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.
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.
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 decades2,4,5. UC-derived MSCs have superior therapeutic potential compared to other sources of MSC, such as bone marrow or adipose tissue6,7.
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 function3,6.
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 properties2,3. 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 diseases9,10,11,12,13,14. 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 proliferation17,18.
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.
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
2. Isolation of Wharton's jelly from the umbilical cord
3. Isolation and culture of UC-MSCs
4. Expression of cell surface markers by flow cytometry
5. Determination of cell growth curve by cell counting method
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 increasingly crawled into a vortex shape. These cells were also identified as P0 and continued to proliferate for subsequent passaging while maintaining uniform size and morphology during proliferation and passaging. The conventional and current methods exhibited negligible differences in terms of cell size and morphology (Figure 3).
The conventional dissection method involves scraping the WJ away from the blood vessels and the inner epithelium of the UC. The WJ yield represented a significant difference between the two methods by weight adjustment. The yield of WJ can be calculated by measuring the weight percentage of the WJ in the 2 cm cord (2.4 g ± 0.2 g). A comparison of the ratio of WJ yield between the current and traditional methods showed a significant increase in WJ yield using the novel approach (Figure 4A). Further analysis by counting the total quantity of WJ-MSCs harvested by both methods from the equivalent weight of UC simultaneously showed a pronounced gap in cell quantity with increasing passage numbers (Figure 4B). The growth curve indicated that the proliferation rate of MSCs in the novel method was significantly higher than in the conventional method under the same inoculation density after 2 days (Figure 4C). These experiments demonstrate the advantages of the current method, which allows an efficient way to increase the efficient supply of the same material.
Following the International Society for Cell and Gene Therapy (ISCT) guideline's minimum criteria for the definition of MSCs, the immunophenotype of MSCs demonstrates positive expression of CD105, CD44, CD90, CD73 (more than 95%), while being absent of CD45, CD19, CD34, CD11b, and HLA-DR ( less than 2%)22. WJ-derived MSCs were investigated via quantitative flow cytometry (Figure 5); the cells isolated by both methods were highly positive for the surface antigens CD105, CD44, CD90, and CD73 (all above 99%), with the expression of the surface molecules CD45, CD19, CD34, CD11b, and HLA-DR were expressed below detectable levels (data not shown). Based on the results of cell surface markers, adherent cells can be finely regarded as MSCs, and their identification and purity can be assured.
Figure 1: Schematic representation of the dissection and isolation of WJ-MSCs from the umbilical cord. Step 1: The human umbilical cord sample is collected from the newborn and rinsed with a saline solution, followed by perfusion of the vein. The cord is then cut into 2 cm pieces. Step 2: Pure Wharton's Jelly (WJ) is obtained using the current method, involving the separation of the arteries, amniotic epithelium, and vein epithelium. The traditional method is also used for comparison. The umbilical cord is dissected accordingly. Step 3: The dissected WJ is cut into 1-3 mm pieces, and the dish is placed upside down in the incubator for 30 min to strengthen the attachment between the pieces and the plastic surface. The WJ pieces are then cultured and passaged at a seeding density of 1 x 104 cells/cm2 until reaching 80% confluence. Step 4: Flow cytometry is used to characterize the P5 cells by analyzing mesenchymal stem cell (MSC) surface markers. Step 5: The proliferative ability of the cells is determined by plotting a cell growth curve using the cell counting method. Please click here to view a larger version of this figure.
Figure 2: Anatomy of the umbilical cord and schematic demonstration of dissection. (A) The cross-section of the UC, highlighting arteries (A), vein (V), Wharton's jelly (WJ), and the subsequent dissection separates UC into different compartments. (B) Schematic demonstration of removing the amniotic epithelial layer. The incision is made along the dashed line on the amniotic epithelium, separating the epithelium beginning with the side corner of the cord. (C) Schematic demonstration of removing the vein epithelial. Flipping the cord inside out (the direction of the arrow indicates the direction of force) exposes the vein epithelium for removal. Please click here to view a larger version of this figure.
Figure 3: Morphological features of primary cultured and passaged WJ-MSCs. Distinguished fibroblastoid cell growth from the explants can be observed around day 4 (denoted by the arrows) and increasingly exhibited a vortex-like growth pattern. The explants were removed and subcultured; the isolated cells were homogeneous in regard to the visualized size and morphology. An insignificant difference was observed in the size and morphology of cells between the two methods. Scale bar: 200 µm. Please click here to view a larger version of this figure.
Figure 4: Graphical comparative analysis of WJ yield and the growth characteristics of the cultured-MSCs. (A) Comparison of WJ yield using 2 cm UC with an initial weight of 2.4 g ± 0.2 g by both methods. WJ yield = (weight of WJ) / (initial weight of the unprocessed cord. Ratio of WJ yield (%) = (WJ yield) / (average WJ yield of current method) × 100. (B) Comparison of cell counting after culture and passage under the same condition by both methods. (C) Growth curves of P5 cells in both methods. All experiments included three biological replicates, and the statistical differences were analyzed by the two-way ANOVA method (***p < 0.001). Please click here to view a larger version of this figure.
Figure 5: Cell surface expression of MSC analyzed by flow cytometry. P5 cells collected by both conventional and current methods are subjected to flow cytometry analysis. The surface antigens CD44, CD105, CD90, and CD73 were highly positive (all above 99%), while the expressions of CD45, CD19, CD34, CD11b, and HLA-DR surface molecules were below the detection limit. Please click here to view a larger version of this figure.
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 application of MSCs lies in attaining sufficient cell numbers24. However, as the passage number increased, the cells began to show a certain degree of aging phenomenon, and the proliferative capacity of MSCs decreased gradually after P524. Optimizing methods to enhance MSC yield is, therefore, imperative.
The most efficient way to isolate WJ-MSCs is to dissect the entire WJ while preserving its integrity6. Research shows that these border cells from umbilical cord border regions have a higher migration characteristic and a higher proliferation rate than intervascular WJ-MSCs23. However, the regions and methods for dissecting WJ have not been standardized, and it remains unclear whether stem cell populations within WJ-MSCs share the same qualities due to the lack of clear histological boundaries between partitions3.
Previous studies have indicated significant differences in the properties of WJ-MSCs derived from various compartments of the umbilical cord, with cells located close to the amniotic surface exhibiting enhanced proliferative ability3. The current unique method of dissecting UC and isolating WJ-MSCs aims to reduce both the loss of the lining WJ and cell damage, which enhances cell yield and viability. This protocol demonstrates a method that facilitated the processing of UC into nearly intact WJ. The primary step of the protocol involves removing the epithelial layer through peeling and blunt dissection. The innovation step is to peel off the epithelial layer lengthways with mosquito clamps. This approach is effective in reducing the adjacent cell damage caused by conventional sharp dissection with scissors25.
The endothelium of the vein is fragile and hard to get rid of. After removing the amniotic epithelium from the umbilical vein, the umbilical cord is flipped through the vein to expose the endothelium. Internal tension created by the WJ is then used to peel off the endothelium of the vein, thereby minimizing interference from human umbilical vein endothelial cells (HUVECs) interference during subsequent cell culture processes.
The explant culture method is adopted as it has a higher yield compared to other available methods18. The unattached cells will be gradually removed through the exchange of the human MSC culture medium, resulting in the loss of cells26. The explants that fail to adhere to the plastic could impede cell outgrowth in previous experience. The impact of water vapor given off from the tissue due to the changes between the inside and outside temperatures of the incubator needs to be considered. To maintain the dry state of the Petri dish, an innovative step of turning the dish upside down for 30 min during incubation with the lid on the bottom was implemented. This inversion of a dish avoided the condensation of water vapor on the contact between tissue and the surface of the Petri dish, which in turn allows for better attachment of exosomes to the plastic surface to improve cell outgrowth.
The current method is compared with the conventional dissecting method by measuring the weight percentage of the obtained WJ and the quantity of cultured and passaged MSCs; a significantly higher yield of WJ and a further quantity of harvested MSCs were shown by the novel method. Though any protocol to isolate stem cell populations from each of these compartments carries the risk of cell contamination from the other compartments, the results demonstrated the purity of isolated MSCs, characterized by high expression of mesenchymal markers (all above 99%) and minimal detection of hematopoietic and endothelial markers (data not shown). The results proved the WJ-MSCs are pure and suitable for various applications, aligning with ISCT guidelines21. The cultured MSCs exhibited fibroblast-like cells in a short period, and the cells increasingly formed into vortex-shaped cells, displaying great proliferative potential and migratory properties.
The protocol outlined in this study presents several shortcomings. Firstly, the proficiency required for the operator is high, as indicated by the separation of the amniotic epithelium. This may hinder its widespread adoption in research or clinical applications. Secondly, the protocol relies heavily on manual manipulation during dissection, which may introduce inconsistency between experiments or operators. Standardization of the dissection process would enhance the reliability and reproducibility of the protocol25. Furthermore, the lack of other comparisons with the conventional method leaves unanswered questions.
In conclusion, the current method provides an efficient way to separate the intact WJ from the amniotic epithelium and vessels, resulting in a harvest that includes the perivascular and subamniotic Wharton’s jelly. The introduction of an inverted incubation step further contributes to improved MSC attachment. Comparative analysis with traditional methods demonstrates the significant advantages of the proposed protocol in terms of obtaining a higher WJ yield and harvesting more MSCs with better proliferative ability. The findings also highlight the purity of the isolated MSCs, as indicated by a distinct pattern of cell surface marker expression. This method could enhance MSC yield and proliferative ability, offering a cost-effective method to augment MSC production.
The authors have nothing to disclose.
This work was financially supported by the National Natural Science Foundation of China (82172107), the Natural Science Foundation of Guangdong Province, China (2021A1515011927, 2021A1515010918, 2020A1515110347), Shenzhen Medical Research Fund (SMRF.D2301015), the Shenzhen Municipal Science and Technology Innovation Committee (JCYJ20210324135014040, JCYJ20220530172807016, JCYJ20230807150908018, JCYJ20230807150915031), and Longgang District Special Fund for Economic and Technological Development (LGKCYLWS2022007).
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 |