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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Abstract

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

Introduction

Hemodynamic forces generated by the blood flow are well known to play major roles in endothelial and vascular function1,2. It is also well known that several types of ion channels acutely respond to changes in shear stress3,4,5 leading to the hypothesis that ion channels can be primary shear stress sensors. More recently, we and others showed that mechanosensitive ion channels play critical roles in several shear-stress sensitive vascular functions, including the vasoactive response to shear stress6,7,8, and developmental angiogenesis9. The mechanisms of the shear-stress sensitivity of ion channels, however, are almost totally unknown. This gap of knowledge is likely to be due to the technical difficulty of performing electrophysiological recordings under well-defined shear stress. In this article, therefore, we provide a step by step detailed protocol routinely performed in our lab to achieve this goal6,7,10,11.

The overall goal of this method is to allow the real-time investigation of ion channel mechanoactivation under well-defined shear stress in the physiological range. This is achieved by modifying a standard parallel plate flow chamber to allow an electrophysiological pipette to be lowered into the chamber and access cells grown on the bottom plate during the real time exposure to flow, providing a unique approach to achieve this goal6,7,11. In contrast, standard parallel plate flow chambers, described in prior publications can be used for the real time imaging analysis of cells exposed to shear forces12 or other non-invasive approaches13,14 but not for electrophysiology. Similarly, the cone and plate apparatus, another powerful approach to expose cells to shear stress15,16 is also not suitable for electrophysiological recordings. Thus, these flow devices do not allow the investigation of shear stress sensitivity of ion channels. The difficulty in performing electrophysiological recordings under flow is the main reason for the paucity of information about the mechanisms responsible for these crucial effects.

In terms of the alternative approaches to achieve the same goal, there are none that are as accurate or controlled. Some earlier studies attempted to record ion channel activity under flow by exposing cells to a stream of liquid coming from another pipette brought to the vicinity of a cell from above17,18. This is highly non-physiological, as the mechanical forces generated under these conditions have little in common with the physiological profiles of shear stress in the blood vessels. Similar concerns apply to the attempts to simulate physiological shear stress by perfusion of open chambers. As discussed in detail in our earlier study10, an open liquid-air interface creates multiple disturbances and recirculation, which are non-physiological. To address all these concerns, we have designed a modified parallel plate (MPP) flow chamber, also referred to as the “minimally invasive flow device” in our earlier studies6,7,10,11, made from acrylic and extensively used in our lab. However, in spite of the fact that the original description of the design has been published almost 20 years ago and is the only flow device that allows performing electrophysiological recordings under well-defined shear stress, this methodology has not been adopted by other labs and there are only very few studies that attempt to record currents under flow. We believe, therefore, that providing a detailed description for using the MPP flow chamber will be of great help to researches who are interested in mechanosensitive ion channels and vascular biology.

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Protocol

The use of animals in our studies is approved by the University of Illinois at Chicago Animal Care Committee (#16-183).

1. Assembly of the Modified Parallel Plate Flow Chamber

NOTE: Please refer to Table 1 and Figure 1 for MPP flow chamber piece IDs. Please refer to Figure 1 for a schematic detailing the orientation of chamber pieces for assembly.

  1. To adhere the rectangular cover glass, piece D, to the bottom of piece C, first make the silicone elastomer solution by thoroughly mixing 500 µL silicone elastomer curing agent into 5 mL of silicone elastomer base.
  2. Apply a thin layer of the silicone elastomer solution around the edges of the rectangular space of piece C and gently place the rectangular cover glass piece D directly on the elastomer solution such that piece D completely covers the open rectangular space of piece C. Carefully wipe away excess silicone elastomer solution.
  3. Repeat step 1.2 for adhering the rectangular cover glass, piece F, to the bottom of piece E and allow the silicone elastomer solution to cure over night at room temperature.
    NOTE: Once cured, the rectangular cover glass will remain adhered for up to six months before needing to be replaced.
  4. Beginning with the bottom chamber piece, piece E, assemble the MPP flow chamber by sequentially placing each piece on top of the previous in the following order: piece E (bottom), piece C, piece B, piece A (top).
  5. Align the screw holes of each piece at the corners and tightly screw the pieces together to prevent leaks from occurring while administering flow to the MPP flow chamber.

2. Cell Preparation and Seeding into the MPP Flow Chamber

NOTE: Follow steps 2.1−2.7 for cultured endothelial cells. Follow the method detailed in steps 2.8−2.14 for isolating endothelial cells from the mouse mesenteric arterial arcade and preparation of freshly isolated endothelial cells.

  1. In a 6-well plate, place four to five 12 mm cover glass circles/well and seed cells between 10% and 30% confluency such that single cells can be accessed for electrophysiological recordings.
  2. Incubate cells under standard culture conditions (5% CO2, 37 °C) for no less than 2 h to allow cells to adhere and no more than 24 h as endothelial cells in authors’ experience become very flat and difficult to patch when seeded at sub-confluency for more than 24 h.
  3. Remove a cover glass containing adhered cells from a well of the 6-well plate, quickly rinse in phosphate-buffered saline (PBS), and transfer to a 35 mm Petri dish containing 2 mL electrophysiological bath solution (Table 2) prior to transfer to the MPP flow chamber.
  4. Transfer the cover glass circle to the rectangular cover glass, piece D, which is adhered to piece C of the MPP flow chamber being sure that adequate solution stays on the cover glass so that cells to not become exposed to air. Add the desired bath solution (~250 µL) to the cells so that the cover glass circle and cells are completely submerged in solution.
  5. Position the cover glass circle such that it rests in the half closest to the vacuum reservoir side so that cells will be in line with the slit openings of piece B. Ensure that the glass cover circle adheres to piece D through solution-glass adhesion so that application of flow to the chamber does not disrupt the position of the cover glass circle.
  6. Assemble the MPP flow chamber by screwing the pieces together in the appropriate order, as described in steps 1.4 and 1.5 and as shown in Figure 1. Transfer the chamber to the microscope stage and immediately perfuse the chamber with bath solution such that solution reaches the vacuum reservoir for aspiration (~10 mL).
  7. Identify a healthy cell for the experiment by identifying a cell with a dark border and obvious nucleus. Avoid cells that appear to be blebbing or cells that are in contact with other cells.
    NOTE: In the authors’ laboratory, human aortic endothelial cells and primary mouse mesenteric endothelial cells in culture are used. However, any other type of adherent cell that is of interest to specific research needs can be used in the same way.
  8. Wash the isolated arterial arcade in dissociation solution (Table 2). Transfer the arcade to a 2 mL centrifuge tube containing 2 mL of pre-warmed (37 °C) dissociation solution (recipe for dissociation solution shown in Table 2) containing neutral protease (0.5 mg/mL) and elastase (0.5 mg/mL). Incubate for 1 h at 37 °C with gentle shaking every 10 min.
  9. Remove 1 mL of the neutral protease/elastase dissociation solution and add 1 mg/mL collagenase type 1. Return the collagenase solution to the solution containing the arteries for a final collagenase type 1 concentration of 0.5 mg/mL. Incubate for 2−3 min at 37 °C.
  10. Using 5 grade forceps, quickly move the arcade onto a 35 mm diameter Petri dish containing a 750 µL drop of fresh, chilled dissociation solution. Further dissociate the tissue by using two 20 G syringe needles to mechanically liberate endothelial cells from enzymatically digested arteries.
  11. Using a 9” disposable Pasteur glass pipet, triturate the cell solution 10x before transferring the cells to a new 1.5 mL centrifuge tube using the glass pipet.
  12. Wash the Petri dish with another 750 µL of dissociation solution and transfer to the same tube. Using the glass pipette, further mechanically disperse the cells by pipetting at least 10x to create a single cell suspension being careful not to introduce bubbles that may damage endothelial cell integrity.
  13. Add 750 µL of the endothelial cell suspension (~500−1,000 cells) directly to piece D of the MPP flow chamber on the half closest to the reservoir vacuum side. Allow the endothelial cells to adhere between 45 min and 1 h at room temperature.
  14. Assemble the MPP flow chamber by screwing the pieces together in the appropriate order as described in steps 1.4 and 1.5 and as shown in Figure 1. Transfer the chamber to the microscope stage and immediately perfuse the chamber with bath solution such that solution reaches the vacuum aspiration (~10 mL). Identify accessible endothelial cells by their rough and round phenotype19,20.
    NOTE: A variety of digestion methods and enzyme cocktails have been used to isolate endothelial cells from different arterial beds. See Table 3 for detailed descriptions of protocols that have been used by a variety of investigators to isolate endothelial cells for patch clamp electrophysiology of mechanosensitive ion channels. These methods are likely suitable for use in combination with the MPP flow chamber.

3. Controlling Shear Stress to the MPP Flow Chamber for Electrophysiological Recordings of Shear-activated Mechanosensitive Ion Channels

  1. Set-up a gravity perfusion system by connecting a 30 mL graduated syringe cylinder to a 3-way luer lock fitted with microbore tubing (internal diameter: 0.05 inch, outer diameter: 0.09 inch) suited for insertion into the 3 mm diameter inlet hole of piece A of the MPP chamber.
  2. Attach the graduated cylinder to the outer face of the Faraday cage surrounding the electrophysiology rig (Figure 2) using double-sided tape. Prior to inserting the tubing in the MPP chamber, pre-fill the syringe and tubing with bath solution (see Table 2 for bath solution used for investigating inwardly rectifying K+ channels in endothelial cells). Insert the tubing into the MPP flow chamber inlet hole of piece A.
  3. Pre-fill the MPP flow chamber with solution such that solution is being removed in the vacuum reservoir. Stop flow to the chamber and refill the graduated cylinder to the top mark. Calculate flow rates manually by allowing the solution to flow through the chamber and using a stop-watch to calculate mL/s at a given syringe cylinder height.
  4. Raise or lower the syringe to alter flow, and thus shear in the chamber, and continue this process until a desired level of shear stress is found.
  5. Calculate shear stress in a parallel chamber using the following equation21:
    τ = 6µQ/h2w
    where µ = fluid viscosity (g/cm·s), Q = flow rate (mL/s), and parallel plate chamber width (w = 2.2 cm) and height (h = 0.1 cm).
    NOTE: In the current gravity perfusion system, at a syringe cylinder height (as measured from the top of the cylinder) of 57 cm above the microscope stage, the flow rate is 0.3 mL/s. The shear calculated in the chamber at this syringe cylinder height and flow rate is 0.7 dyn/cm2. It should also be noted that other perfusion systems, such as a peristaltic pump, can be used to control flow to the MPP flow chamber. However, these devices may add unwanted turbulence and influence stability of the electrophysiology measurements under flow, therefore, using the gravity perfusion system described here is recommended.
  6. Transfer an assembled chamber containing adhered cells to the microscope stage of the electrophysiology rig and insert the tubing pre-filled with bath solution into the hole of piece A. Simultaneously fill the chamber and wash the cells with 10 mL of bath solution by turning the 3-way luer lock such that solution flows to the chamber.
  7. Once the desired patch configuration is successfully obtained allow channel currents to stabilize in a static bath at room temperature. Once currents have stabilized, apply shear in a step-wise fashion allowing increases in current to stabilize prior to the next step increase in shear stress.
    NOTE: The authors find the most success with the perforated patch configuration when studying mechanoactivated ion channels in endothelial cells. To perform perforated whole-cell patch configurations, add 5 µL of a 60 mg/mL stock amphotericin B in dimethyl sulfoxide (DMSO) to 1 mL of 0.2 µm sterile filtered pipette solution. After generating a giga-ohm seal in the cell-attached configuration, perforated whole-cell patches form within 2−5 min.
  8. Remove shear exposure to the cells by stopping flow to the chamber allowing mechanosensitive channel currents to return to baseline currents observed in the static bath.
  9. Isolate mechanosensitive ion channel currents of interest by altering solution valence (e.g., 60 mM K+ in bath solution with 0 Ca2+ in pipette solution to study inwardly rectifying K+ channels. Table 2 shows example solution recipes) and/or pharmacological inhibition of potentially contaminating current sources.

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Results

Multiple photographs showing different views of the MPP flow chamber on the microscope stage (upper panel) and a schematic representation of the MPP flow chamber (bottom panel) are shown in Figure 1. The schematic details the dimensions of the entire device and flow chamber. Figure 2 shows a photograph of the gravity perfusion system to the MPP flow chamber in our laboratory (upper panel). Also shown is a schematic representation...

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Discussion

The vascular system is constantly exposed to active hemodynamic forces, which activate mechanosensitive ion channels3,22 but the physiological roles of these channels in shear stress-induced mechanotransduction is only starting to emerge4,6,8. The mechanisms responsible for the mechanosensitivity of shear stress-activated channels remain unknown. The protocol detailed he...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by the National Heart, Lung, and Blood Institute (R01 HL073965, IL) and (T32 HL007829-24, ISF). The authors would also like to acknowledge the Scientific Machine Shop at the University of Illinois at Chicago for generating our latest MPP flow chambers.

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Materials

NameCompanyCatalog NumberComments
0.2 µm sterile syringe filtersVWR28145-501Used for filtering electrophysiolgoical pipette solution
5 grade forcepsFine Scientific Tools1252-30Used for transferring digested arteries to fresh solution
9" Pasteur PipetFisher Scientifc13-678-20DUsed for mechanically disrupting digested arteries and transferring freshly isolated endohtelial cells 
12 mm diameter Cover glass circlesFisher Scientifc12-545-80For use with studies involving cultured cells and multiple treatments. Cells adhered to the cover glass are used for patch clamp analyses
24 mm x 40 mm Rectangluar Cover glassSigma-AldrichCLS2975224Cover glass to be added to MPP flow chamber pieces C (Figure 1)
24 mm x 50 mm Rectangular Cover glassSigma-AldrichCLS2975245Cover glass to be added to MPP flow chamber E (Figure 1)
20 G syringe needlesBecton Dickinson and Co305175For use in mechanical disruption of digested mesenteric arteries
35 mm Petri dishGenesee Scientific32-103For use in mechanical disruption of digested mesenteric arteries
Amphotericin B solubilizedSigma-AldrichA9528-50MGUsed for generating the perforated whole-cell patch configuration.
Collagenase, type IWorthington Biochemical100 mg - LS004194Enzyme used in our laboratory as a brief digestion following the initial cocktail of neutral protease and elastase
Dimethyl Sulfoxide (DMSO)Fisher Scientifc67-68-5Solvent for Amphotericin B used in perforated whole-cell patch clamp
Elastase, lyophilizedWorthington Biochemical25 mg - LS002290 Enzyme used in our laboratory in a cocktail with neutral protease/dispase to begin digestion of arteries for endothelial cell isolation.
Falcon Tissue culture Plate, 6-well, Flat Bottom with Low Evaporation Lid Corning353046For use with studies involving cultured cells and multiple treatments
Neutral protease/dispaseWorthington Biochemical10 mg- LS02100 50 mg - LS02104Enzyme used in our laboratory in a cocktail with elastase to begin digestion of arteries for endothelial cell isolation
SylGard World Precision InstrumentsSYLG184Silicone elastomer for adhering the rectangular cover slip to the MPP flow chamber pieces C and E (Figure 1)
Tygon ND 10-80 tubingMicrobore TubingAAQ04133ID: 0.05 in, OD: 0.09 in, inlet perfusion tubing for adminsitering flow to the chamber

References

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Electrophysiological RecordingsMechanosensitive Ion ChannelsShear StressModified Parallel Plate Flow ChamberSilicone Elastomer SolutionCell AdhesionEndothelial CellsSingle cell Ion CurrentsFlow Activated Ion ChannelsBAF SolutionExperimental ProceduresCulture Conditions

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