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 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.
Protocol
Representative Results
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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 |
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