By simply culturing the total blood with animal serum free optimized medium. In a 75 square centimeter flask, E CFCs adhere to the flask surface to remove most of the red and white blood cells. Wash the flask on day one and eliminate the remaining cells by changing the total medium on day five.
Keep screening for colonies every other day, or if convenient three times per week. And you should observe the first colonies around day 12. Hi, my name is Nicole Hoffman from the lab of at the stem Cell Research Unit of the Medical University grots.
Today we will show you a simplified process for the isolation and expansion of endothelial colony forming progenitor cells or ECFC from adult human peripheral blood. In our lab, we use this process to study the function and biology of ECFC in vitro and in vivo. So let's get started.
This section begins with the preparation and filter sterilization of endothelial growth medium two, which will be used to culture. The EFCs prewarm 20 milliliters of medium in a 37 degree water bath. Draw five milliliters of adult human peripheral blood from the cubital vein into a six milliliter preservative free sodium heparin blood vacutainer tube.
Blood samples must be processed within two hours of collection in a laminar flow hood. A range 1 75 square centimeter flask with a vented cap, 1 25 milliliter pipette, and two five milliliter pipettes in a laminar flow bench. Use a 25 milliliter pipette to prefill the flask with 15 milliliters of prewarm supplemented E GM two.
Open the blood file and use the five milliliter pipette to directly transfer all five milliliters of blood into the flask. Use the pipette to rinse the empty blood vial with the remaining five milliliters of pre-war E GM two. Add this volume to the flask.
Close the vented cap of the flask, which should now contain 25 milliliters. Place the flask in an incubator set at 37 degrees, 5%carbon dioxide and 95%humidity for 24 hours. The next day prewarm 20 milliliters of supplemented e GM two and 30 milliliters of PBS in a 37 degree water bath and arrange 2 25 milliliter pipettes and three 10 milliliter pipettes in a laminar flow hood.
Remove the flask containing the sample from the incubator and use a 25 milliliter pipette to remove the medium blood mixture which contains non-adherent cells. Take care not to scratch the flask surface with the pipette. To avoid scratching off adherent cells gently add 10 milliliters of prewarm PBS to the inner flask cell adherent surface, and tilt the flask from side to side.
Immediately remove and discard the PBS. Repeat this, wash twice. Next, working quickly and gently.
To avoid detaching the cells, add 20 milliliters of fresh prewarm e GM two to the flask and place it back in the incubator. Taking care not to lead the flask outside the incubator for more than five minutes, use a low magnification light microscope to check for ECFC outgrowth in the flask every other day. Starting on day two on the fifth day after seeding pre-war 20 milliliters of EGM two in a 37 degree water bath and prepare 2 25 milliliter pipettes.
Working quickly to prevent drying. Use these supplies to replace the medium with fresh prewarm supplemented e GM two, which will remove non-adherent cells. Place the flask back into the incubator and repeat the screening process daily.
Replace 30%of the medium with freshly supplemented egm. Two twice a week. Typical colonies with cobblestone morphology should appear around day 12.
Mark the top outer side of the flask to indicate the position of each colony. Allow the colonies to grow until day 28, unless single cells start to detach. Once primary colonies have been identified, they can be harvested between days 14 and 28 to depending on their size.
Prewarm five milliliters of a trypsin EDTA solution and 20 milliliters of PBS to 37 degrees. Taking care not to touch the surface of the flask, transfer all medium into a falcon tube. This is used later to quench trypsin activity and carefully wash the cells with 10 milliliters of pre-war PBS.
Add five milliliters of tripsin EDTA and incubate the solution for approximately five minutes at 37 degrees or until the cells detach. Take care not to incubate for more than seven minutes, as trypsin can become toxic to the cells after incubation, strongly tap the sides of the flask to help the cells detach after the reaction. Quench the tryin protease activity by adding approximately 10 milliliters of the preserved used culture medium.
Transfer the cell suspension into a 50 milliliter falcon tube. Wash the flask once with 10 milliliters of PBS and add the wash solution to the harvested cell suspension. Spin the cell suspension at 300 times G for seven minutes in a refrigerated centrifuge to collect the cells and remove the trypsin, discard the supernatant and immediately resuspend the cell pellet in one milliliter of PBS.
Keep the resuspended cells on ice. Determine cell number as described in another JoVE protocol by Ricardo and Phelan. Then move on to ECFC expansion.
In the event that more than 360, 000 E CFCs are recovered, proceed to the expansion step in the event that less than 260, 000 e CFCs are recovered. Consider further expanding the cells before large scale expansion. Do this by seeding approximately 75, 000 EFCs in 20 milliliters of E GM two in a 75 square centimeter cell culture flask and culture for another week or until confluence is reached.
Supplement 500 milliliters of E GM two as described in the accompanying written protocol and prewarm this mixture in a 37 degree water bath. For each approximately 2, 500 square centimeters of culture space, prepare one four layered cell factory or CA four. In addition, gather two fresh Tyvek caps and a special sterile funnel and store them in the laminar flow hood to seed E CFCs at a starting cell density of 100 cells per square centimeter.
First, calculate the cell number for each CF four 250.2, 800. Cells are resuspended in 500 milliliters of fresh E GM two and poured into the CF four. Using a sterile funnel, use the covered Tyvek cap to close one vent and tilt the CF four to one longitudinal side to allow for the equal distribution of medium between all four levels.
Now the chamber can be tilted back and the lid of the Tyvek cap can be opened. Be sure to protect the open vents using two red caps. These caps can be purchased with the CF four on the way to the incubator.
Place the CF four in the incubator and remove the red caps to enable gas exchange change 20%of the medium with pre-war medium at least once weekly until the CF four is confluent. This process is donor dependent and will likely occur after 10 to 14 days. Finally, cells can be harvested as described earlier by using pre-war Tripsin EDTA and the used medium to quench the protease activity.
In addition, use approximately 200 milliliters of PVS for washing at around day 12. ECFC colonies will begin to appear with a typical cobblestone morphology as you can see in this colony or as a few grouped cells like in this colony found on day 12. As the colonies grow, more cells form the typical cobblestone morphology.
As you can see here, after 15 days and after 18 days by immunochemistry, the isolated e CFCs can be characterized with typical endothelial cell surface markers like CD 1 46, CD 1 44, CD 31 fermenting, and von Willebrand factor. We have just shown you how to isolate and expand an endothelial colony forming progenitor cells from the adult human peripheral blood while doing this procedure. It's important to remember that the success rate of this protocol is donor dependent and ranges from 50 to 70%So that's it.
Thank you for watching and good luck with your experiments.
