Matrix-Bound Nanovesicles: The Effects of Isolation Method upon Yield, Purity, and Function
| Autor: | Hui Li, Catalina Pineda Molina, Neill J. Turner, Lisa J. White, Lina M. Quijano, Stephen F. Badylak, Li Zhang, Juan Diego Naranjo, Joseph G. Bartolacci, Salma O. El-Mossier |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0206 medical engineering
Size-exclusion chromatography Biomedical Engineering Medicine (miscellaneous) Bioengineering 02 engineering and technology Extracellular matrix 03 medical and health sciences Extracellular Vesicles medicine Humans Particle Size 030304 developmental biology Cell Proliferation 0303 health sciences biology Chemistry Stem Cells Proteins Proteinase K 020601 biomedical engineering Endocytosis Enzymes Extracellular Matrix Methods Articles Ultrafiltration (renal) MicroRNAs Biochemistry Solubility Yield (chemistry) Collagenase biology.protein Nanoparticles Ultracentrifuge Peptides Function (biology) medicine.drug |
| Zdroj: | Tissue Eng Part C Methods |
| ISSN: | 1937-3384 1937-3392 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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