This work was supported by the University of Sydney Cardiovascular Initiative Catalyst Grant for Precision CV Medicine (LAJ, FHP), National Heart Foundation of Australia Postdoctoral Fellowship 101285 (LAJ), CSANZ-BAYER Young Investigator Research Grants (LAJ), The Royal College of Pathologists of Australasia Kanematsu Research Award (LAJ, FHP).

Competing interests

The authors have no conflicting interests to declare.

References

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  2. Chatzizisis, Y. S., Jonas, M., Coskun, A. U., Beigel, R., Stone, B. V., Maynard, C., Gerrity, R. G., Daley, W., Rogers, C., Edelman, E. R., Feldman, C. L. and Stone, P. H. (2008). Prediction of the localization of high-risk coronary atherosclerotic plaques on the basis of low endothelial shear stress: an intravascular ultrasound and histopathology natural history study. Circulation 117(8): 993-1002.
  3. Hou, H. W., Petchakup, C., Tay, H. M., Tam, Z. Y., Dalan, R., Chew, D. E., Li, K. H. and Boehm, B. O. (2016). Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus. Sci Rep 6: 29410.
  4. Jackson, S. P. (2007). The growing complexity of platelet aggregation. Blood 109(12): 5087-5095.
  5. Ju, L., Dong, J. F., Cruz, M. A. and Zhu, C. (2013). The N-terminal flanking region of the A1 domain regulates the force-dependent binding of von Willebrand factor to platelet glycoprotein Ibα. J Biol Chem 288(45): 32289-32301.
  6. Ju, L., Chen, Y., Zhou, F., Lu, H., Cruz, M. A. and Zhu, C. (2015). Von Willebrand factor-A1 domain binds platelet glycoprotein Ibalpha in multiple states with distinctive force-dependent dissociation kinetics. Thromb Res 136(3): 606-612.
  7. Ju, L., McFadyen, J. D., Al-Daher, S., Alwis, I., Chen, Y., Tonnesen, L. L., Maiocchi, S., Coulter, B., Calkin, A. C., Felner, E. I., Cohen, N., Yuan, Y., Schoenwaelder, S. M., Cooper, M. E., Zhu, C. and Jackson, S. P. (2018). Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets. Nat Commun 9(1): 1087.
  8. Lane, W. O., Jantzen, A. E., Carlon, T. A., Jamiolkowski, R. M., Grenet, J. E., Ley, M. M., Haseltine, J. M., Galinat, L. J., Lin, F. H., Allen, J. D., Truskey, G. A. and Achneck, H. E. (2012). Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress. J Vis Exp (59) pii: 3349.
  9. Mannino, R. G., Myers, D. R., Ahn, B., Wang, Y., Margo, R., Gole, H., Lin, A. S., Guldberg, R. E., Giddens, D. P., Timmins, L. H. and Lam, W. A. (2015). Do-it-yourself in vitro vasculature that recapitulates in vivo geometries for investigating endothelial-blood cell interactions. Sci Rep 5: 12401.
  10. Morigi, M., Zoja, C., Figliuzzi, M., Foppolo, M., Micheletti, G., Bontempelli, M., Saronni, M., Remuzzi, G. and Remuzzi, A. (1995). Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells. Blood 85(7): 1696-1703.
  11. Nesbitt, W. S., Westein, E., Tovar-Lopez, F. J., Tolouei, E., Mitchell, A., Fu, J., Carberry, J., Fouras, A. and Jackson, S. P. (2009). A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat Med 15(6): 665-673.
  12. Passam, F., Chiu, J., Ju, L., Pijning, A., Jahan, Z., Mor-Cohen, R., Yeheskel, A., Kolsek, K., Tharichen, L., Aponte-Santamaria, C., Grater, F. and Hogg, P. J. (2018). Mechano-redox control of integrin de-adhesion. Elife 7: e34843.
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简介

微流体装置已经成为心血管研究的一个不可或缺的方法,因为它们能够研究生物过程中的剪切力,例如血小板功能和血栓形成。此外,微流体芯片提供了使用少量血液或纯化血小板进行血小板粘附的离体测试的益处。微流体芯片包括连接到注射泵的不同尺寸和几何形状的流动通道。泵通过通道抽取血液或血小板悬浮液,允许通过荧光显微镜成像血小板粘附和血栓形成。芯片可以由各种与血液相容的材料制成。目前的方案使用商业塑料或内部聚二甲基硅氧烷(PDMS)芯片。商用生物芯片具有标准化的优势,而内部芯片具有降低成本和设计灵活性的优势。微流体装置是研究血小板和其他细胞类型的生物流变学的有力工具,具有用于心血管疾病的诊断和监测工具的潜力。
【背景】血小板的主要功能是通过密封任何破坏血管完整性来维持血液循环,否则血管完整性将导致放血。对于这种功能,血小板已经进化出特化的细胞表面受体,允许在不同的剪切速率下粘附到内皮和内皮下,范围从静脉循环中的200 s -1 到> 1 x 10 4 s -1 在狭窄动脉中(Chatzizisis et al。,2008)。剪切应力与动脉粥样硬化病变的发展有关(Chatzizisis et al。,2008)。剪切率和应激影响蛋白表达(Morigi 等,1995)和功能(Yago et al。,2004; Ju et al。 ,2013年和2015年)。因此,流动对细胞功能的影响必须包括在血管研究中。

由Baumgartner(Baumgartner,1973)于1973年引入的流动室结合了剪切速率的参数,剪切速率是整个运动流体中的速度梯度,剪切应力是由于电导率管壁所经受的力。移动流体的摩擦力。微流体技术的应用使血小板生物学得到了重大发现。例如,通过在受控剪切速率下对粘性蛋白灌注血液,发现血小板利用其受体糖蛋白Ibα(GPIbα)粘附于血管性血友病因子(vWF),而他们使用其受体整合素αIIbβ3( α IIb β 3 )粘附纤维蛋白原(Ruggeri,2009; Ju et al。,2018; Passam et al。,2018)。在低中等剪切速率(<1×10 3 s -1 )下,通常在静脉和大动脉中发现,血小板粘附主要由整合素α介导。 IIB β<子> 3 。在高剪切速率下(1 x 10 3 -1 x 10 4 s -1 ),通常见于动脉微循环和动脉狭窄,血小板粘附主要依赖于vWF(Reininger 等人,2006; Ruggeri 等人,2006; Jackson,2007)。由于流体动力学对细胞功能和蛋白质表达的影响,微流体是研究循环细胞的有力工具。

已经开发出商业微流体装置。通过这些装置的生物流体的流变学已得到很好的表征(Lane et al。,2012)。然而,这些装置受到高成本和不灵活的几何设计的限制。该领域的最新研究利用聚二甲基硅氧烷(PDMS)弹性体来制造微流体流动室或“生物芯片”,其中x和y轴的典型通道几何形状为10-1,000μm,z轴为10-200μm。 PDMS是一种透明和弹性材料,已被广泛用于创建具有各种几何形状的微流体通道,如狭窄(Nesbitt 等。,2009; Tovar-Lopez 等。 ,2013),分叉(Tsai et al。,2012),动脉瘤(Mannino et al。,2015),以及用于二维细胞分选的螺旋通道(Hou et al。,2016)。与通常需要毫升样品体积的传统流动室相比,微流体生物芯片仅需要微升样品,使其成为小体积(例如,儿科或稀有样品和小鼠研究)。此外,该方案中描述的生物芯片是可重复使用的,使其成为微流体研究的经济和通用选择。

使用微流体,我们最近表明,巯基异构酶家族成员,内质网蛋白5(ERp5)以剪切依赖性方式调节血小板与纤维蛋白原的粘附(Passam 等,,2018)。目前的协议描述了内部直通道生物芯片的制造和组装。该方案还描述了直通道商业和内部芯片在受控剪切条件下研究血小板粘附的应用。

关键字

材料和试剂

  1. 2毫升玻璃鲁尔锁注射器(Tömopal,目录号:140-1502)
  2. 24 mm x 50 mm x 0.17 mm#1硼硅酸盐矩形盖玻片(Thermo Fisher,目录号:MENCS24501GP)
  3. 150 mm x 20 mm培养皿(SARSTEDT,目录号:82.1184.500)
  4. 医用级Tygon管,内径0.8 mm,外径1.6 mm(Watson-Marlow)
  5. 20毫升Syringe Luer Lok Tip(BD,目录号:302830)
  6. 硅片,P型硼涂料,4英寸(Research and Prototype Foundry,悉尼大学纳米研究所)
  7. 365纳米紫外灯(悉尼大学纳米研究所研究和原型铸造厂)
  8. 1.5毫升转移移液器(Thermo Fisher,目录号:282TS)
  9. Connecta TM 三通旋塞,2母1旋转公鲁尔锁连接器(BD,目录号:394995)
  10. 静脉采血装置(BD,Vacutainer Safety-Lok Blood Collection Set with Pre-Attached Holder,21 G,目录号:368654)&nbsp;
  11. 透明胶带
  12. 全酸性管用酸性柠檬酸葡萄糖(ACD)溶胶A(BD,目录号:364606)
  13. 酸性柠檬酸葡萄糖(储存在4°C,保质期:6个月)(Sigma-Aldrich,目录号:C3821-50ML)
  14. 牛血清白蛋白(在4°C储存干燥的白蛋白,保质期:6个月)(Sigma-Aldrich,目录号:A8531-1VL)
  15. Butan-1-ol(Ajax Finechem,目录号:AJA107-2.5LGL)
  16. Calcein AM(-20°C储存,保质期:6个月)(Thermo Fisher,目录号:C1430)
  17. 与Dylight fluor 488偶联的大鼠抗小鼠糖蛋白Ib抗体(Emfret Analytics,目录号:X488)
  18. DMSO,室温下避光保存(Life Technologies,目录号:D12345)
  19. Extran ® MA 02,室温下储存(默克,目录号:1075532500)
  20. 纤维蛋白原,4 mg / ml(储存在-80°C,保质期:12个月)(Haematologic Technologies,目录号:HCI-0150R)
  21. 血管性血友病因子(vWF),0.3 mg / ml(保存在-80°C,保质期:12个月)(Haematologic Technologies,目录号:HCVWF-0190)
  22. 光刻胶SU-8 2000(MicroChem,目录号:SU-8 2050)
  23. 光刻胶显影剂Microposit稀释剂P型(Rohm and Haas)
  24. 异丙醇(IPA)(Sigma-Aldrich,目录号:W292907)
  25. 边缘去毛刺(MicroChem,EBR PG)
  26. 双蒸<子> 2 0
  27. Sylgard(R)184有机硅弹性体,室温下储存(聚二甲基硅氧烷; PDMS)(Dow Corning,目录号:1317318)
  28. 前列腺素E1(PGE1),5 mg / ml(14 mM),100%乙醇(-80°C保存,保质期:6个月)(Sigma-Aldrich,目录号:P5515-1MG)
  29. HEPES(西格玛奥德里奇,目录号:H3375-100G)
  30. NaCl(Sigma-Aldrich,目录号:S7653-250G)
  31. KCl(Sigma-Aldrich,目录号:P933-500G)
  32. Na 2 HPO 4 (Sigma-Aldrich,目录号:RES20908-A702X)
  33. KH 2 PO 4 (Sigma-Aldrich,目录号:P0662-500G)
  34. NaHCO 3 (Sigma-Aldrich,目录号:S5761-500G)
  35. MgCl 2 (Sigma-Aldrich,目录号:M8266-100G)
  36. CaCl 2 (Sigma-Aldrich,目录号:C5670-100G)
  37. D-葡萄糖(Sigma-Aldrich,目录号:G8270-100G)
  38. 1x PBS(磷酸盐缓冲盐水)(室温储存,保质期:6个月)(见食谱)
  39. 10%Extran ®(在室温下储存,保质期:1年)(见食谱)
  40. 封闭缓冲液(储存在4°C,保质期:2周)(见食谱)
  41. HEPES-Tyrode的葡萄糖缓冲液,在室温下储存HEPES-Tyrode无葡萄糖6个月,每次加入新鲜葡萄糖(见食谱)

设备

  1. 对于内部芯片
    1. 离心机(Eppendorf,目录号:5810000017)
    2. 海德堡台式无掩模对准器(海德堡仪器,产品目录号:MLA100)
    3. 移液器(Eppendorf,目录号:3120000917)
    4. 1和6 mm Harris Uni-Core活检打孔器(World Precision Instruments,目录号:501907,501910)
    5. 水浴(Ratek仪器,目录号:WB20)
    6. Nalgene ®真空干燥器(Sigma-Aldrich,目录号:D2797-1EA)
    7. Heratherm重力对流烤箱(Thermo Fisher,目录号:51028112)
    8. Hotplate(SAWATEC,目录号:HP-150)
      注意:用于此过程的所有热板都是我们的Ritetrack设备的组成部分,该设备还包含用于此项目的涂布机/显影模块。
    9. PHD ULTRA TM 可编程注入/抽出注射泵(Harvard Apparatus,目录号:70-3007)
    10. 超声波仪(Thermoline,目录号:UB-405)
    11. 台式离心机(Thermo Fisher,目录号:75002415)
    12. 奥林巴斯荧光显微镜IX81 60x油浸物镜NA 1.35

  2. 用于商业芯片
    1. 进口和出口销(Cellix,目录号:SS-P-B1IC-B1OC-PACK200)
    2. 用于BiochipConnect的Tygon管(Cellix,目录号:TUBING-TYGON-BIC-B1OCROLL100FT)
    3. Vena8 Fluoro + TM 生物芯片(Cellix,目录号:V8CF-400-100-02P10)
    4. Mirus Evo Nanopump(Cellix)
    5. AxioObserver A1倒置落射荧光显微镜(Zeiss,德国)
    6. ExiBlu CCD相机(加拿大Q成像)

  1. 用于血小板分离
    1. Sysmex KX21血液分析仪(Sysmex America,Inc。Lincolnshire,Illinois,USA)

软件

  1. 对于内部芯片
    1. 斐济/ ImageJ 1.52h( https://fiji.sc/ )
    2. GraphPad Prism(GraphPad软件, https://www.graphpad.com/scientific-software/prism/ )
  2. 用于商业芯片
    1. VenaFlux 2.3成像软件
    2. ImagePro Premier 64位软件图像分析&nbsp;
    3. GraphPad Prism(GraphPad软件, https://www.graphpad.com/scientific-software/prism/ )
    注意:VenaFlux 2.3成像软件和ImagePro Premier 64位图像分析软件预装在Venaflux平台的PC中(Cellix Ltd.,Unit 1,Longmile Business Park,Longmile Road,Dublin 12,Ireland, info@wearecellix.com )。

程序

  1. PDMS生物芯片的制造
    微流体生物芯片可以用PDMS(Sylgard 184试剂盒)制造,所述PDMS通过光刻法从硅晶片上的母模铸造(Qin 等人,,2010)。大多数大学和研究所都有光刻和洁净室设施来制造母模。在该协议中,光刻在研究和原型铸造厂进行。悉尼大学纳米研究所。没有铸造厂就可以进行光刻。所需设备包括:1。空气等离子系统,2。用于PDMS固化的烘箱,3。用于PDMS脱气的干燥器,4。可编程旋涂机,5。紫外灯-LED曝光,6。可编程热板。我们引荐感兴趣的读者:微流体设备设计,制造和测试协议或 Elveflow 。

    1. 将4英寸硅晶片在200℃下脱水20分钟,然后在120℃下施加粘合促进剂30秒。
    2. 用SU-8 2050(高对比度,环氧基)光刻胶旋涂晶圆,使用70和22 xg 的展开周期30秒,显影周期为1,000和0.5 xg <为了在z轴上获得所需的膜厚度(例如,在该方案中为50μm),持续30秒。
    3. 使用边缘珠粒去除溶剂进行边缘珠粒去除循环30秒。
    4. 使用剂量为100mJ / cm 2的<0.05>的紫外光直接将图案写入SU-8膜。
    5. 通过在加热板上烘烤并以5℃/ min的速度升温来使薄膜图案交联,从23℃开始并保持在90℃以干燥溶剂。通过接近和真空接触烘烤步骤实现斜坡轮廓,总持续时间为805秒。
    6. 让薄膜在热板上冷却至室温。
      注意:将薄膜放在加热板上以避免热应力。
    7. 使用新鲜的显影剂溶液在摇臂中显影未曝光的SU-8光刻胶3.5分钟。
    8. 用IPA冲洗晶圆。&nbsp;
    9. 使用加压氮气干燥晶圆。&nbsp;
    10. 在150°C下烘烤晶圆30分钟。
      注意:或者,请按照 永久性环氧负片光刻胶加工指南 。

    注意:可以在研究实验室中执行以下步骤(A11-A17)(这些步骤不需要洁净室)。(图1)
    1. 将硅晶片转移到150毫米培养皿中,SU-8侧朝上,并用透明胶带固定。
    2. 将Sylgard 184试剂盒PDMS碱与试剂盒固化剂按重量比10:1混合。例如,为了制备198克PDMS,将180克PDMS基料与18克固化剂混合。
      注意:
      1. 由于碱和固化剂的粘度,更容易按重量制备试剂盒。重要的是将基料和固化剂完全混合以防止最终产品的固化不一致。彻底混合,直到看不到明显的条纹。此过程将产生大量需要通过脱气去除的小气泡。
      2. 一旦将固化剂添加到PDMS基料中,PDMS将在室温下缓慢开始固化和硬化。这将在4小时后显着。 16小时后,PDMS将变得过于粘稠,几乎无法使用。
    3. 将PDMS混合物倒在预制模具上,形成4-5mm厚的薄膜。
    4. 将PDMS和模具放入Nalgene ®真空干燥器中并脱气30分钟。
      注意:使混合物脱气所需的时间取决于PDMS的体积和表面积。混合物必须完全脱气,否则在固化过程中会形成气泡,这将改变装置的形态。对于置于100 mm直径培养皿中的200 g PDMS,2 h足以使混合物完全脱气。
    5. 将PDMS混合物烘烤(固化)并在80℃的烘箱中模具4小时。
    6. 轻轻地小心地从模具中切出固化的PDMS芯片。
      注意:
      1. 对于本协议中使用的设计,可以从模具中切割生物芯片,并将剩余的PDMS留在模具上,以减少将来制造所需的PDMS量。
      2. 典型的PDMS切割块长4厘米,宽2厘米,高5毫米。避免使用较大的PDMS切口,因为这些切口会在图像采集过程中弯曲盖玻片并遮挡焦点平面。
      3. 在切割生物芯片时注意不要损坏或破坏模具。通过在PDMS中连续线性切割并轻轻地将PDMS从硅晶片上提起来逐渐切断PDMS。
    7. 通道侧朝上,使用直径6 mm的活检冲头在通道的一端切出一个孔。使用1 mm直径的活检冲头在通道的另一端打孔。
      注意:一旦将生物芯片放在盖玻片上,6 mm孔用作保持微流体实验样品的孔。 1 mm孔用于将通道连接到注射泵。


    图1. PDMS生物芯片的制造本协议生产的PDMS生物芯片具有直径6 mm的入口(孔),最多可容纳100μl样品,1 mm直径出口连接通道到泵。通道的长度为12毫米,宽度为0.4毫米,高度为0.05毫米(A,B和C)。制造的硅晶片掩模包含形成通道(D)形状的凹痕。

  2. 清洁生物芯片
    灰尘和其他杂质会影响盖玻片与纤维蛋白原的涂层,并在显微镜下阻碍视野。灰尘还可能干扰PDMS与盖玻片的粘附和血小板与涂层盖玻片的粘附。以下是温和的清洁程序,以清除PDMS和盖玻片表面的灰尘和杂质。
    1. 将生物芯片放入400ml 10%extran中并超声处理15分钟。
    2. 使用压缩空气彻底吹干生物芯片。
      注意:在继续操作之前,请确保生物芯片完全干燥。来自小液滴的残余物可能损害生物芯片和盖玻片之间的接触,导致微流体实验期间的泄漏。
    3. 将生物芯片置于100%丁-1-醇中并超声处理30分钟。
      注意:在Butan-1-ol处理后,PDMS会略微膨胀并显示压痕。这是正常的,一旦在dH 2 O中超声处理,PDMS将恢复其原始形状和大小。
    4. 使用压缩空气彻底吹干生物芯片。将生物芯片置于10%extran中并超声处理10分钟。
    5. 使用压缩空气吹干设备。将生物芯片置于dH 2 O中并超声处理10分钟。
    6. 使用压缩空气吹干设备。
    7. 通道侧朝下,将生物芯片放在#1盖玻片上。用手掌按下盖玻片上的PDMS,用手指抚平任何气泡。
      注意:
      1. 需要在PDMS和盖玻片之间进行良好的密封,以防止细胞从通道侧面泄漏。
      2. 应清洁盖玻片,以清除可能干扰PDMS与盖玻片之间接触的任何灰尘和污迹。可以使用步骤B1和B5清洁盖玻片。
      3. PDMS可在微流体实验后重复使用。按照步骤B再次清洁PDMS。将PDMS存放在远离光线的密封容器中。 PDMS可以重复使用,直到它永久弯曲并且不能正确粘在盖玻片上。根据我们的经验,PDMS可以重复使用至少10次。
      4. 所使用的盖玻片的大小取决于生物芯片的大小。选择最接近生物芯片区域的盖玻片尺寸。

  3. 涂覆生物芯片的通道
    生物芯片涂层所涉及的步骤(C部分)可以在视频1中找到。
    1. 将100μl纤维蛋白原(40μg/ ml最终纤维蛋白原浓度在PBS中,pH7.4)置于生物芯片的6mm入口中。
    2. 使用1.5 ml移液管,将纤维蛋白原通过通道吸入移液管。
      注意:
      1. 必须在转移移液管和1 mm出口之间进行良好的密封,否则纤维蛋白原不会正确地横穿通道。移液器中的气泡表明没有达到良好的吸力。
      2. 不要完全排空进样口或通道。吸取足够的纤维蛋白原溶液,直到您确定通道已充满纤维蛋白原溶液。要从出口取下移液器,在移液管上施加正压,直到液体停止吸入移液管,然后取下移液器。
      3. 要检查通道是否已填充涂层基质(纤维蛋白原),请在光照下来回旋转生物芯片。由于空气和PDMS之间的折射率不匹配,空通道的透明度将略低于充满流体的通道,导致通道在光线下闪烁。
    3. 将生物芯片在25°C孵育2小时。
      注意:孵育期和温度取决于用于涂覆通道的涂层基质。但是,通道孵化的时间越长,通道泄漏的风险就越大。
    4. 去除入口孔中的纤维蛋白原,用100μlTyrode缓冲液或PBS替换,并重复步骤D2以清洗通道。
    5. 从入口孔中取出Tyrode缓冲液或PBS,并用100μl封闭缓冲液替换。重复步骤D2以阻塞具有阻塞缓冲区的通道。在25°C孵育30分钟。
    6. 从入口孔中取出阻塞缓冲液,用100μlTyrode缓冲液或PBS替换,并重复步骤D2以清洗通道。
      注意:清洗后,将Tyrode的缓冲液或PBS保留在通道中,直至获取图像。这将防止在细胞流过生物芯片时在通道中形成气泡。


      视频1.内部PDMS生物芯片的涂层

  4. 从全血中分离血小板
    人体血小板
    通过静脉穿刺抽取人体血液的程序由机构的人体血液采样伦理和协议确定。
    1. 通过静脉穿刺将8ml静脉血吸入ACD管中。&nbsp;
    2. 在200 x g 离心20分钟,无制动,并分离富含血小板的血浆(上清液)。&nbsp;
    3. 让富含血小板的血浆在37℃的水浴中静置30分钟。
    4. 在离心之前立即将PGE1添加到富含血小板的血浆(1μM终浓度)中。
    5. 在800 x g 离心20分钟,无制动,丢弃贫血小板血浆(上清液)。
    6. 用葡萄糖将血小板沉淀重悬于HEPES-Tyrodes缓冲液中。对血液分析仪进行血小板计数,并将血小板浓度调整为3 x 10 5 /μl(Nesbitt 等,2009)。在通过微流体通道灌注前5分钟,加入钙黄绿素至终浓度为1μg/ ml,并将血小板保持在黑暗中直至使用。在一小时内使用标记的血小板,因为钙黄绿素从细胞中流出,细胞荧光将会丢失。

    鼠血小板
    通过静脉穿刺抽取小鼠血液的程序由机构的动物伦理和当地方案确定。
    1. 在CO 2 安乐死后通过心脏穿刺抽取1ml血液或在麻醉下通过静脉穿刺下腔静脉。
    2. 将台式离心机中的血液以400 x g 离心5分钟。
    3. 收集富含血小板的血浆(上清液)和上部红细胞层的1/8。
    4. 将200μl带有葡萄糖的HEPES-Tyrodes缓冲液加入红细胞层并轻轻混合。将红细胞层在400 x g 下离心5分钟。
    5. 收集上清液和上部红细胞层的1/8,并用步骤3中富含血小板的血浆池。
    6. 将合并的富含血小板的血浆在200 x g 离心6分钟。
    7. 收集富含血小板的血浆(上清液)而不干扰红细胞层。
    8. 将200μl带有葡萄糖的HEPES-Tyrodes缓冲液加入红细胞层并混合。将红细胞层在200 x g 下离心6分钟。
    9. 收集上清液而不干扰红细胞层并用来自步骤7的富含血小板的血浆汇集。
    10. 让富含血小板的血浆在室温下静置10分钟。在温育结束时,加入PGE1(0.5μM终浓度)并在500μM离心下离心10分钟。
    11. 丢弃贫血小板血浆(上清液)并将沉淀重悬于含有葡萄糖的HEPES-Tyrode缓冲液中。在血液分析仪上进行血小板计数,并将血小板浓度调整至300×10 3 /μl。
    12. 在进行微流体测定之前,将血小板与抗GPIb 488抗体(3μg/ ml终浓度)在室温下在黑暗中孵育10分钟。
    注意:
    对于人类和小鼠血小板:
    1. 所有离心步骤均为室温。
    2. 血小板悬浮液在任何步骤都不能在4°C下储存。
    3. 血小板应在静脉穿刺后4小时内使用。
    4. 灌注测定在黑暗中进行。

  5. 图像采集
    1. 如下图所示组装注射泵(图2)。将2毫升Luer锁玻璃注射器连接到3向连接器上的水平面对面母鲁尔锁。将硅胶管滑到公鲁尔锁上,并将旋转锁拧在管上。将充满水的20毫升塑料鲁尔锁定注射器连接到最终的母鲁尔锁,并将玻璃注射器放在PHD注射泵上。将玻璃注射器固定到位。


      图2.注射泵的设置 A.装配2 ml玻璃注射器,装有水的塑料20 ml注射器(每次实验后冲洗管道)和管道(直径1 mm)金属适配器)在三通旋塞连接器上。 B.在泵上组装玻璃注射器。 C.旋塞阀的旋转使得能够从管道流到玻璃注射器。 D.微流体芯片设置的示意图。虚线箭头表示流动方向。芯片中的黄色方块代表血小板悬浮液。

    2. 将一个30毫米长的1毫米直径钢管以90°角弯成两半,将另一端插入医用级Tygon管中。将金属管插入生物芯片的1 mm孔出口(图3)。将生物芯片固定在显微镜载物台上,使盖玻片平面聚焦(图4)。
      注意:生物芯片组装到显微镜所涉及的步骤(图2-3)可以在视频2中找到。

      视频2.将内部PDMS生物芯片组装到显微镜载物台上


      图3.微流体系统的最终组装。 A.直径为1 mm的金属管连接到PDMS生物芯片的1 mm孔。 B.然后将系统组装到倒置显微镜上。


      图4.焦点平面。使用60x物镜在Olympus荧光显微镜下放置生物芯片的成像。焦平面显示在盖玻片(A)下方,盖玻片(B)上方和盖玻片(C)上。当盖玻片聚焦时,可以在通道内看到PDMS的锯齿状边缘(制造过程的人工制品)。

    3. 清空进样口并开始采集。为ve +染色的血小板获取DIC通道和荧光通道(激发:488nm,发射:520nm)的时间堆栈(图5),设置采集参数以定期拍摄图像。
    4. 在生物芯片的入口孔中放置100μl的3×10 5个/μlμl洗涤的血小板。
    5. 启动注射泵,以5μl/ min(500 s -1 )的速率通过通道抽取液体5分钟。或者,要将血小板暴露于其他剪切速率和应力水平,请使用下面的简化公式(1,2)计算所需的流速。提供了一个表格作为快速参考,用于计算本协议中使用的芯片的流速剪切速率(表1)。



      哪里,
      Q =流速(μl/ min)
      W =通道宽度(cm)
      h =通道高度(cm)
      γ̇=所需的剪切速率(s -1 )



      哪里,
      W =通道宽度(cm)
      h =通道高度(cm)
      μ =粘度(Pa∙s)
      τ =壁面剪切应力(Pa)

      在37°C
      μ(HEPES-Tyrode缓冲液中含有葡萄糖的洗涤血小板)= 0.71 x 10 -3 Pa∙s
      μ(全血)= 3.8×10 -3 Pa∙s = 0.038泊
      μ(水)= 0.695 x 10 -3 Pa∙s

      1厘泊= 1 mPa∙s = 0.001 Pa∙s 1 Pa = 10 dyn / cm 2 = 1 Newton / m 2



      表1.在直通道PDMS芯片中达到所需剪切速率和剪切应力所需的血小板悬浮液流速



      图5.流动时小鼠血小板对纤维蛋白原的粘附。用抗GPIb-488抗体标记的血小板灌注在涂有纤维蛋白原(40μg/ ml)的剪切速率为500的PDMS通道上小号 -1 。通过落射荧光(顶行)和DIC(底行)显微镜在0,6和20分钟捕获粘附血小板的时间过程。比例尺=20μm。

  6. 使用Vena8 Fluoro +生物芯片进行血小板粘附
    Vena8 Fluoro +生物芯片是由Cellix TM 制造的商业生物芯片。它们是8通道生物芯片,盖玻片宽度为0.17毫米。每个通道的尺寸高度为0.1 mm,宽度为0.4 mm,长度为25 mm。生物芯片上的每个通道的容量为8微升。
    1. 使用20μl移液管吸取10μl纤维蛋白原(PBS pH 7.4中的40μg/ ml)或vWF(PBS中的100μg/ ml,pH 7.4)。
    2. 将移液器的尖端放入Vena8 Fluoro +生物芯片通道的一个开口中(图6)。按下移液管的柱塞,用涂层基质填充通道。


      图6. Vena8 Fluoro +通道的涂层。 A.将10μl移液管放入通道的开口中,并用涂层基底填充通道。填充后,通道将是透明的。 B.生物芯片的前4个通道(标记为绿色)填充有涂层基板。其他4个频道都是空的,显得灰色。

    3. 将生物芯片放入具有用dH 2 O润湿的纸巾的容器中并关闭容器。将生物芯片在4°C孵育过夜。
    4. 从储液器中取出涂层基材。
    5. 用10μl移液管吸取10μl封闭缓冲液,并用封闭缓冲液填充通道。在室温下孵育通道1小时。
    6. 用10μl移液管吸取10μlPBS并填充通道进行清洗。重复步骤F6一次。
    7. 使用出口销将管道连接到生物芯片。
    8. 将通道一端的管子浸没在装有洗过的血小板的Eppendorf管中(图7)。将通道另一端的管道连接到Mirus Evo Nanopump。启动泵以通过生物芯片的通道吸取血小板悬浮液。有关达到所需剪切速率所需流速的表格,请参见 Cellix网站根据技术规范。或者,使用通道的尺寸以及程序E中的公式1和2来计算流速。


      图7. Vena8 Fluoro +生物芯片的设置。 Eppendorf管用作容器以容纳洗过的血小板。通道的另一端连接到管道,管道连接到Mirus Evo Nanopump。

  7. 图像采集
    1. 如下图所示组装注射泵(图8A):将进样针(1)(浸没在含Eppendorf的血小板溶液或全血中)连接到芯片上8个通道之一的入口。将出口销(2)连接到通道的出口和管道(连接到泵)。

      &nbsp;&nbsp;
      图8. Cellix微流体系统的组装。 A.将Vena8 Fluoro +生物芯片插入温度控制台支架。入口引脚(1)连接到芯片上8个通道之一的入口。出口销(2)连接到通道的出口和通向泵的管道。 B. Cellix微流体系统的设置。 (1)硬盘; (2)灯; (3)电机控制; (4)屏幕显示; (5)阶段控制; (6)ExiBlu CCD相机; (7)落射荧光显微镜; (8)温度控制; (9)Mirus Evo Nanopump。

    2. 切换PC Hardrive(1),指示灯(2),电机控制(3),屏幕显示(4),舞台控制(5),ExiBlu CCD相机(6),落射荧光显微镜(7)的“开机”按钮,温度控制(8),Mirus Evo Nanopump(9)(图8B)。启动安装在PC上的VenaFlux Assayx64软件。这将打开Venoflux界面。&nbsp;
    3. 单击“新协议”或“开放协议”(如果已设置参数)。这两个选项都会显示相同的屏幕页面(图9A)和一个命令列表。右键单击鼠标即可激活每个命令,应按显示的顺序进行操作。命令序列为:1。VenFlux设置,2。初始化VenFlux平台,3。启动摄像机预览,4。几何设置,5。更新几何,6。冲洗泵,7。冲洗电缆,8。清洗/连接生物芯片,9。冲洗芯片,10。细胞试验(图9A)。启动摄像机预览后,频道将显示在屏幕上(图9B)。
      注意:
      1. 对于“冲洗泵”和“冲洗电缆”的步骤,卸下出口销。泵将连接的泵溶液(水或PBS)流入电缆,电缆的自由端应放入废液收集器中。
      2. 对于“洗涤/连接生物芯片”的步骤,出口销连接到电缆但不连接到生物芯片的出口通道。出口销的针保持在通道的出口位置上方。当您右键单击“清洗/连接生物芯片”时,泵将向出口孔中喷出100μl泵液,以避免在针插入出口时空气进入通道。如果通过移液已经在井中包含了一些流体,则可省略此步骤。
      3. 对于台阶“冲洗芯片”,出口销必须插入通道的出口。默认设置是以1μl/秒的速度输送40μl泵液,以清洗通道。
      4. 对于“细胞分析”步骤,通过调整剪切单位,采集时间和捕获延迟(如果需要)输入流量参数。输入所有步骤后,右键单击“细胞分析”以启动泵并获取数据。要停止检测,请单击“停止检测”。
      5. 要在屏幕上查看频道,请将显微镜连接切换到相机。切换到目镜,以获得通道的精细对焦。
      6. 数据采集所涉及的步骤(图9-11)可以在Cellix,Ltd的视频网络研讨会中找到:Cellix网络研讨会: VenaFlux平台技术演示文稿 ;
        Cellix网络研讨会: Cellix生物芯片与标准注射泵进行灌注检测


      图9.启动VenaFlux协议。 A.在“从VenaFlux页面开始”中,通过右键单击激活每个命令,应按所显示的顺序执行。 B.启动摄像机预览后,频道将显示在屏幕上。 2条水平黑线是通道的边框(放大20倍)。

    4. Vena8 Fluoro +生物芯片具有嵌入标记,有助于将生物芯片映射到VenaFlux assay x 64软件。每个通道1-8在通道2,3 ... 6上方具有标记位置,其可以在显微镜下分别以2,3和6个点可视化。在VenaFlux分析x 64的对话屏幕上,在屏幕顶部,单击“设置Venaflux”选项。这将带来另一个对话框,显示名为“Set Vena8 / VenaEC origin and channel / position spacing”的x,y坐标。点击Vena8获取生物芯片类型。使用操纵杆移动舞台,将焦点对准通道1,位置2(图10A)。点击左上角的“更新位置”。使用操纵杆将舞台移动到通道8,位置6(图10B)。点击右下角位置的“更新位置”。然后按设置。回到“Start with VenaFlux”页面,您可以点击屏幕右上角显示的生物芯片地图,这将自动将舞台移动到该位置。
      注意:位置1和8的生物芯片上没有点.Vena8芯片的映射基于位置2和6,因此采集范围小于生物芯片的尺寸。


      图10.映射Vena8生物芯片。 A.在“Setup VenaFlux”页面上,移动平台以聚焦在通道1,位置2上,然后单击左上角位置的“更新位置”。 B.将舞台移动到通道8,位置6.单击右下角位置的“更新位置”。然后按下设置。

    5. 要开始测定,请通过调整剪切单位,采集时间和捕获延迟来输入所需的流量参数(图11A)。完成所有步骤后,单击“开始测定”以启动泵和数据采集。要停止测定,请单击“停止测定”。要获取图像,请单击“Setup VenaFlux”页面上的“获取图像到文件”。这将把图像保存在预先指定的位置作为位图图像(图11B)。要获取视频,请单击“获取视频到文件”。视频的图像和图像堆栈的分析可以通过如下所述的ImageJ或使用ImagePro Premier 64位软件来执行。我们更喜欢ImageJ进行分析,因为它非常灵活,可以包含可调节的宏。


      图11.启动VenaFlux检测。 A.在“细胞分析”下(以黄色圈出),右键单击“可变流量分配”选项并在“步骤属性视图”上调整流量参数“图标(以棕色圈出)。 B.单击“获取图像”(在面板A中以红色圈出)以将图像捕获为位图文件。显示的是用钙黄绿素1μg/ ml(白点)标记的人血小板捕获的图像的实例,并在涂有vWF100μg/ ml的Vena8通道上灌注。&nbsp;

数据分析

  1. PDMS生物芯片
    描述了两种手动分析方法,用于分析血小板粘附随时间的变化:血小板计数和荧光强度分析。该协议描述了在PC上使用Fiji / ImageJ进行分析,但对于Mac用户来说,这个过程是相同的。

    计数血小板粘附分析
    血小板计数和血小板总和荧光数据分析(图13-14)中涉及的步骤可以在视频3中找到。


    视频3.计算血小板粘附的ImageJ分析

    1. 打开斐济/ ImageJ版本1.52h。&nbsp;
    2. 单击工具栏上的“多点”工具。双击“多点”工具,在“点工具”窗口中,选中“标签点”和“在所有切片上显示”复选框(图12)。


      图12. ImageJ多点工具。 A.多点工具位于ImageJ窗口的工具栏上(以红色圈出)。 B.双击多点工具将打开点工具窗口。此窗口可用于计算放置的标记,并调整放置在图像上的标记的显示选项。

    3. 单击视野中的每个血小板以计算粘附的血小板数量(图13)。通过确保血小板在488 nm通道上染色来验证细胞类型。


      图13.使用ImageJ多点工具计数血小板。 A.血小板在DIC通道上计数。 B.使用荧光通道验证血小板以检查抗GPIb-488染色。单击图像,选择多点工具,将在图像上留下带数字的标记。按住“alt”然后单击标记将删除标记。随后的每次点击或删除都会将标记上的数字分别增加或减少一个。使用此功能可计算图像上的血小板数。或者,在点击图像上的每个血小板后,使用点工具窗口上的计数器(图12)来确定计数的血小板数量。

    4. 在GraphPad prism或Microsoft Excel上输入血小板计数。
    5. 移动到图像堆栈的下一帧,单击已粘附的其他血小板,取消选择不再存在的血小板,如步骤3中所述。记录血小板计数(图13)并对图像堆栈中的每个帧重复。
    6. 绘制血小板计数与时间的关系(图14)。


      图14.随时间推移粘附于纤维蛋白原通道的血小板数量。将野生型C57BL / 6小鼠血小板洗涤并流过涂有纤维蛋白原40μg/ ml的PDMS生物芯片,剪切速率为500 s -1 。每3分钟测定视野中的血小板计数。使用GraphPad棱镜将粘附血小板的总数相对于时间作图。点表示特定时间点的平均血小板计数,误差条表示n = 3次实验的平均值的标准误差。

    荧光强度分析血小板粘附分析
    血小板总荧光数据分析中涉及的步骤(图15-22)可以在视频4中找到。


    视频4.荧光强度对血小板粘附的ImageJ分析

    1. 打开斐济/ ImageJ。
    2. 单击“分析”选项卡,然后选择“设置测量”(图15)。


      图15.访问分析参数。可以从设置的测量菜单中选择将在测量读数上显示的参数。要访问菜单,请选择“分析”选项卡(以蓝色突出显示),然后单击“设置测量...”(以红色圈出)。

    3. 选中“Area”,“Mean grey value”,“Integrated density”和“Median”复选框(图16)。
      注意:这些测量中的每一个都提供了血小板的描述。对于单个血小板,“区域”描述了在血小板周围绘制的感兴趣区域占据的区域。 “平均灰度值”和“中值”都是量化单个血小板或血小板聚集体的平均荧光强度的方法。 “综合密度”量化了所选区域的总强度。


      图16.用于测量血小板荧光强度的分析参数可以选择各种参数来测量感兴趣区域中的荧光。为了分析该方案中的血小板荧光强度,我们选择了“面积”,“标准偏差”,“平均灰度值”和“综合密度”。

    4. 单击“分析”选项卡,单击“工具”,然后选择“ROI管理器...”(图17)。


      图17. ImageJ上的感兴趣区域(ROI)管理器。可以通过选择“分析”然后选择“工具”(以蓝色突出显示)并单击“ROI管理器...”来访问ROI管理器(用红色圈出来。

    5. 单击工具栏上的“徒手选择”工具(图18)。

      &nbsp; &nbsp;
      图18. ImageJ上的徒手选择工具。徒手选择工具(用红色圈出)可用于追踪血小板或血小板聚集体周围的感兴趣区域。然后可以将感兴趣的区域存储在ROI管理器上,并且可以测量在步骤B3中设置的参数。

    6. 对于时间堆栈上的每个帧,在488 nm通道上,圈出血小板或血小板聚合,然后在ROI管理器窗口中单击“添加”(图19)。


      图19.在ROI经理上存储感兴趣的区域。使用徒手选择工具绘制的感兴趣区域可以通过按键盘上的“t”或点击“存储”来存储在ROI管理器上在ROI经理上添加“(以红色圈出)。

    7. 单击并将感兴趣的区域拖动到原始位置附近没有血小板的区域(图20),然后单击ROI管理器窗口上的“添加”。这可以测量背景荧光。


      图20.感兴趣的“背景”区域。在步骤B6中绘制的感兴趣区域可用于选择新的感兴趣区域以减去背景荧光强度测量值。单击并将感兴趣的区域拖离血小板/血小板聚合体,并将新区域添加到ROI管理器。

    8. 在ROI管理器窗口中选择两个区域,然后单击“测量”(图21)。


      图21.感兴趣区域的测量。选择所需的感兴趣区域(以蓝色突出显示),然后单击“测量”(以红色圈出),将打开一个新窗口,显示测量在步骤B3中选择的参数。

    9. 从相应的血小板测量值中减去背景荧光测量值的Mean / Median / RawIntDen。在GraphPad prism或Microsoft Excel上输入数据。
    10. 对框架中的每个血小板和血小板聚集重复步骤5-9,对荧光求和,并记录该值。对图像堆栈中的所有帧重复此操作。
    11. 在GraphPad prism或Microsoft Excel上绘制荧光强度随时间变化(图22)。


      图22.随着时间的推移粘附于纤维蛋白原通道的血小板的荧光。将野生型C57BL / 6小鼠血小板洗涤并流过涂有纤维蛋白原40μg/ ml的PDMS生物芯片,剪切速率为500s -1 。每3分钟测定视野中血小板荧光的总和。使用GraphPad棱镜绘制血小板荧光的总和对时间的曲线。点表示特定时间点的荧光平均值,误差棒表示n = 3次实验的平均值的标准误差。

    血小板粘附随时间变化的统计分析
    1. 打开GraphPad Prism。
    2. 选择“分组”选项卡。
    3. 检查“并排列中输入_____重复值”,输入实验中执行的最大重复次数。
    4. 在列标题中,输入要分析的时间点。确保每个点之间的时间相等。
    5. 在第一行的相应时间点下输入实验数据。
    6. 单击“分析”,在“列分析”下拉菜单下,选择“单向ANOVA(和非参数)”。
    7. 根据所需分析选择分析参数:
      1. 对于具有高斯分布的数据,在“假设高斯分布?”部分下,单击“是,使用ANOVA”选项(注释1和2)。
      2. 对于没有高斯分布的数据,例如分布偏斜的数据,在“假设高斯分布?”部分下,单击“否,使用非参数测试”(注释1和2)。
    8. 选择“确定”,然后在页面左侧的“结果”下拉菜单中,选择数据分析并检查 P - 值。 P - 值&lt; 0.05表示统计学显着性。

    特定时间点血小板粘附治疗效果的统计分析
    1. 打开GraphPad Prism。
    2. 选择“分组”选项卡。
    3. 检查“并排列中输入_____重复值”,输入实验中执行的最大重复次数。
    4. 在列标题中,输入要分析的处理的标题。
    5. 输入第一行中特定时间点的治疗数据。
    6. 单击“分析”,在“列分析”下拉菜单下,选择“ t - 测试(和非参数测试)”。
    7. 根据所需分析选择分析参数:
      1. 对于具有高斯分布的数据,在“假设高斯分布?”部分下,单击“是,使用参数测试”选项(注释1和2)。
      2. 对于没有高斯分布的数据,例如,分布偏斜的数据,在“假设高斯分布?”部分下,单击“否,使用非参数检验”(注释1和2) 。

  2. 商用生物芯片
    使用随附的VenaFlux 2.3成像软件捕获图像,并以每5秒1帧(30帧)的速率捕获视频。在通道的位置2,4和6(位于距血小板悬浮液进入部位6,14和22mm处)处分析图像。这些位置代表离血小板悬浮液进入通道最近,中间和最远的流量。使用Image J或ImagePro Premier 64位软件 http,每隔3分钟测量血小板计数和血小板荧光总和://www.mediacy.com/imagepro 。数据导出到Excel或GraphPad Prism中进行统计分析。

食谱

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4
    pH 7.4
    注意:提前准备并在室温下保存长达1年。
  2. 10%Extran
    40毫升Extran ® MA 02
    360 ml ddH 2 O
    pH 7.0
    注意:使用前准备新鲜的10%稀释液,使用后丢弃。
  3. 阻塞缓冲区
    2%牛血清白蛋白
    1x磷酸盐缓冲盐水
    pH 7.4
    注意事项:提前准备白蛋白2%溶液,并以-20℃的等分试样储存,直至使用1年。避免冻融等分试样超过2次。
  4. HEPES-Tyrode的葡萄糖缓冲液
    20 mM HEPES
    134 mM NaCl
    0.34mM Na 2 HPO 4
    2.9 mM KCl
    12 mM NaHCO 3
    1 mM MgCl 2
    1 mM CaCl 2
    在洗涤血小板之前加入5mM D-葡萄糖 pH 7.4
    注意:事先准备好HEPES-Tyrode的缓冲液(不含D-葡萄糖),并在室温下保存长达1年。将D-葡萄糖加入等份的HEPES-Tyrode缓冲液中,24小时内使用。

致谢

我们感谢Ethel Ilagan,Fangyuan Zhou,Shaun P. Jackson和他的实验室进行了有益的讨论。这项工作得到了悉尼大学心血管计划催化剂药物精准CV医学(LAJ,FHP),澳大利亚国家心脏基金会博士后奖学金101285(LAJ),CSANZ-BAYER青年研究者助学金(LAJ),皇家学院的支持。澳大利亚Kanematsu研究奖(LAJ,FHP)的病理学家。

利益争夺

作者没有相互冲突的利益申报。

参考

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Copyright Dupuy et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Dupuy, A., Ju, L. A. and Passam, F. H. (2019). Straight Channel Microfluidic Chips for the Study of Platelet Adhesion under Flow. Bio-protocol 9(6): e3195. DOI: 10.21769/BioProtoc.3195.
  2. Passam, F., Chiu, J., Ju, L., Pijning, A., Jahan, Z., Mor-Cohen, R., Yeheskel, A., Kolsek, K., Tharichen, L., Aponte-Santamaria, C., Grater, F. and Hogg, P. J. (2018). Mechano-redox control of integrin de-adhesion. Elife 7: e34843.
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