Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage
July 5th, 2024
This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.
Our research used human stem cells to create various organoids. They can mimic human organs. We developed unique techniques for methadone and endoderm co-differentiation.
They enable these organoids with rich blood networks. These organoids will promote organ development, regenerative medicine, disease model, drug screening, and the personalized medicine, such as organoid transplantation. Our protocol addresses a research gap in optimizing the isolation of intact Wharton's jelly from the umbilical cord while preserving its paravascular and sub-otic components.
This method aims to enhance the yield, viability and proliferative ability of mesenchymal stem cells, offering a cost-effective approach to augment MSC production. The advantage of our protocol lies in its ability to obtain a higher Wharton's jelly used and harvest more MSCs with better proliferative ability compared to traditional methods. 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.
Overview
This study presents a unique blunt dissection procedure to optimize the isolation of intact Wharton's jelly (WJ) from the umbilical cord. The method preserves the integrity of WJ, resulting in less damage and a greater yield of viable mesenchymal stem cells (MSCs).
Key Study Components
Area of Science
- Stem Cell Research
- Regenerative Medicine
- Organoid Development
Background
- Wharton's jelly is a rich source of mesenchymal stem cells.
- Conventional sharp dissection methods can damage WJ and reduce cell viability.
- There is a need for improved techniques to isolate WJ while preserving its components.
- This study addresses the gap in optimizing WJ isolation methods.
Purpose of Study
- To develop a blunt dissection technique for WJ isolation.
- To enhance the yield and viability of MSCs harvested from WJ.
- To compare the effectiveness of the new method against traditional techniques.
Methods Used
- Blunt dissection procedure for WJ isolation.
- Assessment of MSC yield and viability.
- Comparison with conventional sharp dissection methods.
- Evaluation of proliferative ability of harvested MSCs.
Main Results
- The blunt dissection method resulted in less damaged WJ.
- Higher quantity of viable MSCs was obtained compared to sharp dissection.
- The new technique demonstrated superior proliferative ability of MSCs.
- Results indicate potential for improved applications in regenerative medicine.
Conclusions
- The blunt dissection technique is effective for WJ isolation.
- It enhances the yield and viability of MSCs, supporting further research.
- This method could advance the field of regenerative medicine and organoid development.
