The overall goal of this procedure is to isolate endothelial progenitor cells from human umbilical cord blood. This method describes the isolation of endothelial progenitor cells from cord blood for their potential use as autologous cells for the tissue engineering of vascular grafts. The main advantage of this technique is that the isolation procedure can be done in specialized media without the need for any amino sorting.
The isolated endothelial progenitor cells can be used to study neovascularization, and reports have also suggested that these cells can be used as biomarkers to identify patients with risk for cardiovascular diseases. Before beginning the isolation procedure, pre-coat six well plates with two milliliters of freshly prepared rat tail one collagen solution per well. Equilibrate the plate in a 37 degree Celsius and 5%CO2 incubator for at least one hour.
Then dilute approximately 25 milliliters of cord blood with HBSS at a one-to-one concentration. Next add 20 milliliters of density gradient medium reagent to a 50 milliliter conical tube, and carefully dispense 20 milliliters of diluted cord blood down the wall of the tube to layer the blood onto the medium. Separate the cells by centrifugation, followed by careful removal of the plasma layer.
Using a syringe fitted with an 18-gauge needle, collect the mononuclear cell containing buffy coat into a new 50 milliliter tube. And add an equal volume of endothelial basal medium to the cells. Wash the cells by centrifugation and lyse the red blood cells in the resulting palate with five milliliters of ammonium chloride solution on ice.
After five to 10 minutes with occasional shaking, collect the cells by centrifugation and resuspend the white pellet in fresh endothelial growth medium. After counting, aspirate the collagen from each well of the six well plate, and wash the wells three times in PBS. Seed the mononuclear cells at a one times 10 to the seventh cells per two milliliters of medium per well concentration.
And return the plate to the incubator for 24 hours. The next day, rinse the cells one time with fresh endothelial growth medium. Then replace the wash with three milliliters of fresh endothelial growth medium, and return the cells to the cell culture incubator.
Using a bright field microscope, obtain daily images of the cells to monitor the progression of the endothelial cell colonies, marking the plates where the colonies arise to track their growth. When the endothelial cell colonies reach about three millimeters in size, coat a T25 culture flask with fresh rat tail one collagen for at least one hour in the cell culture incubator. Then harvest the cells with 150 microliters of 0.05%Trypsin-EDTA per well in the cell culture incubator for two to three minutes.
Gently tapping the plate to dislodge the attached cells. When the cells begin to detach, immediately add two milliliters of fresh endothelial growth medium, and pull the cells in a single 15 milliliter conical tube. Collect the cells by centrifugation, and resuspend the pellet in fresh endothelial growth medium.
After counting, seed the cells in the collagen coated flask at a five times 10 to the fifth cells in three milliliters of medium per flask density, and mark the flask as passage one. Then return the flask to the incubator, reseeding the cells whenever the colony reaches 90%confluency. After seeding onto the collagen treated plates, the first colony outgrowth is typically observed between days five to nine.
The colonies continue to grow and to acquire a spindle-shaped cell morphology in the early stages of culture, which later progresses to a cobblestone-like morphology. The total number of cells harvested at each passage increases in direct correlation to the total number of days in culture. Western blot analysis reveals an early expression of CD 31 and CD 34 by the cultured endothelial cells, which appears to decrease with subsequent passaging.
The expression of vascular endothelial growth factor receptor two, however, is maintained over time, although at a slightly lower level of expression than that observed after the first passage. Of note, endothelial progenitor cells are not positive for the mesenchymal cell marker alpha smooth muscle actin, unlike for example, valvular interstitial cell lysates, which can be included as a positive control. The entire isolation procedure can be completed in less than five hours depending on the cord blood unit obtained.
After isolating and seeding the mononuclear cells in endothelial growth medium, it usually takes between five and nine days for the EPC colonies to show up in the culture. The proposed method of isolation uses specialized media for culturing the endothelial progenitor cells, and avoids the usage of a flow cytometer, as far as the requirement for having endothelial markers. This method also provides an efficient protocol for obtaining high quantities of endothelial progenitor cells over a short span of time.
After watching this video, you should have a good understanding of how to isolate endothelial progenitor cells from human umbilical cord blood units. Don't forget that human umbilical cord blood can be hazardous, and proper disposal of waste blood and cell culture products should be followed.
