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

Isolamento escalável e purificação de vesículas extracelulares de Escherichia coli e out...

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

Isolamento escalável e purificação de vesículas extracelulares de Escherichia coli e outras bactérias

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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.

As bactérias secretam vesículas extracelulares (EVs) de tamanho nanométrico que transportam moléculas biológicas bioativas. A pesquisa EV se concentra na compreensão de sua biogênese, papel nas interações micróbio-micróbio e hospedeiro-micróbio e doença, bem como suas potenciais aplicações terapêuticas. Um fluxo de trabalho para isolamento escalável de EVs de várias bactérias é apresentado para facilitar a padronização da pesquisa de EV.

Este protocolo é importante porque fornece uma maneira de isolar vesículas extracelulares de uma variedade de bactérias de uma maneira altamente reprodutível. A grande coisa sobre esta técnica é que ela pode ser usada para isolar EVs de ambientes bastante grandes de culturas bacterianas, o que permitirá estudos in vivo. Embora os dados que mostramos reflitam aplicações pré-clínicas, é previsível que este protocolo possa ser adaptado para a fabricação de EVs bacterianos para terapia.

Este método pode ser aplicado a qualquer sistema de cultura de células escalável com pequenas modificações. Devido à escalabilidade do protocolo, é importante ter filtros de tamanho apropriado e colunas SEC para lidar com os volumes iniciais desejados de cultura de células bacterianas. Demonstrando os procedimentos estão Sadie Johnson, uma tecnóloga assistente no laboratório do Dr. Justin Lathia, Dionysius Watson, um bolsista de oncologia no laboratório do Dr. Justin Lathia, e Akeem Santos, um tecnólogo de pesquisa no meu laboratório.

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