Name: the assembly and application of 'shear rings': a novel endothelial model for orbital, unidirectional and periodic fluid flow and shear stress
Uploaded: 2016-10-31T14:00:00
Description: Watch this Scientific Journal Video about The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress at JoVE.com

The authors would like to acknowledge the assistance of Mr. Christopher Nguyen, Aaron Hunter and the Shreveport Jumpstart, SMART, and Biostart training programs as well as the Centenary College of Louisiana department of Biophysics, Shreveport, LA.

1. Construction of 150 mm Diameter Shear Rings (Figure 1)
NOTE: Shear rings may be constructed to create many different dimensions by varying the outer and inner Petri dish sizes, resulting in devices with different total surface areas, cell yields and developed ranges of shear forces. This report describes a 150 mm dish combined with an inner 100 mm dish for a total surface area of 98 cm2 (Figure 2).
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<ol>
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The construction of the shear ring system for exposing endothelial cells to shear is a simple approach to performing shear stress studies. Nevertheless, there are a few steps that are critical for obtaining superior shear rings and better results. A complete seal should be made between the inner and outer ring to prevent media from leaking which could create inconsistent shear stress among samples. If a complete seal is not made, a minimal amount of methylene chloride should be added to the edge between the inner and out...

Fluid shear stress has been shown to modulate endothelial gene programs1-5 through activation of cis-regulatory elements6, histone acetyltransferase activity7 and shear stress response elements (SSRE)8. Shear stress influences endothelial contributions towards coagulation by modulating tissue factor9 and tissue plasminogen activator (tPA)10 expression. Shear stress also influences control of angiogenesis11 and vessel remodeling by regulating PDGF-B synthesis and responsiveness8. The endothelial derived vasoactive mediators adrenomedullin, endothelin-1, urotensin II and relaxin are also regulated by shear12. Transcription of endothelial nitric oxide synthase production and nitric oxide production are both shear dependent10. Shear also controls endothelial ICAM-1 expression13. Flow-induced shear stress can therefore powerfully influence a large variety of endothelial responses. Importantly, vascular pulsations now also appear to play important roles in the pathophysiology of both normal vascular aging and forms of vascular dementia14 and may even contribute to other neurodegenerative diseases, such as multiple sclerosis15.
Venous and arterial endothelial cells are inherently exposed to diverse hemodynamic flow patterns in vivo, and many different endothelial cell phenotypes can be exhibited16. Depending on the magnitude and periodicity of flow, effects on endothelial cells may include inflammatory cell activation and apoptosis, which may reflect changes in gene or protein expression17,18. Studies on endothelial cell responses to shear phenomena therefore remain complicated by the difficulties in producing in vitro models that adequately produce such shear patterns.
Many different experimental protocols have been developed to apply fluid shear stress to endothelial cell monolayers. One of the most commonly used systems is the parallel plate flow chamber, which creates uniform laminar flow within the chamber19-21. A peristaltic pump is typically connected to create periodic flow, which can recapitulate flow characteristics typically found in many locations in vivo22. Another common set-up uses the &#39;cone and plate&#39; model, where fluid shear stress is determined by the rotational speed of the cone23. Both systems, and other arrangements similar to them, can be tedious to set up and require components that can be relatively expensive and inaccessible to many laboratories.
Another major limitation of these current models is the relatively low number of replicate studies that can be simultaneously performed, each with a relatively low surface area. This increases the time and complexity of such approaches. Therefore, an ideal model that induces unidirectional and periodic shear might be one where a high number of study replicates can be easily set up, each with a relatively large surface area. Furthermore, the aforementioned models require a fairly sophisticated setup, which may be cost-prohibitive for many users. A model that can produce fluid shear disturbances using basic laboratory materials might have several advantages.
A simple and highly economical method of applying unidirectional, periodic shear stress involves the placement of circular dishes on an orbital shaker24. This protocol is very simple and can be scaled up to achieve high numbers of study replicates, each with a relatively large surface area, as needed. However, cells located in the center of the dish are exposed to different flow patterns than cells along the periphery, yielding mixed cellular phenotypic responses in the same dish.
In this current report, we describe the construction and use of &#39;shear rings&#39;, our model for creating unidirectional and periodic shear stress. The design for the shear ring effectively limits &#39;mixed&#39; cellular shear-induced phenotypes by restricting the flow pathway within a circular culture dish to the periphery through the placement of an inner ring. The construction and operation of the shear ring is simple and economical and can be easily scaled to accommodate a wide range of orbital shakers using widely available tissue culture supplies. This model can be applied in endothelial cell experiments to provide unidirectional and periodic flow patterns within physiological and pathophysiological levels.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">100 x 20 mm plastic tissue culture dish</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">430167</td>
			<td style="width:195px;">The dishes must be polystyrene</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">150 x 25 mm plastic tissue culture dish</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">430599</td>
			<td>The dishes must be polystyrene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm glass petri dish&#160;</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">3160150BO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15ml polystyrene tissue culture plastic tubes</td>
			<td>Falcon</td>
			<td align="right">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Methylene chloride</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">D65100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">silicone rubber sealant</td>
			<td style="width:119px;">DAP</td>
			<td align="right" style="width:119px;">7079808641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ethanol</td>
			<td style="width:119px;">Decon</td>
			<td align="right" style="width:119px;">2701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">3 mL transfer pipette</td>
			<td style="width:119px;">Becton-Dickinson</td>
			<td align="right" style="width:119px;">357524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">printer paper</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">scissors</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">gloves</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool and set</td>
			<td style="width:119px;">Dremel</td>
			<td style="width:119px;">4000-6/50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool cutting head</td>
			<td>Dremel</td>
			<td>EZ476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool drill head</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">distilled water</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">orbital shaker</td>
			<td style="width:119px;">VWR</td>
			<td style="width:119px;">57018-754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">incubator</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat retinal microvascular endothelial cells</td>
			<td>Cell Biologics</td>
			<td>RA-6065</td>
			<td></td>
		</tr>
	</tbody>
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the assembly and application of 'shear rings': a novel endothelial model for orbital, unidirectional and periodic fluid flow and shear stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Here we present representative results from both hCMEC/D3 brain endothelial cell and rat retinal microvascular endothelial cell monolayers, cultured in shear rings.
After allowing hCMEC/D3 brain endothelial cell monolayers to grow to confluence in complete EBM, the shear rings were placed on an orbital shaker for 72 hours. Using the equation from step 3.5, the calculated maximal shear stress <img alt="Equation 6" src="/files/ftp...

Watch this Scientific Journal Video about The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress at JoVE.com

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

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.

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