In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

The overall goal of the following experiment is to observe the effects of different pulsatile flow pathologies on endothelial cells. In vitro. This is achieved by first chemically treating glass slides and coating them with protein to ensure cell attachment during exposure to flow.

As a second step, the pulsatility index of the flow is determined, which allows for comparison of in vitro flow to values described in the literature. Next cell seated slides are connected to the flow system in order to measure cell response to flow pathologies. The results show changes in morphology and protein expression based on flow profiles using optical microscopy and immunofluorescent staining.

The main advantage of this technique over the peristaltic or pulsatile pump is the ability to adjust flow pulsatility to mimic diseased or healthy response. This can help answer key questions in the field of arterial disease. To begin place new glass slides measuring 75 millimeters by 50 millimeters by one millimeter into a clean glass slide staining dish.

Then add enough of a 20%sulfuric acid solution to fully immerse the glass slides and incubate them in the acid solution overnight the following day. Wash the slides three times by immersing them in fresh deionized water for five minutes each time. Once rinsed, immerse the slides in acetone for 30 minutes, and then overnight in a solution of 6%three amino propyl trixi seline in acetone.

Next, rinse the slides three times in pure acetone for five minutes changing acetone after each wash. Then wash slides three times in deionized water for five minutes, changing the deionized water after each wash. Next immerse slides in 1.5%glutaraldehyde and leave them submerged for 60 minutes.

Once cross-linked, wash the slides twice in deionized water, changing the deionized water After each wash, then place the slides into 70%ethanol for 30 minutes to sterilize them. Remove the ethanol and allow any remaining residue to evaporate from the slides. Once dry, rehydrate the slides by placing them briefly in sterile deionized water.

Next, remove the slide holder and place it into a 100 millimeter by 100 millimeter square Petri dish. In the laminar flow hood coat the functionalized slide with one milliliter of a 25 microgram per milliliter fibronectin solution, covering the approximate area that will be exposed to the cell culture chamber. Then cover the dish and incubate the slides at 37 degrees Celsius for one to two hours After incubation, aspirate the remaining solution and seed 600, 000 endothelial cells in one milliliter of DMEM with 10%FBS onto the coated side of the slide.

Cover the chamber and then incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 120 minutes. Then cover the cells with additional medium and continue to incubate them until they are 70 to 80%confluent. Connect the flow circuit as described in the accompanying text protocol and as shown here, ensure that the damping chamber and the ultrasonic flow meter are arranged in the correct flow direction.

Next, fill the flow, circuit and reservoir with deionized water. Ensure that the reservoir outlet tube is submerged within the reservoir volume. Then visualize the flow waveform using a flow meter.

Open the air release valve on the damping chamber to change the fluid to air volume ratio. Close the air release valve at different fluid level intervals and calculate the pulsatility index. To calculate the index, evaluate the flow waveform as the peak represents the VM max.

The trough represents the v min and the V mean is the mean of the signal record. The puls index values in a notebook. Mark the resulting fluid level and pulsatility index on the damping chamber using a felt tipped permanent ink pen for future use.

Determine the desired pulsatility index levels from pathology literature referenced in the accompanying text protocol while preparing the flow chamber warmup. DMEM supplemented with 1%FBS in a 37 degree Celsius water bath. Take one of the cell coated slides out of the media once they are 70 to 80%confluent and place a gasket on top of the cell seated side.

Next, align the vacuum channel of the flow chamber with the holes in the gasket. Then attach vacuum tubing to the vacuum ports, ensuring that no media leaks to the vacuum. Place a polycarbonate sheet underneath the glass slide and clamp the flow chamber assembly with one spring clamp on each of the short sides and two spring clamps along the long sides of the flow chamber.

Next, connect the flow chamber, inlet and outlet to the flow circuit. Place the flow system in an incubator set at 37 degrees Celsius and 5%carbon dioxide and fill the circuit with the prewarm media by filling the reservoir with the media and pumping it in at low pulsatility. Maintain this flow for four hours to precondition the cells.

Then adjust the fluid volume in the damping chamber to the pre-marked pulsatility index level and culture. The cells for the desired duration. The endothelial cells shown here were cultured with no flow, steady flow, and pulsatile flow.

Changes in morphology may be seen from the typical endothelial cell morphology to a more spindle like morphology after 24 hours of exposure to high pulsatile flow. While both static and steady flow show little or no difference in morphology. Smooth muscle cells are also affected by co-culture with endothelial cells exposed to high pulsatile flow as indicated here by different levels of alpha smooth muscle actin and myosin heavy chain.

Throughout the varying flow conditions, cells that were exposed to pulsatile flow showed the greatest amounts of both proteins, which is indicative of hypertrophy and contractility. While attempting this procedure, it's important to remember to maintain all seals and ensure fluid levels remain constant following this procedure. Other methods such as endothelial cell, vascular cell co-culture may be performed in order to answer additional questions such as cell to cell interactions.

After watching this video, you should have a good understanding of how to assemble the flow circuit, control the index, and expose endothelial cells to pulsatile flow.