Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

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 phenotype^19,20.
    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.