Ethics code: IR.TMI.REC.1403.018
Abstract: (8 Views)
A B S T R A C T
Background and Objectives
Exosomes are a subclass of extracellular vesicles (EVs) released via the endosomal pathway, ranging in diameter from 30 to 150 nm. They carry a diverse of biological macromolecules such as DNA, RNA, and proteins. Platelet-derived exosomes are the most abundant nanoparticles in the bloodstream. Given the need for simple and efficient isolating techniques, this study evaluates a method based on polymer precipitation for exosome isolation.
Materials and Methods
This experimental interventional study utilized platelet concentrate samples prepared by the platelet-rich plasma method, obtained from the Iranian Blood Transfusion Organization after one day after collection. On day three of storage, two different protocols differing in centrifugation steps and based on polymer precipitation were used to isolate platelet exosomes. The size of the isolated particles was compared by dynamic light scattering (DLS) (n=2). Additionally, the concentration of the isolated particles was determined using the Bradford assay (n=2), and Western blot analysis was performed for presence of the exosomal marker CD63.
Results
DLS analysis showed that Protocol A produced particles with a broader size distribution, with over 73% of particles exceeding the accepted exosome size range (>150 nm). In contrast, Protocol B which included multiple consecutive centrifugation steps, 100% of particles were within the 30-150 nm range, demonstrating more uniform size distribution. Bradford assay results showed that the average protein concentration of the extracted nanoparticles was 0.424±0.12 mg/mL for protocol A compared to 2.62±0.19 mg/ml for protocol B. Western blot analysis confirmed the expression of exosomal marker CD63 in the isolated particles.
Conclusions
Our findings demonstrate that performing multiple consecutive centrifugation steps into the polymer-precipitation method significantly enhances the purity and size of isolated platlet exosomes. This study introduces an optimized protocol for isolating high-quality exosomes based on polymer-precipitation.
Background and Objectives
Exosomes are a subclass of extracellular vesicles (EVs) released via the endosomal pathway, ranging in diameter from 30 to 150 nm. They carry a diverse of biological macromolecules such as DNA, RNA, and proteins. Platelet-derived exosomes are the most abundant nanoparticles in the bloodstream. Given the need for simple and efficient isolating techniques, this study evaluates a method based on polymer precipitation for exosome isolation.
Materials and Methods
This experimental interventional study utilized platelet concentrate samples prepared by the platelet-rich plasma method, obtained from the Iranian Blood Transfusion Organization after one day after collection. On day three of storage, two different protocols differing in centrifugation steps and based on polymer precipitation were used to isolate platelet exosomes. The size of the isolated particles was compared by dynamic light scattering (DLS) (n=2). Additionally, the concentration of the isolated particles was determined using the Bradford assay (n=2), and Western blot analysis was performed for presence of the exosomal marker CD63.
Results
DLS analysis showed that Protocol A produced particles with a broader size distribution, with over 73% of particles exceeding the accepted exosome size range (>150 nm). In contrast, Protocol B which included multiple consecutive centrifugation steps, 100% of particles were within the 30-150 nm range, demonstrating more uniform size distribution. Bradford assay results showed that the average protein concentration of the extracted nanoparticles was 0.424±0.12 mg/mL for protocol A compared to 2.62±0.19 mg/ml for protocol B. Western blot analysis confirmed the expression of exosomal marker CD63 in the isolated particles.
Conclusions
Our findings demonstrate that performing multiple consecutive centrifugation steps into the polymer-precipitation method significantly enhances the purity and size of isolated platlet exosomes. This study introduces an optimized protocol for isolating high-quality exosomes based on polymer-precipitation.
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