Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

Summary

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.

Abstract

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

Introduction

Since the first isolation of Mesenchymal Stem Cells (MSCs) from Wharton's jelly (WJ) in 1991, these multipotent stem cells have gained significant attention from researchers due to their regenerative properties and multilineage differentiation capacity¹. MSCs can be isolated from various sources, including bone marrow, peripheral blood, dental pulp, adipose tissue, fetal (human abortion), and birth-related tissues². The umbilical cord (UC) has emerged as a promising reservoir due to its non-invasive nature, abundant cell yield and differentiation capacity, exhibiting a high rate of proliferation, differentiation potential, and immune modulation properties³. Fetal MSCs exhibit strong stemness and immune properties, making them the primary focus of clinical trials and basic research conducted over the past two decades^2,4,5. UC-derived MSCs have superior therapeutic potential compared to other sources of MSC, such as bone marrow or adipose tissue^6,7.

The UC is composed of amniotic epithelium, three vessels (two arteries and one vein), and the gelatinous substance known as WJ³. Intriguingly, the UC constitutes a simple vasculature, consisting only of the endothelium and mesothelium, but not the tunica adventitia; the WJ does not contain lymph or nerves⁸. The UC presents a unique structure ideal for segmental separation. UC-MSCs are primarily located in the WJ. MSCs could be isolated from different compartments of the WJ, including amnion, subamnion (the amnion and subamnion also designated as cord lining region), and the perivascular area of the WJ⁸. Each region of the WJ has its own structure, immunohistochemical characteristics, and function^3,6.

MSCs isolated from the WJ of the UC are widely regarded as having superior clinical utility compared to those from other regions³. WJ-MSCs have been extensively studied in preclinical and clinical settings for the treatment of various diseases due to their multi-line differentiation potential, immunomodulatory properties, paracrine effects, anti-inflammatory effects, and immune-privileged properties^2,3. WJ-MSCs have been proven to hold promise in treating a range of diseases, including graft-versus-host disease (GvHD), graft rejection, Crohn's disease, autoimmune diseases, and cardiovascular diseases^{9,10,11,12,13,14}. As clinical demand for WJ-MSCs continues to increase, the shortage supply of umbilical cords is currently an impediment to their widespread applications.

The yield of WJ-MSCs is dependent on the method used for cell extraction¹⁵. While WJ-MSCs can be isolated through explant culture or enzyme digestion, the latter method has a longer propagation time that may increase the risk of cell damage and decrease cell viability¹⁶. However, numerous studies have shown that the explant culture method increases cell yields and viability, and that paracrine factors released from explant tissues also help promote cell proliferation^17,18.

This study applied a unique dissection approach to obtain whole WJ, yielding MSCs with enhanced proliferative capacity, viability, and quantity, while minimizing damage to the WJ. This innovative method offers a streamlined strategy for isolating WJ-MSCs, addressing critical needs in MSC applications.

Protocol

Samples were obtained from the consenting, healthy donors from the Shenzhen Longgang District Maternity and Child Healthcare Hospital, Guangdong, China. The use of human samples for the study was approved by the Ethics Committee of Shenzhen Hospital, Beijing University of Chinese Medicine (SZLDH2020LSYM-095) and Medical Ethics Committee of Shenzhen Longgang District Maternity and Child Healthcare Hospital (LGFYYXLLS-2020-005). All experiments were conducted according to the approved guidelines. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Collection of human umbilical cord

1. Obtain human umbilical cord samples from the cooperative hospital under sterile conditions.
   NOTE: Choose a donor under 35 years of age, preferably a primipara, with no history of hepatitis, sexually transmitted diseases, or genetic disorders. The admission examination results for hepatitis B virus, hepatitis C virus, and cytomegalovirus need to be negative. Choose cesarean section as the mode of delivery.
2. Select a straight and intact piece of UC of approximately 10-15 cm in length.
3. Close both ends with hemostatic forceps and ligate with surgical knots at the inner end between the forceps right after the delivery of a full-term newborn by cesarean section. (collected UC as shown in Figure 1, step 1).
   NOTE: The operation of the cesarean section ensures the collection is sterile. Use surgical knots of size 0 or 2-0 suture for better tensile strength.
4. Cut the tied umbilical cord between the forceps and knots using surgical scissors (Figure 1, step 1).
5. Rinse it with saline solution to remove any contaminating blood clots on the surface and transfer promptly to a sterile 50 mL plastic tube containing 20 mL of sterile phosphate-buffered saline (PBS).
   NOTE: No antibiotic was used during the transfer.
6. Seal the tube. Place it on ice in a polystyrene box and store it at 4 °C before transporting it to the laboratory.
   NOTE: Dissect the cord within 4 h of collection.

2. Isolation of Wharton's jelly from the umbilical cord

1. Process the umbilical cord following the steps below:
   1. Prepare a sterilized tray with surgical instruments, aseptically packaged 90 mm dishes, 1000 µL pipettes, sterile PBS, 75% ethanol solution, and xeno-free human MSC culture medium.
      NOTE: The toothed forceps and mosquito clamps permit the slithery cord to be dealt with efficiently.
   2. Surface decontaminate the required items and work zone before placing the items inside the biosafety cabinet.
      NOTE: Allow the work zone air to purge for a few minutes before commencing work.
   3. Disinfect the surface of the tube and remove the sample into a Petri dish in the biosafety cabinet. Immerse the sample with the knots in 75% ethanol for 30 s and then rinse it multiple times with sterile PBS in a new Petri dish.
      NOTE: The cord must be ligated before immersing in 75% ethanol, and the time of immersing should be limited to 30 s to 1 min to fully disinfect the cord without damage.
   4. Cut the knots off and cut the cord between two knots transversally into short pieces about 2 cm in length with surgical scissors and forceps.
      NOTE: The ends of the cord with knots should be disposed of. The length of the UC shouldn't be too long or too short; both conditions may increase the difficulty of the operation.
   5. Perfuse the vein (larger vessel) with PBS using 1000 µL pipettes until the fluid coming out of the other end of the cord becomes completely transparent¹⁹.
2. Dissect the umbilical cord.
   1. Transfer one of the already cut pieces to a new Petri dish.
      NOTE: Two arteries and one vein should be exposed visibly on the cross section⁸ (Figure 2B).
   2. Add the appropriate amount of PBS to keep the cord wet throughout the operation.
   3. Grasp the cord with forceps and pull out the arteries along its major axis with the help of mosquito clamps.
      NOTE: Try to pull from the other end of the cord if the artery has been torn off, as the artery wall has great elasticity.
   4. Fix the cord horizontally with the thicker side facing up by inserting one blade of the forceps into the umbilical vein and then clamping tightly, ensuring the forceps clamp on the thicker side.
   5. Make a linear incision on the layer of amniotic epithelium from one end to the other along the edge of the other blade of the forceps using a scalpel (Figure 3).
      NOTE: Preserve the integrity of the Wharton's jelly by cutting as little WJ as possible.
   6. Begin at one corner of the cutting gap. Lift the corner of the amniotic epithelium with toothed clamps and continue to cut horizontally a little further with corneal scissors along the inner surface of the epithelium.
      1. Separate the Wharton's jelly and amniotic epithelium with clamps and gradually expand to the whole circle of one end of UC. Peel off the epithelial layer lengthways (Figure 3).
         ​NOTE: The Wharton's jelly is a mucous connective tissue enclosed by the dense amniotic epithelium⁸. The whole operation should be smooth, but mosquito clamps could be used for blunt dissection if one encounters difficulties during peeling.
   7. Insert both blades of the forceps inside the vein, clamp a portion of WJ, and then flip it inside out to expose the epithelium of the umbilical vein (Figure 3).
   8. Peel off the epithelium of the vein easily, as the internal tension creates a separation between the epithelium of the vein and the perivascular WJ (Figure 3).
3. Dissect the UC using the conventional method for comparison²⁰.
   1. Choose another already cut sample of nearly the same weight as the previous cord and transfer it to a new Petri dish.
   2. Cut along the umbilical vein longitudinally with scissors and spread the UC to expose the vessels and WJ.
   3. Remove the umbilical vein and two arteries, and scape the WJ off the endothelial layer with scissors and scalpel.

3. Isolation and culture of UC-MSCs

1. Place the collected WJ in a pre-labeled 90 mm Petri dish and cut it into 1-3 mm pieces with scissors and forceps.
2. Invert the dish with the cap on the bottom and place it in a 5% CO₂ incubator at 37 °C for 30 min to strengthen the attachment between the pieces and the plastic surface.
3. Turn the dish over, and gently add an appropriate amount of human MSC culture medium for further culture.
   NOTE: Use the lowest speed to ensure the pieces are still attached.
4. Change the human MSC culture medium every 3 days. Exchange two-thirds of the medium and observe the appearance of outgrowth of fibrous cells every 2 days in the first 7 days, change the full medium, and observe it on a daily basis thereafter.
   NOTE: Avoid moving the Petri dish for the first 7 days. The distinguished outgrowth of fibroblast-like cells was observed in about 4 days.
5. Subculture until confluence reaches 80%.
   NOTE: This step takes 7-10 days.
   1. Pre-warm the PBS, 0.25% trypsin, and xeno-free human MSC culture medium to 37 °C in a water bath.
   2. Rinse with PBS after aspirating the supernatant with pipettes, and detach the cells with pre-heated 0.25% trypsin until the cells can be dislodged by tapping the Petri dish.
   3. Inactivate the trypsin by mixing pre-heated MSC culture medium at a ratio of 4:1, collect the cell suspension, and centrifuge at 300 x g for 5 min at 4 °C.
      NOTE: These cells are considered passage 0 (P0).
   4. Discard the supernatant with a pipette, resuspend the cells in the human MSC culture medium, and inoculate into new Petri dishes at a seeding density of 1x10⁴ cells/cm² for propagation.
6. Using a hemocytometer, count the total cell number of the two methods after amplification at first, second, and third passage. Determine the cell morphology under the microscope in the first, third, and fifth passages.
   NOTE: The morphology of these cells should be similar to the P0 cells.
7. Use cells at the fifth passage for flow cytometry and other experiments.

4. Expression of cell surface markers by flow cytometry

1. Detach passage 5 (P5) cells with 0.25% trypsin, wash the cells with cell staining buffer, and centrifuge at 300 x g for 5 min at 4 °C. Resuspend the pellets with cell staining buffer to 100 µL each.
2. Label each tube and add the appropriate amounts of antibodies, which were conjugated with Allophycocyanin (APC), fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD44-APC, CD73-APC, CD90-FIFC, CD105-FIFC, CD11B-PE, CD19-PE, CD43-PE, HLA-DR-PE following the manufacturer's instructions²¹. Incubate for 15-20 min in the dark at room temperature.
3. Centrifuge the stained cells at 300 x g for 5 min at 4 °C, aspirate the supernatant with a pipette, and wash once with cell staining buffer.
4. Repeat the centrifugation and resuspension of the cells with 300 µL of the cell staining buffer.
5. Run the cells through the flow cytometer and analyze the antibody-stained cells according to the manufacturer's instructions.

5. Determination of cell growth curve by cell counting method

1. Prepare a single-cell suspension of P5 cells using two methods by diluting them in a human MSC culture medium.
2. Seed 8,000 cells per well in a 24-well plate, with a total of 21 wells of each method being seeded.
   NOTE: Mix the cells gently by blowing and suctioning with pipettes before seeding.
3. Harvest cells from three wells after 24 h of seeding. Perform cell counting using a hemocytometer to calculate the mean value.
   NOTE: Shake the plate before the cell counting. The duration of shaking can be extended to prevent the formation of cell aggregates.
4. Repeat the cell counting every 24 h for a total duration of 7 days. Plot a growth curve using time (d) on the X-axis and cell count (x10⁴/mL) on the Y-axis.

Results

The procedures for collecting and culturing UC-MSCs, as well as their subsequent analysis, are summarized in Figure 1. The UC was neatly dissected into several sections using the unique method; the specific operation diagram of the main procedures is illustrated in Figure 2. The outgrowth of cells from explant cultures was routinely monitored and recorded. Adherent spindle-shaped cells were observed approximately 4 days after culturing the explants and increasin...

Discussion

MSCs represent a dynamic area of research with profound implications for regenerative medicine²². Their unique properties make them a focal point for scientific inquiry and hold the potential to revolutionize the treatment of a wide range of diseases and injuries⁷. WJ-MSCs are a distinct subset of MSCs, which can be obtained from the gelatinous connective tissue within the UC situated between the intervascular and amniotic epithelium²³. The clinical ...

Materials

Name	Company	Catalog Number	Comments
APC anti-human CD44 Antibody	Biolegend	338806
24-well cell culture plates	Thermo Scientific	142475
APC anti-human CD73 (Ecto-5'-nucleotidase) Antibody	Biolegend	344006
APC Mouse IgG1, κ Isotype Ctrl (FC) Antibody	Biolegend	400122
Autoclave	HIRAYAMA	HVE-50
Automatic Cell Counter	Countstar	FL-CD
BAMBANKER Cryopreservation Solution	Wako	302-14681
Cell Staining Buffer	Biolegend	420201
Centrifugal Machine	Eppendorf	5424R
Clean Bench	Shanghai ZhiCheng	C1112B
CO2 Incubator	Thermo Scientific	HERAcell 150i
D-PBS	Solarbio	D1040
Electro- thermostatic Blast Oven	Shanghai JingHong	DHG-9423A
FITC anti-human CD105 Antibody	Biolegend	323204
FITC anti-human CD90 (Thy1) Antibody	Biolegend	328108
FITC Mouse IgG1, κ Isotype Ctrl (FC) Antibody	Biolegend	400110
Flow Cytometry	Beckman	CytoFLEX
hemocytometer	Superior Marienfeld	640410
Intracellular Staining Permeabilization Wash Buffer (10×)	Biolegend	421002
Inverted Biological Microscope	ZEISS	Axio Vert. A1
Liquid Nitrogen Storage Tank	Thermo Scientific	CY50935-70
Normal saline (NS)	Meilunbio	MA0083
PBS	Solarbio	P1032
PE anti-human CD11b Antibody	Biolegend	393112
PE anti-human CD19 Antibody	Biolegend	392506
PE anti-human CD34 Antibody	Biolegend	343606
PE anti-human CD45 Antibody	Biolegend	368510
PE anti-human HLA-DR Antibody	Biolegend	307606
PE Mouse IgG1, κ Isotype Ctrl (FC) Antibody	Biolegend	400114
PE Mouse IgG2a, κ Isotype Ctrl (FC) Antibody	Biolegend	400214
Precision Electronic Balance	Satorius	PRACTUM313-1CN
Snowflake Ice Machine	ZIEGRA	ZBE 30-10
steriled 50 mL plastic tube	Greniner	227270
Thermostatic Water Bath	Shanghai YiHeng	HWS12
Trypsin 1:250	Solarbio	T8150
UltraGRO-Advanced	Helios	HPCFDCGL50
Ultrapure and Pure Water Purification System	Milli-Q	Milli-Q Reference
Xeno-Free Human MSC Culture Medium	FUKOKU	T2011301