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

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and ...

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

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

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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

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.

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

This protocol is important because it provides a way to isolate extracellular vesicles from a variety of bacteria in a highly reproducible manner. The great thing about this technique is it can be used to isolate EVs from quite large environments of bacterial cultures, which will enable in vivo studies. While the data we show reflects preclinical applications, it is foreseeable that this protocol could be adapted to manufacturing bacterial EVs for therapeutics.

This method can be applied to any scalable cell culture system with minor modification. Because of the protocol scalability, it's important to have appropriately-sized filters and SEC columns to handle the desired starting volumes of bacterial cell culture. Demonstrating the procedures are Sadie Johnson, an assistant technologist in Dr.Justin Lathia's lab, Dionysius Watson, an oncology fellow in Dr.Justin Lathia's lab, and Akeem Santos, a research technologist in my laboratory.

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Extracellular Vesicles Escherichia Coli Bacterial Cultures Scalable Isolation Purification Protocol In Vivo Studies Therapeutic Manufacturing Luria-Bertani Broth Centrifugation Filtration Device Ultrafiltration Protocol Scalability Viable Cells Removal