The saphenous vein is suddenly exposed to arterial conditions after coronary artery bypass surgery. Understanding its adaptation response to mechanical stress may help designing therapeutic tools to prevent vein graft failure. This endothelial cell isolation protocol is very simple and only requires incubation with collagenase.
With minimal vessel manipulation, this results in reduced contamination with smooth muscle cells and fibroblasts. The protocol for isolating human SVECs and culture them under shear stress and cyclic stretch is essential to understand the contribution of mechanical forces in endothelial cell dysfunction associated with saphenous vein graft failure. Prior endothelial cell culture is very demanding in terms of cell media supplements.
It is mandatory to add growth factors to successfully isolate a culture, the human SVECs. For isolating primary human saphenous vein endothelial cells or human SVECs, collect at least a two-to three-centimeter long saphenous vein segment. Working under a sterile laminar flow hood, transfer the vein segment to a Petri dish filled with pre-warmed PBS.
To remove all the blood from inside the lumen, insert a pipette tip attached to a pipette and filled with PBS into one end of the vein and gently flush it. Flush it until the washing flow becomes completely clear. Then close one end of the vein with a sterile cotton suture.
Carefully fill the vessel with one milligram per milliliter collagenase type 2 solution and close the other end of the vessel with a cotton suture. Incubate the vessel with collagenase solution in a humidified atmosphere of 5%carbon dioxide at 37 degrees celsius for one hour. Then cut one end of the vessel over a 15-milliliter tube to collect the collagenase solution before cutting the other end.
Flush the luminal surface with one to two milliliters of PBS to collect all the detached endothelial cells inside the tube with collagenase. Centrifuge the tube at 400G in room temperature for five minutes. After removing the supernatant, re-suspend the cell pellet in three to four milliliters of complete endothelial cell medium containing an additional heparin solution.
Plate the cells in a 60-millimeter cell culture dish pre-coated with 3%weight per volume gelatin. Incubate the cells in a humidified atmosphere for four days without changing the medium. After that, change the medium every other day without adding the heparin supplementation.
Examine the plate under the microscope to ensure that the red blood cells were removed. After one to four days post extraction, depending on the size and diameter of the vein segment, visualize the human SVECs by phase contrast microscopy to evaluate the degree of confluence and their cobblestone morphology. Continue changing the media every two days until the cells reach 70%to 80%confluency.
Coat the flow chamber slide with 0.1%weight per volume gelatin and incubate for at least 30 minutes at 37 degrees Celsius. To equilibrate the fluidic unit and the sterile perfusion set having 10 millimeter reservoirs, incubate those overnight in a humidified atmosphere. For the shear stress experiment, seed 200, 000 endothelial cells suspended in 100 microliters of culture medium into the gelatin-coated flow chamber slide.
For attaching the cells to the culture surface, incubate the cells for four hours. Place the perfusion set on the fluidic unit. Fill in the reservoirs and the perfusion set with about 12 milliliters of pre-warmed complete endothelial cell medium.
Remove air bubbles from the perfusion set to equilibrate the level of both reservoirs at five milliliters. Place the fluidic unit on the mounted perfusion set without the chamber slide in the incubator and connect its electric cable to the computer-regulated pump. By running the pre-defined protocol in the pump control software, remove all air bubbles from the reservoirs and the perfusion set.
Take the fluidic unit with the mounted perfusion set to the laminar flow hood and attach the slides having a confluence cell monolayer. After placing the whole unit with the slides in the incubator, connect its electrical cable to the pump. In the pump control software, adjust the fluidic unit setup by selecting the type of slide and setting the viscosity of the medium.
In flow parameters, enter the shear stress value and set the cycle duration and flow type. Then click the play button to start the experiment. Remember to maintain the slide with cells treated with the same media without exposure to flow as static controls.
Apply silicone-based lubricant to the tops and sides of the 25-millimeter 6-well equibiaxial loading station. Seed 400, 000 cells suspended in three milliliters to each well of the 6-well flexible-bottomed culture plates coated with collagen one. Incubate the cells in humidified air until they reach 100%confluency.
Prior to starting the stretch protocol, wash the cells once with pre-warmed PBS and add three milliliters of fresh culture medium per well. Insert the base plate with all lubricated loading stations in the incubator and appropriately connect the tubing with the controller equipment of the tension cell stretching bioreactor system. Attach each culture plate to one red gasket provided by the bioreactor system.
Then put them into the base plate in the incubator. Place all four culture plates in the base plate to achieve proper vacuum and stretching load. Turn on the controller equipment and the computer system.
Open the FlexCell control software and configure the desired regimen including the percentage of elongation, waveform shape, frequency, and time. Save the regimen. Turn on the vacuum system, then select the regimen and click start on the computer screen to run the stretching.
Check that the silicone membrane is moving and stretching the cells. The adhered endothelial cells could be observed 4 to 10 days after the extraction. They initially form cell clusters displaying a typical cobblestone morphology.
They expressed the EC markers CD31 and VE-cadherin. The hSVECs when cultured under shear stress aligned in the direction of the flow. The shear stress of 20 dynes per square centimeter for 72 hours induce the expression of typical mechanosensitive genes, KLF2, KLF4, and NOS3, indicating the effectiveness of the shear stimulus.
The cyclic stretch outcome depended on the intensity applied to the human SVECs. Cells under low stretch showed a cortical F-actin pattern similar to static cells. Without change in the nitric oxide release for up to 72 hours, our arterial levels of stretch remodeled the actin cytoskeleton after 24 hours and decreased NO release after 72 hours.
Besides mechanical stress studies, researchers can use the isolated cells to perform various methods to expand knowledge of endothelial cell function and dysfunction. Following the demonstrated protocol, it's possible to study any other type of endothelial cells under shear stress and stretch.
