This method can help answer key questions in the deliberation of UCMSC, such as the best source of USMSC for cell therapy. The main advantage of this technique is that it enables consistent isolation and vigorous proliferation of UCMSC from pre-term and term infants. The implications of this technique extend toward therapy of intractable diseases.
Though this method can provide insight into the deliberation of UCMSC, it can also be applied to other MSC sources, such as adipose tissue, synovium, skin, dental pulp, and so on. Generally, an individual new to this method will struggle because there are so many variation in the steps of umbilical dissection and mesenchymal stem cell isolation. Visual demonstration of this method is critical as these steps are difficult to learn because there are several points prone to be overlooked.
To begin dissecting the umbilical cord, first prepare a metal tray, sterilized scissors, and forceps, ten milliliter pipettes, 25 milliliter pipettes, and two 60 millimeter tissue culture dishes. Warm up PBS purified enzyme blends, a 500 milliliter of reduced serum medium and culture medium, to room temperature. Remove previously isolated umbilical cord from a 50 milliliter tube in alpha MEM, and place it in the plastic tray.
Weigh the umbilical cord, and then use sterilized scissors to dissect five grams of the cord. To sterilize the umbilical cord, use ten milliliter pipette to pour ten milliliters of 70 percent ethanol over it. Then wash the cord twice by adding ten milliliters of PBS with ten milliliter pipette.
Place the washed umbilical cord in the sterilized 60 millimeter tissue culture dish and add ten milliliters reduced serum medium and 0.5 milliliters purified enzyme blends. Use sterilized scissors and forceps to cut the cord into two to three millimeter pieces. Place the dish in a five percent carbon dioxide incubator and incubate at 37 degrees Celsius for 30 minutes.
Then cut the partially digested pieces into smaller pieces, and incubate them at 37 degrees Celsius in a five percent carbon dioxide incubator until the homogenate becomes viscous. Finally, make sure that the homogenate flows easily through a 25 milliliter pipette. First prepare four, 50 milliliter plastic tubes in a 500 milliliter bottle of culture medium.
Use a 25 milliliter pipette to divide the umbilical cord homogenate into two, 50 milliliter plastic tubes with approximately 7.5 milliliters in each tube. Then add 20 milliliters of culture medium into each tube and mix well. Collect each homogenate with 25 milliliter pipette and filter through a 100 micrometer cell strainer placed on top of a new 50 milliliter collection tube.
Centrifuge two tubes with filtrates at 1000g for five minutes at room temperature. After finished centrifugation, carefully aspirate the supernatant and discard it. Add five milliliters of culture medium to each tube, and re-suspend the cell pellets.
Transfer the cell suspension into a new 60 millimeter plate and culture at 37 degrees Celsius in a five percent carbon dioxide incubator. To culture the UCMSC's, warm the PBS, trypsin EDTA, and culture medium, to room temperature. When cells are attached to the plate, remove the medium, and wash twice with five milliliters of PBS to remove debris and red blood cells.
Add the culture medium and incubate at 37 degrees Celsius in a five percent carbon dioxide incubator. Replace the medium twice per week, and culture the cells until they reach 90 to 100 percent confluency. Then wash the cells twice with five milliliters of PBS, add 0.5 milliliters of trypsin EDTA, and incubate at 37 degrees Celsius for five to ten minutes.
When cells become rounded and detached, add nine milliliters of culture medium, and mix well to inactivate trypsin. Add the cell suspension into a new 100 millimeter plate. Grow the cells at 37 degrees Celsius in a five percent carbon dioxide incubator until they reach 90 to 100 percent confluency.
Repeat trypsinization and dividing into two new plates, until the tenth passage. Surface marker expression of UCMSC's was used as criterion for their characterization. When incubated with appropriate antibodies and analyzed by flow cytometry, they were found positive for MSC's signature markers, but negative for monocyte macrophage, negative for hematapoietic stem cell and ephithelial cell, negative for B cell, negative for pan leukocytes, and negative for major histocompatibility complex markers.
To evaluate trilineage differentiation capacity of USCMSC's from preterm and term infants, the cells were induced to differentiate into adipocytes, osteocytes, and chondrocytes. UCMSC's successfully differentiated into all three types of mesodermal cells confirming their differentiation capacity. While attempting this procedure, it's important to remember that there are four critical steps.
Cutting the cord into two to three centimeter pieces, cutting into smaller pieces, making sure that the homogenate flows easily through a 25 milliliter pipette, and filtering the homogenate through a 100 micrometer cell strainer.
