The human umbilical cord is an easily accessible source of progenitor cells. In this article, we describe a protocol for the isolation and subsequent expansion of endothelial progenitors e CFCs, a subpopulation of cells within the vessel wall and mesenchymal progenitor cells. MSCs derived from a single human umbilical cord.
First using scissors longitudinally, cut along the human umbilical vein and scraped the cells from the inner vessel surface. Transfer the cells from the scraper into a flask and wait for colony outgrowth. Some of the scraped cells will exhibit a progenitor phenotype and will proliferate heavily to forming large colonies.
Once these cells have proliferated, the e CFCs can be expanded in new flasks. For further analysis, a mesenchymal cell population can also be isolated from the human umbilical cord. Cut parts of the cord into small pieces and transfer them to a sterile culture plate.
After a few days, the stromal cell outgrowth will be visible around the intervascular cord tissue pieces. Transfer these cells into new flasks and expand them for further analysis. This protocol allows you to obtain two types of lineages of progenitor cells from a single starting material cord within three to four weeks.
Hi, I'm Andreas Danish from the lab of Dr.T Strong in a stem cell research unit at the Medical University of Grads in Austria. Today we'll show you a procedure for the isolation, expansion and analysis of human mesenchymal and endothelial channel cells from one umbilical code. Just one note about terminology.
Our cells are called multipotent, mesenchymal stromal cells, or MSEs and endothelial colon forming pro cells or e CSCs. We use this procedure in our lab to study non hematic progenitor cell function and biology. So let's get started.
Prior to cell isolation steps, wipe down the laminar flow tissue culture hood with inadine and gather sterile cell culture plates. Cell culture flasks, PBS sterilized surgical instruments, cell scrapers, a tube holder, 25 milliliter strip pets, and a five milliliter syringe fitted with a blunt end needle. Remember to prewarm the alpha MEM and EGM two cell culture medium in a 37 degree water bath.
Now transfer the umbilical cord into the laminar flow and wash it twice in PBS. To remove contaminating blood, use a sterile five milliliter syringe to cannulate the cord vein on one side. Now rinse the umbilical vein with PBS until the fluid flowing out.
The other end of the cord becomes completely transparent with no red tint. Begin endothelial colony forming progenitor cell isolation by filling a 75 square centimeter flask with 15 to 20 milliliters of prewarm EGM two medium. Place the cord into a new plate filled with sterile PBS and use surgical scissors to cut the cord into two pieces that are each about five centimeters long.
Now insert one blade of the surgical scissors and cut the vein longitudinally. At this point, an assistant can use forceps to hold the vein while further cuts are made. Place the cord with the open vein into a new plate.
Without PBS, use a cell scraper to rub the inner surface of the vein over an area of at least one centimeter. Wash the scraper in the EGM two medium to release the rubbed vessel cells into the flask. This procedure will need to be repeated up to 10 times.
Close the flask and put it into an incubator set at 37 degrees, 5%carbon dioxide, and 95%humidity. Now that the e CFCs have been isolated, let's move on to the MSCs. Now that the endothelial layer has been scraped off, use a sterile scalpel to cut the cord into very small pieces.
One to two millimeters. Maximum use sterile forceps to transfer these pieces into a pre-labeled culture plate. Leave the plate open for at least five minutes before filling it with medium to allow the pieces to adhere to the plastic surface.
Using the lowest speed possible gently add 30 to 35 milliliters of prewarm alpha modified MEM. Close the plate and place it in an incubator set at 37 degrees with 5%carbon dioxide and 95%humidity. Outgrowth of mesenchymal stromal cells from the cord pieces will take three to seven days after which the cells can be expanded.
Meanwhile, ECFC can be expanded, which will show you in the next step. The next day, use an inverted microscope to examine the cells. If the scraping procedure was done properly, the first proliferating cell clusters will be visible completely.
Replace the E GM two medium to remove all non-attached cells and particles. Keep expanding the cells and exchanging one third of the medium twice a week. After 10 to 12 days, very large endothelial colonies will regularly be observed.
Now, remove the medium and wash with PBS to get rid of remaining protein use. 0.05%trypsin 0.7 millimolar EDTA to detach the cells. When transferring cells to new culture vessels, be sure to choose appropriately sized flasks to achieve a low seeding density.
This will assist with further expansion. Our lab usually recedes progeny from more than 20 large colonies into four two hundred and twenty five square centimeter flasks. Don't forget to replace one third of the medium twice a week.
A similar protocol is used for MSC expansion in the next section. Once three to seven days have passed, use an inverted microscope to check for the appearance of spindle shaped cells near the attached tissue. When there is sufficient cell outgrowth, the pieces tend to attach as they lose contact with the plastic.
After 10 to 12 days, remove the tissue pieces. Detach the MSCs from the plastic using a trypsin EDTA solution and to transfer the cells to new medium prefilled culture flasks that are the appropriate size and number to guarantee low seeding density during the cell expansion. It will be necessary to exchange one third of the medium twice a week once both cell types have been expanded.
Staining followed by flow cytometry can be performed to detect cell surface marker expression indicative of progenitor phenotypes and determine that there is no contamination with hematopoietic cells. Seed the pure harvested e CFCs at very low plating density of three or 10 cells per square centimeter in cell culture plates to guarantee single colony growth Exchange one third of the medium twice a week after 14 days. The end of culture.
Remove medium. Wash twice with PBS and fix the colonies with ice cold fixation solution For 15 minutes in the refrigerator, discard the fixation solution and leave the plates open to dry for 10 minutes. Rehydrate the cells with distilled water for 10 minutes.
Add Harris Hematin solution and stain the fixed colonies for 12 minutes. Remove hematin solution and rinse the plates with tap water. To get rid of the remaining staining solution.
Take photos of each colony by using a stereo microscope and import jpeg or TIFF format files onto your computer. Open the photos with image J software and analyze the accurate cell number of each colony as described in the following animation. Open the image J software and import a colony image using the file open function in the pull down menu.
Proceed by using the process. Subtract background function. Use the elliptical or brush selection function in the image J menu to mark the colony margin.
Clear the surrounding image using the edit clear outside function and crop the picture by selecting image crop. Adjust the threshold manually using the image adjust threshold function so that all cells are completely colored in.Red. Count the cell number of the colony by selecting.
Analyze, analyze particles. Choose appropriate settings optimized for each cell type and select okay If performed correctly. This protocol allows for the isolation of a virtually pure population of cells that share the characteristics of human endothelial and mesenchymal progenitor cells, which are autologous to each other because they are derived from the same cord.
Beginning on day five, spindle shaped mesenchymal cells will appear from below the piece of the cord attached to the culture plate. After one to two days, the first clusters of endothelial colony forming progenitor cells or e CFCs will become visible under an inverted microscope. This rapidly growing huge colony of e CFCs formed after 11 days.
Both the MSCs and the E CFCs can be passaged and further expanded. We recommend a subsequent analysis of all culture products using flow cytometry to confirm the appropriate phenotype. Please remember that both endothelial and mesenchyme lineages react to antibodies specific for CD 73, CD 1 46, CD 29, and CD 1 0 5.
Note that the MSCs expressed thigh one, which was detected using an anti CD 90 antibody. The E CFCs are positive for CD 31, which is also called platelet endothelial cell adhesion molecule or pcam. It's well established that CD 34 immunoreactivity expression in cord by E CFCs can be reduced through repeated culture.
Blood cell markers such as CD 45 H-L-A-D-R, and CD 14 remained negative for both cell types. Progenitor cell hierarchy among E CFCs can be evaluated using Image J software by counting accurate cell numbers of each single colony. We've just shown you how to isolate, expand, and analyze maces and ECEs from a human umbilical code when doing this procedure.
It's important to work in sterile conditions and to check for phenotype and purity before using the cells in subsequent studies. So that's it. Thanks for watching and good luck with your experiments.
