Ethics code: IR.SBMU.ENDOCRINE.REC.1400.140
Abstract: (35 Views)
A B S T R A C T
Background and Objectives
Mesenchymal stem cells (MSCs) and their derived small extracellular vesicles (sEVs)- based therapies have attracted considerable attention as novel therapeutic strategies in preclinical studies and clinical trials. sEVs exhibit therapeutic effects similar to their parental cells while offering higher safety, making them a promising alternative to direct cell transplantation. Consequently, identifying optimal cellular sources and developing efficient methods for sEV isolation are of critical importance. Human umbilical cord Wharton’s jelly–derived MSCs (WJ-MSCs), represent a valuable source for sEV production due to ease accessibility; high proliferative capacity, and potent immunomodulatory properties. This study aimed to established a practical, streaight forward, and cost-effective protocol for the isolation and characterization of WJ-MSC-derived exosomes to enhance precision and reproducibility of laboratory processes.
Materials and Methods
In this Experimental study, MSCs were isolated from two independent human umbilical cord Wharton’s jelly specimens using the explant method. Following phenotypic characterization via assessment of surface markers expression (CD73, CD105, CD34 and CD45) and differentiation assays, sEVs were isolated from conditioned medium. Per batch, 15 mL of conditioned medium was collected from multiple culture flasks maintained under identical conditions, using a commercial kit. The morphological characteristics of the isolated sEVs were evaluated by scanning electron microscopy (SEM), and their protein concentration was determined using the Bradford assay.
Results
Flowcytometric analysis revealed high expression of MSC-associated markers CD73 (98.8%) and CD105 (93.8%), while hematopoietic markers CD34 and CD45 were expressed at levels below 2%. The osteogenic and adipogenic differentiation potentials of the cells were also confirmed. SEM images demonstrated preserved membrane integrity and spherical morphology of the isolated sEVs, with a size distribution falling within the expected range of approximately 40–60 nm. The protein concentration of the isolated sEVs was quantified at 500 µg/mL based on the Bradford assay (n = 3).
Conclusions
These findings indicate that the explant method, in conjunction with density gradient centrifugation–based commercial kits, provides a simple, cost-effective, and efficient approach for the isolation of WJ-MSCs and production of high-quality sEVs, thereby providing a practical platform for future preclinical and translational applications.
Background and Objectives
Mesenchymal stem cells (MSCs) and their derived small extracellular vesicles (sEVs)- based therapies have attracted considerable attention as novel therapeutic strategies in preclinical studies and clinical trials. sEVs exhibit therapeutic effects similar to their parental cells while offering higher safety, making them a promising alternative to direct cell transplantation. Consequently, identifying optimal cellular sources and developing efficient methods for sEV isolation are of critical importance. Human umbilical cord Wharton’s jelly–derived MSCs (WJ-MSCs), represent a valuable source for sEV production due to ease accessibility; high proliferative capacity, and potent immunomodulatory properties. This study aimed to established a practical, streaight forward, and cost-effective protocol for the isolation and characterization of WJ-MSC-derived exosomes to enhance precision and reproducibility of laboratory processes.
Materials and Methods
In this Experimental study, MSCs were isolated from two independent human umbilical cord Wharton’s jelly specimens using the explant method. Following phenotypic characterization via assessment of surface markers expression (CD73, CD105, CD34 and CD45) and differentiation assays, sEVs were isolated from conditioned medium. Per batch, 15 mL of conditioned medium was collected from multiple culture flasks maintained under identical conditions, using a commercial kit. The morphological characteristics of the isolated sEVs were evaluated by scanning electron microscopy (SEM), and their protein concentration was determined using the Bradford assay.
Results
Flowcytometric analysis revealed high expression of MSC-associated markers CD73 (98.8%) and CD105 (93.8%), while hematopoietic markers CD34 and CD45 were expressed at levels below 2%. The osteogenic and adipogenic differentiation potentials of the cells were also confirmed. SEM images demonstrated preserved membrane integrity and spherical morphology of the isolated sEVs, with a size distribution falling within the expected range of approximately 40–60 nm. The protein concentration of the isolated sEVs was quantified at 500 µg/mL based on the Bradford assay (n = 3).
Conclusions
These findings indicate that the explant method, in conjunction with density gradient centrifugation–based commercial kits, provides a simple, cost-effective, and efficient approach for the isolation of WJ-MSCs and production of high-quality sEVs, thereby providing a practical platform for future preclinical and translational applications.
Type of Study: Research |
Subject:
Stem cells
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