Isolation of extracellular vesicles from human plasma samples: The importance of controls.
Biotechnol J
; 18(6): e2200575, 2023 Jun.
Article
en En
| MEDLINE
| ID: mdl-36988156
ABSTRACT
BACKGROUND:
Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. RATIONAL Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. METHOD ANDRESULTS:
We performed a comparative study to evaluate the effect of EV isolation methods focusing on (a) pretreatment of plasma with additives, which include rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000 g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks.CONCLUSION:
Appropriate controlsare required when adding any additives during EV isolation as even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Plasma
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Precipitación Química
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Ultracentrifugación
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Métodos Analíticos de la Preparación de la Muestra
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Vesículas Extracelulares
Límite:
Animals
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Humans
Idioma:
En
Revista:
Biotechnol J
Asunto de la revista:
BIOTECNOLOGIA
Año:
2023
Tipo del documento:
Article
País de afiliación:
Canadá