Mesenchymal Stem Cells (MSCs) have a wide range of sources. Bone marrow-derived MSCs have been the most extensively studied, but their proliferative capacity is relatively weak and decreases with donor age. Adipose-derived MSCs are easy to obtain, have better immunomodulatory ability than bone marrow-derived ones, and exhibit strong proliferative capacity in the early stage. Umbilical cord-derived MSCs, known for their "zero-age cell" characteristics and low immunogenicity, are highly favored and have become a research hotspot. Therefore, in clinical transformation and application, the use of umbilical cord-derived and adipose-derived MSCs is more commonly carried out.
Sample processing, as the "first step" in MSC in vitro culture, directly determines the success or failure of subsequent experiments. This guide will mainly focus on common problems in the selection and processing of umbilical cord samples, helping you have a smooth start before culture.
Q: How to select qualified umbilical cords for culturing umbilical cord-derived MSCs?
The selection of umbilical cords is also a crucial link for the smooth conduct of the primary isolation experiment of umbilical cord MSCs.
Conditions of a high-quality umbilical cord:
No obvious edema or congestion in the umbilical cord;
Thick Wharton's Jelly (WJ) in the umbilical cord;
Fresh umbilical cord, preferably used immediately after collection, and shall not be stored for more than 48 hours;
The donor mother has no infectious diseases.
Storage and transportation conditions of umbilical cords:
The umbilical cord should be stored in preservation solution at 4℃. If transported in a dry state, it is prone to edema and slow cell migration out of the tissue;
The umbilical cord should be used within 48 hours after collection; there is a certain probability that no cells will migrate out of umbilical cords stored for more than 72 hours;
The umbilical cord must be stored in preservation solution containing 1% antibiotics; otherwise, there is a high risk of contamination.
Q: What issues should be noted when selecting umbilical cords?
Edema of the umbilical cord and transparent or translucent Wharton's Jelly may result in fewer and slower cells migrating out of the tissue blocks;
Twisted or atrophic umbilical cords may lead to slow cell migration out of the tissue blocks;
Severe congestion of the umbilical cord may cause slow cell migration out of the tissue blocks, and a large number of red blood cells can be seen in the culture dish;
Improper operation during cutting and removal of the umbilical cord from the delivery room may lead to contamination.
Recommended solutions:
Select plump umbilical cords without edema or severe congestion, and cut off the small edematous, atrophic, or congested parts. If the laboratory does not frequently experience contamination, the umbilical cord can be rinsed 3-5 times with saline solution containing 1% double antibodies (penicillin and streptomycin) or 25mg/L gentamicin. If contamination is frequently detected, rinse the umbilical cord with 75% ethanol for 40-60 seconds first, then wash it 10-20 times with a large amount of saline solution containing 1% double antibodies.
Q: What are the classifications of umbilical cord mesenchymal stem cells?
The umbilical cord is a cord-like tissue connecting the fetus and the placenta, responsible for gas exchange, nutrient supply, and excretion of metabolic products between the mother and the fetus. Its basic characteristics are as follows:
Development time: Starts to develop 4-8 weeks after human embryo formation;
Morphological data: Average length 55cm, diameter 14.42mm, weight 40g;
Structural composition: Covered with amniotic membrane (off-white) on the surface, it contains 1 umbilical vein and 2 umbilical arteries inside. Surrounding the blood vessels is a colorless and translucent colloidal tissue—Wharton's Jelly (WJ), whose main function is to protect the umbilical blood vessels.
Mesenchymal stem cells can be obtained from four different compartments of the umbilical cord and are classified into the following four types based on their sources: Wharton's Jelly Mesenchymal Stem Cells (WJ-MSC), Perivascular Mesenchymal Stem Cells (PV-MSC), Subamniotic Mesenchymal Stem Cells (SA-MSC), and Amniotic Mesenchymal Stem Cells (AM-MSC). Mesenchymal stem cells from these four compartments have different isolation methods.
Q: What are the advantages of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs)?
Compared with mesenchymal stem cells from other compartments of the umbilical cord, WJ-MSCs perform better in terms of proliferative capacity, differentiation potential, safety, etc.:
Quantity and isolation advantages: Wharton's Jelly accounts for a large proportion of the umbilical cord, has the largest surface area and volume, and contains far more stellate mesenchymal stem cells than other regions. Its isolation does not require complex enzyme treatment, and sufficient cells can be obtained through short-term culture, which not only ensures viability and purity but also reduces the risk of microbial contamination during culture.
Stronger differentiation potential: WJ-MSCs have more prominent osteogenic and chondrogenic differentiation abilities, higher survival rate and proliferation rate, as well as stronger proliferation and regeneration capabilities.
Higher safety: Low immunogenicity, does not express MHC-Ⅱ; non-tumorigenic and possesses anti-inflammatory properties.
Q: How to isolate Wharton's Jelly from the umbilical cord?
Use medical scissors to cut the umbilical cord into 2cm tissue segments, and incise each segment along the umbilical vein. Use tissue forceps to peel off the intima of the umbilical vein, the outer skin, and the two umbilical arteries to fully expose the Wharton's Jelly.
The Wharton's Jelly should be completely stripped of the three blood vessels and the outer epidermis. If not fully stripped, epithelial-like heterologous cells will migrate out, which will occupy the growth space of MSCs and result in a reduced number of harvested cells. These cells are difficult to be digested by mild enzymes and can be removed through passage. The processed umbilical cord can be used for subsequent inoculation via the implantation method.
Q: What issues should be noted during inoculation via the implantation method?
Cutting into blocks
The isolated Wharton's Jelly should be cut into small pieces of about 3mm. If the tissue pieces are too small, they will be difficult to adhere to the wall, resulting in no or too few cells migrating out. If the tissue pieces are too large or not completely cut, no cells will migrate out either. If the tissue pieces are completely minced with scissors, it is likely to cause the failure of primary cell culture.
Inoculation
Tissue pieces should be inoculated on a suitable medium according to the size of the culture dish/flask, and an appropriate amount of culture medium should be added. Too little culture medium will prevent cells from migrating out of the tissue pieces, while too much will cause the tissue pieces to float, and cells will still not migrate out.
After inoculation, the tissue pieces should be avoided from movement as much as possible. It is best to keep them completely undisturbed in the incubator for the first 7 days except for medium supplementation. Frequent movement will cause the tissue pieces to suspend, making it impossible for cells to migrate out.
