The overall goal of this model is to create a simple and economical method for In Vitro periodic fluid flow, and shear stress studies on endothelial cells. This method can help answer key questions in the field of Endothelial Cell Physiology. Such as, how does periodic shear affect endothelial cell shape, structure, and functioning.
The main advantage to this technique is that it uses a simply assembled tissue culture device, which provides relatively large surface areas for evaluation of cellular responses. Demonstrating this procedure, will be Mr.Luke White, a medical student in my laboratory. Begin this procedure by using presentation software toe create a shear ring construction template with a 150 millimeter outer edge profile and a 100 millimeter internal diameter placed in the center of the 150 millimeter circle.
Print the template on a sheet of A4 white paper. Working under a fume hood with adequate ventilation and wearing gloves pipette 10 millimeter methylene chloride into a 150 millimeter glass Petri dish. It is critical that the dish is glass as methylene chloride solubilizes most plastics.
Align a 150 millimeter plastic Petri dish onto the outer shearing template. Use a rotary tool to drill a three 3 millimeter hole in the center of a 100 millimeter dish. Remove any plastic shavings produced from the drilling.
While holding the 100 millimeter Petri dish with a gloved hand invert and dip the top edge of the dish into the pool of methylene chloride for approximately three seconds. Allow excess methylene chloride to drip off the 100 millimeter dish and then quickly transfer the wetted dish edge down onto the center of the 150 millimeter dish. Carefully aligning the 100 millimeter dish onto the marked template.
Gently rotate the 100 millimeter dish clockwise and counterclockwise a few times to ensure good adhesion to the 150 millimeter dish. Once the methylene chloride has dried, flip the newly bound Petri dishes over and carefully inspect the points of contact. If a tight seal has not completely formed inject 0.5 milliliters of methylene chloride through the 3 millimeter hole with a transfer pipette.
Pick up the dish and gently rotate it to allow methylene chloride to reach the edge. After ensuring that a tight seal has been formed between the two Petri dishes, seal off the hole in the 100 millimeter dish by applying a 3 millimeter bead of silicone rubber sealant. Using a rotary tool fitted with a flat cutting head, cut off the top 3 centimeter portion of a 15 millimeter conocole polystyrene tissue culture tube, leaving at least one centimeter of the tube below the cap.
Use the flat side of the cutting head to polish the edge of the cut tube until smooth and flat. Remove any plastic shavings produced by grinding. With a marker trace the cut off 15 milliliter concole tube onto the lid of the 150 millimeter dish approximately 0.5 centimeters away from the edge of the dish.
Drill a hole inside the drawn circle, leaving a margin approximately one to two millimeters from the edge of the circle. Place the cut off conocole tube top over the drilled hole. Using a transfer pitette apply approximately a quarter milliliter, or 250 microliters of methylene chloride to the cut off edges of the conocole tube to bind the conocole tube to the 150 millimeter lid.
Finally, seal the 150 millimeter lid onto the 150 millimeter dish by applying a one millimeter thick bead of silicone rubber sealant around the inner edge of the top Petri dish. A schematic of an assembled sear ring is shown. The blue represents the area of plated endothelial cells.
The outer an inner red arrows indicate the orbital motion of the shear ring and media inside the shear ring when placed on an orbital shaker. Prepare for the sterilization by pipetting approximately 10 millimeters of PBS into the newly formed shear ring, through the 15 milliliter conocole tube port. Swirl around to remove any debris inside the shear ring.
Remove the PBS with a Pasteur pipette and vacuum aspirator. Repeat the PBS wash until all debris is removed. To sterilize the shear ring a combination of a 70%ethanol rinse and UV irradiation will be used.
Unscrew the vented cap, pipette approximately 10 milliliters of 70%ethanol through the access port and screw the cap back on. Rotate and flip over the sear ring multiple times ensuring that the inside of the sear ring is thoroughly washed. Under a tissue culture hood, aspirate the excess 70%ethanol.
With a spray bottle, thoroughly spray the access port and cap with 70%ethanol. Next place the shear ring and cap under UV radiation in the tissue culture hood until completely dry. Once the shear ring is dry screw the cap back on.
Store the sterilized shear rings at room temperature until they are used in cell culture plating. To prepare for a shear stress study, first plate endothelial cells in sterilized shear rings following the standard cell culture protocol. Allow the culture to reach confluency prior to initiation of flow studies.
When the cells have reached confluency place the experimental shear rings on an orbital shaker that is placed on the bottom shelf in the incubator to minimize shelf stress and vibration. Place static control group shear rings inside the same incubator. Leave the shear rings on the shaker for the desired duration of shear stress application.
After the cell layers have been exposed to shear for the desired length of time, remove the shear rings from the incubator. Pull off the shear ring lid and retrieve the cells and or media for the desired end point analysis. Note that after opening the shear rings investigators can harvest cells and medium for analysis using several methods.
Shown are representative results of the appearance of human CMEC D3 Brain Endothelial cell mono layers in shear rings following 48 hours of periodic fluid shear or static exposure it was observed that the alignment of the culture is not always parallel to the flow direction indicated by the arrows. Some responses to periodic shear are shown in Rat Retinal Microvascular Endothelial cell mono layers. 72 hours of periodic fluid shear in shear rings resulted in a significant reduction in Platelet-endothelial cell adhesion molecule relative to the Beta-actin loading control.
Once mastered several Shear Rings can be constructed in about three hours if it's performed properly While attempting this procedure it's important to remember to carefully align inner dish with the outer dish, and that tight seal forms between them. After watching this video you should have a good understanding of how to construct and use shear rings as a simple method to perform In Vitro periodic fluid flow and shear stress studies on endothelial cells. Don't forget that working with methylene chloride can be hazardous and precautions, such as working under a fume hood should always be taken while performing steps that require it.
