Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

The overall goal of this protocol is to reliably generate blood outgrowth endothelial cells from human peripheral blood. This method can help answer key questions in the field of vascular biology, such as how endothelial cell dysfunction contributes to cardiovascular diseases including pulmonary arterial hypertension or von Willebrand disease. The main advantage of this technique is that it consistently generates a stable population of blood outgrowth endothelial cells from a small volume of adult peripheral blood Demonstrating procedure will be Christopher Wang, the graduate student from my laboratory and laboratory Professor Nicholas Morrell.

To begin this procedure. For each donor, add three milliliters of sodium citrate to each of the two 50 milliliter conical centrifuge tubes. Then add 30 milliliters of the collected blood to each tube.

Gently invert the tubes two to three times for mixing. Next, dilute 60 milliliters of blood with DPBS at a ratio of one to one tilt the tube containing the density gradient. Centrifugation medium at an angle of 20 degrees with the work surface using a pipette aid and a 25 milliliter serological pipette.

Slowly layer 21 milliliters of diluted blood on top of the medium. Afterward, centrifuge the samples at 400 times G for 35 minutes at room temperature with the accelerator and the break off During the centrifugation repair a collagen solution at 50 micrograms per milliliter by diluting stock. Type one collagen in 10 milliliters of 0.02 molar acetic acid sterile.

Filter the collagen suspension prior to use. Next, add 7.5 milliliters of collagen solution to a T 75 cell culture flask. Allow the flask to be coated for one hour at room temperature.

After one hour of coating, aspirate the collagen solution and pipette 10 milliliters of DPBS in the flask to wash away the residual acetic acid. Aspirate off the DPBS and repeat the wash once more. Then add five milliliters of BOEC generation medium to the flask to keep the collagen coating from drying out until the cell suspension is ready for plating.

Following the density gradient centrifugation carefully collect the buffy coat layer using a sterile plastic transfer pipette and avoid transferring the density gradient medium. The collection of buffy coat should yield approximately 20 milliliters of cel suspension and plasma from each tube. Afterward, dilute the mononuclear cell suspension in DPBS at a ratio of one to one and invert to mix then centrifuge at room temperature for 20 minutes at 300 times G with the brake and accelerator set at maximum after centrifugation, aspirate the supernatant and resuspend the cells by adding one milliliter of BOEC generation medium to each pellet and pipetting up and down repeatedly pull all the cell suspensions together and top up the total volume to 10 milliliters with the remaining medium.

To get an estimation of the total cell number dilute 10 microliters of the cell suspension with 490 microliters of Turk solution to ly the red blood cells. Then count a 10 microliter sample of the diluted cell suspension using a hemo cytometer. Subsequently plate the cell suspension into a single collagen coated T 75 flask.

Top the medium volume up to 15 milliliters per flask and culture. The cells at 37 degrees Celsius in a humidified atmosphere containing 5%CO2. Now change the medium every two days by adding 15 milliliters of fresh BOEC generation, medium to the culture for weekends, medium changes can be delayed to every three days.

Monitor the BOEC culture flask on day seven to 14 for the appearance of outgrowth colonies. Identify the colonies as circular groups of cells exhibiting the classic endothelial cobblestone morphology. Once a colony or multiple colonies are identified in the flask, continue with medium changes and allow the colonies to grow to approximately 1000 to 2000 cells per colony.

Before packaging, determine the cell number per colony through a rough visual estimate. Then passage the cells by rinsing the flasks twice with 10 milliliters of DPBS. Add five milliliters of one x tripsin EDTA and incubate in a 37 degrees Celsius incubator for five minutes.

After five minutes, neutralize the trypsin with 10 milliliters of medium containing FBS and bring the cells into suspension with repeated pipetting. Next centrifuge the suspension at 300 times G for five minutes. Resuspend the cells in 15 milliliters of fresh BOEC generation, medium, and plate the entire cell suspension into a new T 75 flask without collagen coating.Continue.

Medium changes until the cells are confluent and passage. The cells as described previously for continued packaging plate. No fewer than 750, 000 cells per T 75 flask as low cell densities can cause the BO eecs to stop proliferating.

In this procedure, trypsin eyes the cells and centrifuge them at 300 times G for five minutes. After that, aspirate the snat and resuspend the cells in 10 milliliters of medium. Collect a 10 microliter sample of the cell suspension and perform a manual cell count.

Next, centrifuge the sample again and resuspend it in ice cold cryo-preservation medium at two times 10 to the six cells per milliliter. Add 0.5 milliliters of cell suspension to each vial and place the vials in an ice cold isopropanol cryo-preservation vessel. Then place the vessel in a negative 80 degrees Celsius freezer for at least two hours before transferring to liquid nitrogen.

To thaw the cells, add 10 milliliters of prewarm medium to a 15 milliliter conical centrifuge tube. Remove the cells from liquid nitrogen and thaw in a 37 degree Celsius water bath with gentle agitation until only a small ice crystal is left in the vial. Then add the contents of the vial dropwise to the conical centrifuge tube and spin down at 300 times G for five minutes.

Subsequently, aspirate the supernatant and resus suspend the cell pellet. Add the cell suspension to the T 75 flask and top up the medium to 15 milliliters. In this image, the outgrowth colonies, which appear as collections of endothelial like cells are arranged in a cobblestone monolayer and proliferate radially out from a central point surrounding the outgrowth colonies are the adherent monocytic cells that make up the vast majority of cells in early cultures Following packaging, the highly proliferative bo Oecs take over the culture as the non-proliferative monocytic cells either die off or fail to reattach.

Here is a representative immunofluorescence image of Bo Oecs immunos stained with an antibody against endothelial cell surface marker CD 1 44. And in this image B oecs were immuno stain with an antibody against the blood glycoprotein von Willebrand factor. Once master, this technique could be done in less than two hours if performed properly.

While attempting this procedure, it's important to remember process the blood samples as soon as possible and preferably within two hours of collection. Following this procedure, cells can be used to study endothelial cell function in health and disease or reprogramed into induced flury potent stem cells for use in differentiation studies, disease modeling, or cell therapy After its development. This technique paved the way for us to study the impact of mutations in the type two bone morphogenetic protein receptor on the pathogenesis of pulmonary arterial hypertension in both patient blood outgrowth endothelial cells and IPSC derives mood muscle cells.

After watching this video, you should have a good understanding of how to generate and characterize stable blood outgrowth endothelial cells from a small volume of peripheral blood. Don't forget that working with unscreened human blood can be extremely hazardous and personal protective equipment, including gloves and a lab coat should always be worn while performing this procedure.