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

Summary

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.

Abstract

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.

Introduction

Fluid shear stress has been shown to modulate endothelial gene programs^1-5 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 expression^17,18. 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 chamber^19-21. 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 'cone and plate' 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 'shear rings', our model for creating unidirectional and periodic shear stress. The design for the shear ring effectively limits 'mixed' 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.

Protocol

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).

Figure 1. Shear ring assembly. The top portion of the figure shows a partially assembled shear ring. Inject 0.5 ml of methylene chloride through the 3 mm hole with a transfer pipet if a tight seal has not completely formed between the inner and outer dishes. The shear ring is sealed shut by applying a 1 mm thick bead of silicon rubber adhesive around the inner edge of the shear ring top. The bottom portion of the figure shows the assembled shear ring. The blue represents the area of plated endothelial cells. The outer and inner red arrows indicate the orbital motion of the shear ring and media inside the shear ring when placed on an orbital shaker. Please click here to view a larger version of this figure.

Figure 2. Surface measurements for a 150 mm shear ring (not drawn to scale). Top panel shows centrifugal shifting of fluid towards outer ring in response to orbital rotation. Please click here to view a larger version of this figure.

1. Create a shear ring construction template by generating a 150 mm outer edge profile in presentation software with a 100 mm internal diameter placed in the center of the 150 mm circle. Print the template on a sheet of A4 white paper.
2. Pipet 10 ml of methylene chloride (dichloromethane) into a 150 mm glass Petri dish. It is critical that the dish is glass and not polystyrene (or other vinyl plastic), as methylene chloride solubilizes most plastics and is used here to join plastic components.
   CAUTION: Use gloves for all construction with adequate hood ventilation. Methylene chloride is a contact irritant and is hepatotoxic.
3. Align a 150 mm plastic Petri dish onto the outer shear ring template.
4. Drill a 3 mm hole in the center of a 100 mm dish using a rotary tool. Remove any plastic shavings produced from the drilling.
5. While holding with a gloved hand, invert and dip the top edge of the 100 mm Petri dish from the previous step into the pool of methylene chloride for approximately 3 sec.
6. Transfer the "wetted" 100 mm dish edge-down onto the center of a 150 mm dish, carefully aligning the 100 mm dish onto the marked template. The methylene chloride will fuse the plastic, joining the 100 mm and 150 mm dishes. Gently rotate the 100 mm dish clockwise and counterclockwise a few times to ensure good adhesion to the 150 mm dish.
   NOTE: Take great care to ensure proper alignment of the inner dish to the center of the outer dish. Eccentric alignment may result in variations of the shear stress at different locations in the shear ring. Take care to not allow methylene chloride to accidentally drip onto the outer track portion of the 150 mm Petri dish during the placement of the 100 mm dish. This will melt the plastic on the bottom surface where cells will be growing, creating an uneven surface that may cause flow disturbances.
7. Once the methylene chloride has dried, flip the newly bound Petri dishes over and carefully inspect the points of contact to ensure that a tight seal has been formed between the two Petri dishes.
8. If a tight seal has not completely formed, inject 0.5 ml of methylene chloride through the 3 mm hole with a transfer pipet. Pick up the dish and gently rotate it to allow the methylene chloride to reach the edge.
   NOTE: If rotated too quickly, the methylene chloride can spill into the outer portion of the 150 mm dish, deforming the surface where cells will grow and ruining the shear ring. Leaking shear rings should be discarded.
9. Seal off the hole in the 100 mm dish by applying a 3 mm bead of silicon rubber sealant.
10. Using a rotary tool fitted with a flat cutting head, cut off the top 3 cm portion of a 15 ml conical polystyrene tissue culture tube, leaving at least 1 cm of the tube below the cap. Polish the edge of the cut tube until smooth and flat using the flat side of the cutting head. Remove any plastic shavings produced by grinding.
11. Trace the cut off 15 ml conical tube onto the lid of the 150 mm dish with a marker, approximately 0.5 cm away from the edge of the dish. Drill a hole inside the drawn circle, leaving a margin approximately 1-2 mm from the edge of the circle.
12. Place the cut off conical tube top over the drilled hole. Using a transfer pipet, apply approximately 250 µl of methylene chloride to the cut off edges of the conical tube to bind the conical tube to the 150 mm lid.
13. Seal the 150 mm lid onto the 150 mm dish by applying a 1 mm thick bead of silicon rubber sealant around the inner edge of the top Petri dish.

2. Sterilization of Shear Rings

1. Pipet approximately 10 ml of phosphate buffered saline into the newly formed shear ring through the 15 ml conical tube port. Swirl around to remove any debris inside the shear ring. Remove the phosphate buffered saline with a Pasteur pipet/vacuum aspirator.
2. Repeat the previous step until debris is removed.
3. To sterilize the shear ring, use a combination of a 70% ethanol rinse with UV irradiation.
   1. Unscrew the vented cap, pipet approximately 10 ml of 70% ethanol through the access port, and screw the cap back on. Rotate and flip over the shear ring multiple times, ensuring that the inside of the shear ring is thoroughly washed.
   2. Under a fume hood, aspirate out excess 70% ethanol. With a spray bottle, thoroughly spray the cap and access port with 70% ethanol.
   3. Place the shear ring and cap under UV radiation within the tissue culture hood until completely dry.
4. Once dry, screw the cap back on and store sterilized shear rings at room temperature until used in cell culture plating.

3. Shear Stress Studies

1. Plate endothelial cells in sterilized shear rings following the standard cell culture protocol typically using a 1:4 split ratio for transformed endothelial cell lines.
   NOTE: Rat retinal microvascular endothelial cells were obtained commercially. Human brain endothelial cells (hCMEC/D3) cells were provided as a generous gift from Dr. Pierre-Oliver Couraud (Inserm, France) and were cultured in complete endothelial basal medium (EBM).
2. Allow cultures to reach confluence prior to initiation of flow studies. Use 30 ml of tissue culture media (10% fetal calf serum, Dulbecco's modified Eagle's medium with 1% penicillin/streptomycin/amphotericin). Change cell culture medium every 3 days and maintain cells at 37 °C with 7.5% CO₂ and 92.5% room air.
3. Place the orbital shaker in the incubator.
   Note: Orbital shakers are typically heavy (>10 kg). Place the orbital shaker on the bottom shelf of the incubator to minimize shelf stress and vibration.
4. Place the experimental shear rings on the orbital shaker. Place static control group shear rings inside the same incubator.
5. Estimate the maximum shear stress within the shear ring with the equation where is the radius of rotation for the orbital shaker (in cm), is the viscosity of the medium (in poise), is the density of the medium (in g/ml), and is the frequency of rotation (in rotation/sec)²⁴.
6. Initiate the rotation setting on the orbital shaker to the desired rpm (e.g., 90 rpm), leaving the shear rings on the shaker for the desired duration of shear stress application (e.g., 72 hr).
   NOTE: Orbital shakers can produce heat, so the incubator temperature should be monitored initially to ensure 37 °C is maintained throughout the duration of the experiment. Alternatively, shear rings can be transferred into environmental chambers (e.g., modular incubator chamber) and then placed within a rotating incubator.
7. After cell layers have been exposed to shear for the desired length of time, remove shear rings from the incubator. Pull off the shear ring lid and retrieve cells and/or media for examining desired endpoint analyses (e.g., Western blotting, fluorescence activated cell sorting, etc.)²⁵.

Results

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

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 x 20 mm plastic tissue culture dish	Corning	430167	The dishes must be polystyrene
150 x 25 mm plastic tissue culture dish	Corning	430599	The dishes must be polystyrene
150 mm glass Petri dish	Fisher	3160150BO
15 ml polystyrene tissue culture plastic tubes	Falcon	352099
Methylene chloride	Sigma-Aldrich	D65100
silicone rubber sealant	DAP	7079808641
ethanol	Decon	2701
3 ml transfer pipette	Becton-Dickinson	357524
printer paper
scissors
gloves
rotary tool and set	Dremel	4000-6/50
rotary tool cutting head	Dremel	EZ476
rotary tool drill head
distilled water
orbital shaker	VWR	57018-754
incubator
Rat retinal microvascular endothelial cells	Cell Biologics	RA-6065