The overall goal of this method is to isolate and culture primary endothelial cells from dogs to study cardiovascular disease in vitro. This method can help answer key questions in the field of cardiovascular research such as the behavior of endothelial cells during angiogenesis, tumor angiogenesis, and atherosclerosis. The main advantage of this technique is that a pure population of primary endothelial cells can be obtained and cultured even from very small blood vessels.
Generally, individuals new to this method will struggle because the inversion and suturing of the vessel can seem difficult, but are in fact easily learned in practice. To isolate and culture primary canine endothelial cells or CAPECs, use 0.5%gelatin to pre-coat six-well plates and incubate in a humidified chamber at 37 degrees Celsius and 5%CO2 for two hours. After aseptically removing blood vessels of interest from a canine cadaver according to the text protocol, transfer the blood vessels to Petri dishes filled with ice cold HBSS.
Then using surgical scissors, remove any adherent tissue and fat from the outside of the vessel making sure the vessel itself is visible at all times when cutting and keeping the vessel itself intact. Use ligatures to close any branches of the vessel and then use surgical scissors or a scalpel to remove the branches. With a curved Halsted mosquito forceps, carefully enter a vessel end, clamp the tip of the forceps at the other end of the vessel and then retract, slowly inverting the vessel until it is completely inside out with the endothelial cell layer on the outside.
It is important to keep the blood vessel moistened with HBSS to facilitate the inversion process. Next, place purse string sutures at both ends of the vessel to close it completely to prevent exposure of any non-endothelial vascular tissue to digestion. Then using the ligature ends to manipulate the inverted vessel, transfer the vessel to a 50 milliliter tube and use HBSS to rinse it twice to remove erythrocytes or residual freezing medium in case of thawing.
Add 30 milliliters of collagenase type two and dispase in canine endothelial cell growth medium or CECGM and incubate the tube at 37 degrees Celsius with gentle agitation to digest the vessel. Remove the vessel from the tube and centrifuge the cell suspension at 250 times g for five minutes. Then resuspend the cell pellet in CECGM and seed two milliliters of the suspension per well in one to three wells depending on the vessel size.
Culture the cells at 37 degrees Celsius in a humidified atmosphere with 5%CO2 in air and change the culture medium twice a week. When the cells reach 70 to 80%confluency, use pre-warmed HBSS to wash away dead or non-attached cells. Then add 200 microliters of recombinant cell dissociation enzyme to each well and place the dish back into the incubator for five minutes or until the cells detach.
Transfer the cells to a 15 milliliter tube and stop trypsinization by adding 10 milliliters of CECGM with 10%FCS, centrifuge for five minutes at 250 times g. Then continue the culture in a gelatin pre-coated plate or flask. To characterize canine aortic endothelial cells or CnAOECs, culture the cells in CECGM on a 0.5%gelatin pre-coated T75 flask.
When the CnAOECs reach 70 to 80%confluency, trypsinize the cells as demonstrated earlier in this video. Then centrifuge the cell suspension at 250 times g for five minutes. Discard the supernatant and use one milliliter of culture medium to resuspend the cells.
Dilute a 10 microliter aliquot of the cell suspension one to one with 0.4%Trypan Blue. Then use an automated cell counter to count the cells and plate four times 10 to the fifth viable cells in a new T75 flask. Add 10 milliliters of pre-warmed CECGM to the flask and culture at 37 degrees Celsius in a humidified atmosphere with 5%CO2.
To isolate RNA from passage one of CAPECs and CnAOECs, collect at least one times 10 to the three cells as a pellet. Add 20 microliters of sample preparation reagent and incubate for one minute to lyse the cells. After the incubation, store the sample at negative 70 degrees Celsius.
Measure gene expression and assess EC functionality according to the text protocol. Using the protocol demonstrated in this video, it was possible to dissect and invert aorta, vena porta, vena cava, and coronary artery from healthy dogs and two congenital portosystemic shunts. Between one and two weeks post-isolation, adhered endothelial cells were visible in the culture plate.
CAPECs from aorta, vena cava, and vena porta had a polygonal shape and grew in patches and reached 80%confluency after 10 days in culture. On average, the cells could be maintained a maximum of eight passages and then stopped growing. As seen here by qPCR, isolated endothelial cells expressed the endothelial cell marker CD31.
The expression in endothelial cells derived from aorta, vena cava, and vena porta from four dogs exhibited comparable CD31 expression to a control culture of CnAOECs. This figure shows that CAPECs derived from aorta, vena cava, and vena porta displayed branching six hours after incubation on the angiogenesis slide. Once mastered, this technique can be done in three hours.
While attempting this procedure, it's important to remember to work aseptically. Following this procedure, other methods like angiogenesis assays can be performed in order to answer additional questions like whether gene silencing or certain drugs will influence endothelial cell behavior. After watching this video, you should have a good understanding of how to isolate primary endothelial cells from blood vessels, a technique that can also be applied to normal and abhorrent blood vessels from other species.
