This method can help answer key questions in the field of endothelial biology, such as why do blood vessels have regional differences in the development of atherosclerosis? The main advantage of this technique is it allows the simultaneous study of multiple conditions under shear stress or multiple shear stress conditions. The implications of this technique extend toward understanding endothelial gene regulation in both the arterial and venous systems because various flow rates and patterns can be used.
Well here, we demonstrate a system that uses steady laminar flow. The setup can be adapted for other flow patterns, including disturbed flow. Generally, individuals new to this method will struggle to maintain a continuous monolayer of cells throughout the duration of the experiment and to obtain consistent results between experiments.
24 hours before the analysis, count the human endothelial cells at an early passage and seed approximately one times 10 to the sixth cells in one milliliter of medium onto one fibronectin coated glass slide per well of a four-well cell culture plate. Allow the cells to adhere to the slide for 15 minutes at 37 degrees Celsius. Then cover each slide with three milliliters of medium and place the plate in the cell culture incubator of 37 degrees Celsius and 5%CO2 for 24 hours.
On the day of the experiment, fill the water tray of a large, heated, built-in environment with adjustable CO2 and heat, or beach incubator, with multiple shelves, internal electricity access, and glass doors, and confirm that adequate carbon dioxide is available for the experiment, and that the CO2 monitor is functional. To set up the flow chamber, insert a 1/8 inch female lure into one end of a number 16 soft tube, and attach a four-way stopcock to the lure. Attach the free end of the tube to the inflow side of the top plate and insert a 1/8th inch male lure into one end of a second number 16 soft tube.
Attach a four-way stopcock to the male lure, and attach the free end of the tube to the outflow side of the top plate. Insert a 1/8th inch male lure and a 1/8th inch female lure into each end of a third number 16 soft tube, and attach the tubing to a bubble trap via the 1/8th inch male lure. Then attach a four-way stopcock to the other end of the tubing via the female lure.
Using sterile tweezers, transfer one cell seeded glass slide to the recess on the bottom plate, cell seeded side up. Use a 10 milliliter syringe to add 10 milliliters of warm medium to the bottom plate within the red gasket line around the plate. Allowing medium to flow through the slide and cover the cells.
Gently place the top plate onto the bottom plate taking care to align the sides and screw the plates together tightly. To remove air bubbles from the bubble trap, first open the inflow and bubble trap stopcocks and close the outflow stopcock. Then use a 30 milliliter syringe to gently flush 20 milliliters of warm medium through the plate.
To remove bubbles from the chamber, close the bubble trap stopcock and open the outflow stopcock. Elevate the outflow side of the chamber to a 45 degree angle and use the 30 milliliter syringe to gently flush 20 milliliters of warm medium from the inflow side of the chamber. It is important to flush at an appropriate rate in the direction of the flow and to use microscope to confirm that a confluent monolayer of cells remains before exposing the cells to the laminar flow.
Then close the stopcocks on both sides of the chamber and cap and confirm that the cells are still attached to the slide under a light microscope. Place the flow chamber and a previously assembled flow loop system into the beach and slowly decrease the pump speed of the flow loop assembly before pausing the pump altogether. Close the stopcocks on the flow loop system to prevent leakage and connect the chamber and loop system together via stopcocks.
When all of the loops have been connected, reopen all of the stopcocks and slowly increase the pump speed while monitoring the setup for any leakage or blockage. Place the flow sensor on the chamber bridge tubing of the inflow side of the chamber with the sensor oriented in the direction of flow and adjust the pump speed to obtain the flow rate for the shear stress rate of interest. Then turn on the CO2 tank to achieve a 5%C02 concentration.
Once the experimental flow period is complete, slowly decrease the peristaltic pump speed to zero and turn off the power. Quickly close all of the open stopcocks and transfer the chamber and the attached tubing with a stopcock on each side to a clean bench top. Carefully unscrew all of the screws to remove the top plate and use needle nose tweezers to transfer the glass slide from the bottom plate to a 100 millimeter diameter dish.
Wash the slide in 10 milliliters of cold PBS and check the cells under a microscope to confirm cell adherence and alignment in the direction of flow. After aspirating the PBS, transfer the slide to a clean 100 millimeter diameter dish and add 350 microliters of lysis buffer supplemented with 1/100th of Beta-Mercaptoethanol from an RNA extraction kit to the slide. Use a polyethylene bladed cell scraper to scrape the cells off the slide and tilt the tissue culture dish to allow the buffer to pool at the bottom.
Use forceps to remove the slide and pipette the cell lysate into a 1.5 milliliter tube on ice. Then add 10 microliters of diluted luciferase RNA to the cell lysate for downstream RNA isolation and analysis according to standard protocols. A successful linearization of the luciferase plasmid using restriction enzymes can be confirmed by running the digested products on an agarose gel and the size of the linearized product can be confirmed with DNA ladders and by comparison with an uncut plasmid.
Manufacturing processes can lead to small variations in the chamber height, thus the flow rate must be calculated for each chamber to achieve the same shear stress. For experiments incorporating laminar shear stress in mammalian endothelial cells, firefly luciferase RNA can be used as an exogenous RNA spike-in. Normalization of the endogenous reference gene, the exogenous reference gene, and both endogenous and exogenous reference genes together yields similar results.
Of note, in these representative experiments using two simultaneously running flow chambers with shear stress at one Pascal for each experiment, Kruppel like factor two knockdown via SI-RNA yielded a similar knockdown efficiency between the tested biological samples. While attempting this procedure it is important to remember to establish an even monolayer of cells on the slide and to profuse the flow loop system prior to attaching the flow chamber. Following this procedure, other methods, like RNA sequencing, can be used to assess genome wide RNA expression and other readouts, such as DNA and protein, can be used for further molecular analysis.
After its development, this technique paved the way for researchers in the field of endothelial biology to explore the relationship between shear stress and angiogenic potential in human endothelial cells.
