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 pote....

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 reagent.......

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