The overall goal of this video article is to describe a technique to subject adherent cells to laminar flow conditions and evaluate the response to well quantifiable fluid shear stresses. This is accomplished by first assembling the flow circuit without the flow chamber. The circuit includes the reservoir pulse, dampener, hard tubing, soft tubing, four-way stop cocks and connected lure adapters, as well as an air filter.
The next step is to fill the circuit with the desired profuse eight, such as cell medium under sterile conditions, and then start the pump to fill the pulse dampener and purge the tubing of air. This is followed by insertion of the cell seated slide into the bottom plate of the flow chamber. The final step of the procedure is the complete assembly of the flow circuit with the flow chamber in line.
Ultimately results can be obtained that show alignment of fluorescently labeled endothelial progenitor cells under fluid shear stress exposure through the transparent chamber using a fluorescent microscope To study the functional behavior of cells. It is often necessary to imitate in vivo physiology by subjecting the cells to fluid shear stress. We have designed a parallel plate flow chamber and continuous flow circuit, which allow us to study the cells under flow conditions and observe them through the transparent chamber.
This technique can serve as a valuable research tool for the fields of medicine and engineering. Our flow chamber allows for intermittent or real-time visualization of cells under flow using either upright or inverted microscope. Furthermore, Dr.Ach Nick's design allows researchers to image cells on either transparent or radiopaque surfaces using standard microscope lenses.
Further, it can as an important tool in pharmacology research to study the effect of drugs on cells under flow conditions. Visualization of the setup is critical because several of the steps are difficult to master by reading a protocol alone. I'll give step by step instructions on how to set up the flow chamber and flow circuit commonly used in Dr.Ock Next's laboratory To assemble the flow circuit.
Start by attaching a 36 inch segment of hard tubing to one end of a pulse dampener to the other end. Attach an 18 inch segment of soft tubing. Place a one eighth inch male lure adapter at the end of the 18 inch soft tubing section.
Connect the male lure to a one eighth inch female lure adapter. Attach the female lure to a new 18 inch segment of soft tubing. Drill three holes into a 250 milliliter glass beaker cap.
In order to fit the tubing, insert a 1.5 inch segment of soft tubing into one hole. As an air vent. Insert the free ends of the hard and soft tubing through the other two holes in the cap into the 250 milliliter glass bottle, which will act as a reservoir.
Ensure that the hard tubing reaches the bottom of the reservoir. The maintenance of sterility throughout the setup of the circuit is crucial to the success of this procedure. Therefore, all the parts must be sterilized and you must wear sterile gloves throughout.
In addition to the assembled parts, these parts must be sterilized. One complete flow chamber, three by two inch segments of soft tubing, one male and one female, one eighth inch lure adapter, four one half inch flathead screws, 6 5 16 inch, eight 30 seconds. Inch tracks per inch, flathead screws, one pair of tweezers, one glass slide, and two towels sterilized by steam.
Autoclaving at 121 degrees Celsius for 60 minutes. Place the flow circuit flow chamber four by four way stop cocks one by one way, stop cock and all the sterilized parts onto this sterile field. While wearing sterile gloves, connect the two four-way stop cocks.
Place caps on the open sample ports. Detach the male and female lure adapters attaching the two soft tubing segments of the flow circuit and insert the connected stop cocks. Attach the soft tubing inserted into the reservoir into a 30 milliliter syringe and remove the syringe piston.
Fill the bottle with 125 milliliters of EPC medium. Reconnect the soft tubing to the stop cock. Place a sterile syringe filter into the air vent.
Now transfer the flow circuit into a humidified incubator at 37 degrees Celsius, 5%carbon dioxide. Clamp the hard tubing into the master flex roller pump head indicated for use with this type of coal Palmer tubing. Ensure that the tubing does not overlap with the roller pump mounting tracks.
Mark the tubing where it exits the pump head on either side with a marking pen. After ensuring that all stop cocks are closed off towards the sample ports and open along the flow circuit, start the pump. Verify the direction of flow.
The PERFUSE eight should move from the glass reservoir through the hard tubing into the pulse dampener. For flow chamber assembly, use sterile gloves to connect one two inch soft tubing segment to a one eighth inch female lure adapter. Connect another two inch soft tubing segment to a one eighth inch male lure adapter.
Attach the two inch soft tubing with female lure adapter to the inflow port and the two inch soft tubing with male lure adapter to the outflow port. Attach four-way stop cocks to both of these lure adapters. Connect the remaining two inch soft tubing piece to the side port connector coming off the bubble trap and attach a one-way stop cock using sterile tweezers.
Remove the cell seeded slide from the cell culture vessel and place it into the recess on the bottom plate of the flow chamber. Make sure that the slide is placed with the cell seated side facing up using a syringe or pipette place 10 milliliters of warm EPC medium over the cell seated slide. Allow the medium to cover the slide in flow chamber, but do not spill the medium over the O-ring on the bottom plate.
Place the top plate of the flow chamber onto the bottom plate, aligning the screw holes. Keep the two plates parallel so as not to introduce air bubbles into the chamber screw plates.Together. Using an automatic battery operated screwdriver de air the inflow bubble trap by opening the one-way stop cock attached to the inflow side bubble trap port.
Use a 10 milliliter syringe to gently flush the inflow tubing with EPC medium and then close off this stop cock and cap it. Now de air the chamber channel by opening the outflow port four-way stop cock and gently flushing EPC medium through the flow chamber. Again using a 10 milliliter syringe cap all of the four-way stop cock connections and close them off.
Transfer the flow chamber into the humidified incubator with the assembled flow circuit. Pause the flow circuit pump close off the flow circuit. Stop cocks in the direction of flow to prevent leakage.
Connect the flow chamber to the flow circuit After the flow chamber has been connected to the flow circuit, open the stop cocks in the direction of flow. Resume the flow pump and ensure that the PERFUSE eight flows in the correct direction. Adjust the flow rate to the desired sheer stress.
During the flow experiment, the flow chamber can be removed from the circuit to image the cells via light or fluorescent microscopy. To do this, disconnect the flow chamber from the flow circuit at the stop cock to stop cock connections. To collect perfuse eight samples for analysis.
First, pause the pump, close the stop cock located closest to the pulse dampener. Remove its protective cap, placing it upside down to ensure it is not contaminated. Insert a small syringe into the sample port.
Close the stop cock off in the direction of the flow chamber, keeping the sample port and circuit in the direction of the pulse dampener open. Draw the desired amount of sample from the circuit, close off the stopcock towards the sample port before removing the syringe. Store the profuse eight sample in a labeled vial and freeze at minus 80 degrees Celsius.
Recap the sample port and ensure that all stopcocks are open before commencing flow. Using this method, human blood derived endothelial progenitor cells can be seeded onto titanium slides within 15 minutes and adhere under physiological shear forces. As shown in this figure, EPCs spread under the influence of flow from about 210 square microns immediately after seeding to about 657 square microns after three hours and about 1, 152 square microns after 48 hours of fluid shear stress of 15 dines per square centimeter.
This light microscope image illustrates the random orientation of human EPCs. After a static seating period of six hours after three hours of flow at 100 dines per square centimeter, the cells have spread but remain in a random orientation. After 48 hours of fluid shear stress at 100 dines per square centimeter, EPCs are aligned and oriented in the direction of flow.
Here EPCs, were labeled with CTO and cell nuclei stained with herx to die after flow. For comparison, this image shows porcine EPCs on titanium slides in alignment with the direction of flow. After 48 hours of flow and 15 dines per square centimeter of shear stress exposure, cell borders were stained with anti pcam stain and nuclei with cytoxin nucleic acid stain.
This method also allows for obtaining samples of the PERFUSE eight at predetermined time points for the analysis and or quantification of secreted cell metabolites. Shown here is an example of the production of nitrite during a 48 hour flow experiment with porcine EPCs. While attempting this procedure, it is important to maintain sterility throughout.
In addition, it is important to take the necessary steps in order to reduce the number of air bubbles in the flow chamber prior to the start of continuous flow as they may tear off the cells. We also recommend checking every single connection in the flow chamber and circuit prior to profusion to prevent leakage of PERFUSE eight during an experiment. This chamber is designed with built-in O-rings that allow for complete opposition of top and bottom plates and provide a leakproof seal without requiring vacuum pumps.
This feature also ensures a constant chamber height between experiments and eliminates the need for rubber gaskets or adjustments. Our flow chamber employs standardized microscope slides. Therefore, a large number of cells are available for further experiments.
For example, RNA and DNA from cells after flow can be evaluated for protein synthesis or gene expression. Our circuit design allows for collection of perfuse eight samples for analysis and quantification of cell metabolites such as nitrite, a primary oxidative metabolite of nitric oxide, which is secreted by cells under fluid shear stress. After watching this video, you should have a good understanding of how to assemble our flow chamber and sterile flow circuit to subject adherence cells to fluid shear stress.
