Dataset Information

Purity and yield of melanoma exosomes are dependent on isolation method.

ABSTRACT: Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58-fold more exosomes than ultracentrifugation, up to 836-fold lower concentrations of co-purified soluble factors when adjusted for exosome yield, and a greater than two-fold increase in PD-L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co-eluted soluble factors.

SUBMITTER: Shu S

PROVIDER: S-EPMC6882439 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Purity and yield of melanoma exosomes are dependent on isolation method.

Shu Shin La S Yang Yunchen Y Allen Cheryl L CL Hurley Edward E Tung Kaity H KH Minderman Hans H Wu Yun Y Ernstoff Marc S MS

Journal of extracellular vesicles 20191120 1

Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine prof ...[more]

PMID: 31807236

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Project description:Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application. Impact statement Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) has raised questions regarding their biologic functions and their potential theranostic application. This study demonstrates that the harvesting methods used can result in samples with physical and biochemical properties that are unique to the isolation and solubilization methods used. Consequently, developing harvesting methods that minimize sample contamination with ECM remnants and/or solubilization agents will be essential in determining the theranostic potential of MBV in future studies.

Dataset Information

Purity and yield of melanoma exosomes are dependent on isolation method.

Publications

Purity and yield of melanoma exosomes are dependent on isolation method.

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