The protocol provides highly proliferative blood outgrowth endothelial cells that can be used for various tissue engineering, cell therapy, and disease modeling investigations. This technique consistently yields many cells from a single blood draw. For this technique to be successful, it is crucial to quickly get the cells into the culture medium and incubator environment as soon as possible.
First, dilute heparin solution to 100 units per milliliter in sterile saline. Add three to four milliliters of heparin solution to each of two 50-milliliter conical tubes and two 60-milliliter syringes. Then, draw undiluted 1, 000 units per milliliter heparin solution into an extension tube.
Connect a 19-gauge needle to one end of the heparin-filled extension tube, and a heparin-containing 60 milliliter syringe to the other end of the extension tube. Next, clean the femoral groin area of an anesthetized pig with a Betadine scrub solution. Puncture the femoral vein or artery with a 19-gauge needle and at one to two milliliters per second, slowly draw 50 milliliters of blood into the syringe.
Leaving the needle in place, immediately kink the extension tube and disconnect the 60 milliliter syringe. Slowly transfer the blood to a heparin-containing 50-milliliter conical tube. Tightly cap the conical tube, gently invert the tube a few times to mix, and place it on ice.
Connect the remaining heparin-containing 60-milliliter syringe to the extension tube. Unkink the extension tube, and slowly draw additional 50 milliliter blood into the syringe. Immediately remove the needle from the artery or vein and slowly draw the remaining blood from the extension tube into the syringe.
Disconnect the 60-milliliter syringe from the extension tube and slowly transfer the blood to the remaining heparin-containing 50-milliliter conical tube. Tightly cap the 50-milliliter conical tube, gently invert a few times to mix, and place on ice. Apply pressure hemostasis to the pig's femoral groin area.
Dilute the blood one-to-one with PBS in four 50-milliliter conical tubes. Add 25 milliliters of room temperature, ready-to-use density gradient solution to eight 50-milliliter conical tubes. Slowly pipette 25 milliliters of blood saline solution inside each 50-milliliter conical tube containing density gradient solution by holding the tube at a sharp angle to the pipette tip.
Centrifuge the tubes for 30 minutes at 560 g and room temperature. Carefully collect the buffy coat, which is the cloudy layer of mononuclear cells above the density gradient solution and below the clear plasma, from each tube with a thin pipette and distribute it evenly into two new 50-milliliter conical tubes. Discard the density gradient solution containing tubes.
To coat a six-well plate with fibronectin, make a stock solution of human plasma fibronectin by diluting it to one milligram per milliliter in sterile water. Make 3.6 milliliters of a working solution of fibronectin by diluting 600 microliters of fibronectin stock solution in three milliliters of PBS. Transfer 600 microliters of the fibronectin working solution into each well of a six-well plate and gently rock until evenly coated.
And gently aspirate the solution out of each well. To wash the mononuclear cells while waiting for the fibronectin to coat, top up the tubes with up to 45 milliliters of PBS, centrifuge for five minutes at 560 X g and four degrees Celsius with low break. Aspirate the supernatant from both tubes.
Resuspend each cell pellet in 25 milliliters of PBS. Resuspend each cell pellet in five milliliters of PBS and add 15 milliliters of 0.8%ammonium chloride solution to each tube. Wash the cells as before by adding PBS.
Aspirate the supernatant from both tubes. Resuspend each cell pellet in 25 milliliters of PBS. Resuspend each cell pellet in six milliliters of EGM-2 culture medium supplemented with 10%FBS and 1X antibiotic-antimycotic solution.
Gently transfer two milliliters of the cell suspension to each well of the fibronectin coated six-well plate and gently rock until evenly coated. 24 hours after seeding the cells, gently add one milliliter of fresh culture medium to each well. The following day, gently aspirate 1.5 milliliters of culture medium from each well, and replace it with 1.5 milliliters of fresh culture medium.
For the next five days, gently change the culture medium with two milliliters of fresh culture medium per well. Regularly visualize the cell cultures under low power 4X objective light microscopy. Morphology of the cultured cells was observed from the start of the culture until BOEC colonies were observed.
A smaller population of adherent cells started to attach to the culture dishes and grow, while non-adherent cells were removed with culture medium changes. Colonies first appeared on day six as a collection of endothelial-like cells proliferating radially outward from a central point. As the culture progressed, cell colonies became more dense and displayed a cobblestone morphology similar to mature endothelial cells.
Two-dimensional phase contrast microscopy was used to image tube formation in serum-free medium and complete growth medium. Cells in both media conditions underwent morphological differentiation and rapidly organized into extensive networks of capillary tube-like structures. BOECs were further characterized by the expression of mature endothelial cell marker CD31 or platelet endothelial cell adhesion molecule one.
BOECs showed uniform expression of CD31. Furthermore, flow cytometry analysis of BOECs with CD14 AF 700 antibody compared to positive control confirmed the absence of EPCs, as the cell stained negative for monocyte marker CD14 compared to the positive control group of PBMCs. Positive CD14 staining shown in blue, and unstained cells shown in red.
During blood collection, it is essential to collect as much of the buffy coat layer as possible while collecting as little of the adjacent layers as possible. This technique has enabled researchers to study new strategies for endothelializing implantable cardiovascular devices using a pig model with autologous blood outgrowth endothelial cells.
