Scalable Isolation and Purification of Extracellular Vesicles from <em>Escherichia coli</em> and Other Bacteria

Dionysios C. Watson; Sadie Johnson; Akeem Santos; Mei Yin; Defne Bayik; Justin D. Lathia; Mohammed Dwidar

doi:10.3791/63155

Aislamiento escalable y purificación de vesículas extracelulares de Escherichia coli y o...

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Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Aislamiento escalable y purificación de vesículas extracelulares de Escherichia coli y otras bacterias

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09:56 min

October 13, 2021

DOI: 10.3791/63155-v

Dionysios C. Watson^1,2,3, Sadie Johnson¹, Akeem Santos^1,4, Mei Yin⁵, Defne Bayik¹, Justin D. Lathia^1,3, Mohammed Dwidar^1,3,4

¹Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute,Cleveland Clinic, ²University Hospitals Cleveland Medical Center, ³Case Western Reserve University, ⁴Center for Microbiome & Human Health,Cleveland Clinic, ⁵Electron Microscopy Core, Lerner Research Institute,Cleveland Clinic

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Overview

This study addresses the isolation of extracellular vesicles (EVs) from various bacterial cultures to enhance the reproducibility of EV research. The protocol offers a scalable approach suitable for large volumes, paving the way for in vivo studies and potential therapeutic applications of bacterial EVs.

Key Study Components

Research Area

Extracellular vesicle isolation
Microbe-microbe and host-microbe interactions
Therapeutic applications of bacterial EVs

Background

Importance of bacterial EVs in biological interactions
Need for standardized methods in EV research
Potential for therapeutic manufacturing of EVs

Methods Used

Scalable isolation protocol for EVs from Escherichia coli
Filtration and size exclusion chromatography techniques
Use of specialized equipment like centrifuges and filters

Main Results

Successfully isolated EVs from large volumes of bacterial cultures
Validated the absence of viable cells post-filtration
Adapted methods for different cell culture systems

Conclusions

The study demonstrates a reproducible method for EV isolation.
It outlines significant potential for bacterial EVs in therapeutic applications.

Frequently Asked Questions

What are extracellular vesicles?

Extracellular vesicles (EVs) are nanometer-sized particles secreted by bacteria that carry biological molecules and play essential roles in communication and interaction.

Why is scalable isolation of EVs important?

Scalable isolation allows researchers to obtain sufficient quantities of EVs for meaningful in vivo studies and therapeutic applications.

Which bacteria were used in this study?

The study focused on isolating EVs specifically from Escherichia coli.

What methods were utilized to ensure the purity of the EVs?

Filtration and size exclusion chromatography were employed to ensure that the EVs were free from viable cells and other contaminants.

How can this protocol be adapted for other organisms?

The protocol can be modified slightly to accommodate various scalable cell culture systems.

What are the potential therapeutic applications of bacterial EVs?

Bacterial EVs may be developed for use in therapeutics, including drug delivery or as vaccines.

What are the benefits of using size exclusion chromatography?

Size exclusion chromatography enables efficient separation of EVs based on size, ensuring higher purity and concentration for further studies.

Las bacterias secretan vesículas extracelulares (EV) de tamaño nanométrico que transportan moléculas biológicas bioactivas. La investigación EV se centra en comprender su biogénesis, su papel en las interacciones y enfermedades microbio-microbio y huésped-microbio, así como sus posibles aplicaciones terapéuticas. Se presenta un flujo de trabajo para el aislamiento escalable de vehículos eléctricos de varias bacterias para facilitar la estandarización de la investigación de vehículos eléctricos.

Este protocolo es importante porque proporciona una forma de aislar vesículas extracelulares de una variedad de bacterias de una manera altamente reproducible. Lo mejor de esta técnica es que se puede utilizar para aislar EV de entornos bastante grandes de cultivos bacterianos, lo que permitirá estudios in vivo. Si bien los datos que mostramos reflejan aplicaciones preclínicas, es previsible que este protocolo pueda adaptarse a la fabricación de EV bacterianas para terapia.

Este método se puede aplicar a cualquier sistema de cultivo celular escalable con modificaciones menores. Debido a la escalabilidad del protocolo, es importante tener filtros y columnas SEC de tamaño adecuado para manejar los volúmenes iniciales deseados de cultivo celular bacteriano. Demostrando los procedimientos están Sadie Johnson, tecnóloga asistente en el laboratorio del Dr. Justin Lathia, Dionysius Watson, becario de oncología en el laboratorio del Dr. Justin Lathia, y Akeem Santos, un tecnólogo de investigación en mi laboratorio.

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