To begin, place all the sterile surgical instruments on the working platform. In the biosafety cabinet, disinfect the surface of the tube containing the previously collected human umbilical cord with 75%ethanol. Remove the sample into a Petri dish.
Immerse the sample with the knots in 75%ethanol for 30 seconds. Then, in a new Petri dish, rinse the sample multiple times with sterile PBS. Using surgical scissors and forceps, cut the knots off, then cut the cord between two knots transversely into short pieces.
Perfuse the vein with PBS using a one milliliter pipette until the fluid coming out of the other end of the cord becomes completely transparent. Transfer one of the already cut pieces of umbilical cord to a new Petri dish. Add the appropriate amount of PBS to keep the cord wet throughout the operation.
Grasp the cord with forceps and pull out the arteries along its major axis with the help of mosquito clamps. To fix the cord horizontally with the thicker side facing up, insert one blade of the forceps into the umbilical vein and clamp it tightly, ensuring the forceps clamp on the thicker side. Using a scalpel, make a linear incision on the amniotic epithelium layer from one end to the other, along the edge of the other blade of the forceps.
From the cutting gap corner, lift the corner of the amniotic epithelium with tooth clamps, and continue to cut horizontally a little further with corneal scissors along the inner surface of the epithelium. Separate the Wharton's jelly and amniotic epithelium with clamps, and gradually expand to the whole circle of one end of the umbilical cord. Peel off the epithelial layer lengthways.
Insert both blades of the forceps inside the vein. Clamp a portion of Wharton's jelly, and then flip it inside out to expose the epithelium of the umbilical vein. Peel off the epithelium of the vein easily.
Place the collected Wharton's jelly in a pre-labeled 90 millimeter Petri dish and cut it into one to three millimeter pieces with scissors and forceps. Invert the dish with the cap on the bottom and place it in a 5%carbon dioxide incubator at 37 degrees Celsius for 30 minutes. Change the human mesenchymal stem cell culture medium every three days.
Compared to the conventional approach, the unique blunt dissection procedure showed a significant increase in Wharton's jelly yield. Further analysis by counting the total quantity of Wharton's jelly mesenchymal stem cells by both methods showed a pronounced gap in cell quantity with increasing passage numbers. The proliferation rate of mesenchymal stem cells in the novel method was significantly higher after two days.
