Extracellular
vesicle isolation
methods: rising
impact of
size-exclusion
chromatography
Presented By
0423022303 – KAZI
TOWFIQUL ISLAM
Content
• Procedure Background
• Outline of The Paper
• Outcome of The paper based on Data or
Figure.
• Conclusions and perspectives
Presentation title
20XX
2
Procedure Background
SIZE EXCLUSION CHROMATOGRAPHY
Definition
History
Principal
Also known as Molecular
Sieve Chromatography
First recognized 60 years ago
by biochemists for analyzing
complex biological samples.
The separation of molecules
based on size or weight.
A separation method of eluting
desired molecules in a
solution mixture, based on
their size and molecular
weight by passing them
through physically, chemically
stable and inert porous matrix.
Presentation title
Howard Barth traces the early
development of SEC
Basis in HPLC Techniques,
Filtration of gel composing of
spherical beads with specific
pore sizes;
The small size particle
reaches the interior of the
pores in the stationary phase,
whereas the large size
particles are excluded, being
unhindered completely.
20XX
3
SIZE EXCLUSION CHROMATOGRAPHY
Advantages
Disadvantages
Applications
Effective Separation
Buffer Independence
Necessity for Mobile Phase
Filtering
Well-Defined Separation
Limited Resolution of Peaks
Short Analysis Time
Poor Selectivity
Analysis of Proteins Purification, identification, and
fractionation (Desalting and
Copolymerization studies)
No Sample Loss
Low Sample Load Capacity
Adjustable Flow Rate
Molecular weight
determination of
macromolecules
High Sensitivity
Molecules Separation
Minimal Mobile Phase
Requirement
Presentation title
20XX
4
Outline of The Paper
Introduction
Extracellular Vesicles (EVs): Diverse membrane-bound
nanovesicles from secreting cells, composed of proteins,
lipids, metabolites, and nucleic acids and protection from
degradation through lipid bilayer
Research Interest and Potential: Valuable carriers of
physiological and pathological information, arising curiosity
because of functioning in human conditions and diseases
and as a source for novel biomarkers in liquid biopsy and as
cell-free therapeutic agents for regenerative medicine.
Advantages of EVs: Stability, ease of handling, and
sterilizability
Schematic representation of EV biogenesis and
secretion:
a) Exosome Formation b) Exosome Release c) Microvesicle Formation and Release and d) Apoptotic Body
Generation
Presentation title
Focus on Size Exclusion Chromatography (SEC): A
potential method for isolating well-purified and defined EV
preparations and a promising avenue for advancing EV
research and application.
20XX
5
Methods for
EV isolation
Presentation title
•
Ultracentrifugation (dUC): Initial Biofluid Composition, pellet Formation and further Purification ( Disc-Uc and DG –UC ,utilization of
sucrose cushion and sucrose or iodixanol gradients respectively to separate proteins and extracellular vesicle (EV) populations.
•
Ultrafiltration: The dead-end filtration system separates molecules based on their molecular weight cutoff
•
Size-Exclusion Chromatography (SEC): Elution of molecules first and later , Intermediate entrance slowly in matrix bead pores.
•
Asymmetrical Flow Field-Flow Fractionation (AFFFF):Separation of molecules through hydrodynamic radius (size) based on
crossflow and longitudinal laminar flow
•
Precipitation-Based Isolation: Concentrating particles into a pellet through PEG.
•
Immunoaffinity Isolation: Recognition of EV-specific markers using specific antibodies, Coupling of separation through centrifugation
or magnetic capture
20XX
6
Outline
• Extracellular vesicles (EVs) are small vehicles released by cells,
carrying bioactive molecules for intercellular communication and
showing promise for immunomodulation, tissue repair, and
biomarker discovery.
• Isolating EVs accurately is challenging due to their small size
and heterogeneity.
• While ultracentrifugation has been standard, size exclusion
chromatography (SEC) has emerged as superior.
• SEC efficiently removes soluble proteins, is user-friendly, and
preserves EV integrity and content.
• It holds potential for biomarker discovery and therapeutics with
scalability and high-quality.
Presentation title
20XX
7
OUTCOME OF THE PAPER
• 1. Analysis of EV isolation protocols from articles in
the EV-TRACK database focused on EV function (n =
937):
• Density gradient (DG) ultracentrifugation is primarily
utilized as a validation technique or in combination with
dUC for downstream functional assays.
• SEC (size exclusion chromatography) is not among the
preferred methods.
• 2. Analysis of EV isolation protocols from articles
published in 2017 (n = 34):
• In publications from 2017, SEC emerges as the second
most used method for EV isolation, often in combination
with other techniques.
Presentation title
20XX
8
Result
Presentation title
•
Purity: Higher scores imply better separation and purification of EVs.
•
Resolution: Higher scores suggest better capability to resolve distinct EV
populations
•
Recovery: Higher scores indicate higher yields of EVs obtained.
•
Functional Studies: Higher scores imply the ease of using the isolated EVs for
functional assays.
•
Ease of Use: Lower scores indicate higher complexity and specialized training
requirements.
•
Time: Lower scores indicate shorter processing times.
•
Scalability: Higher scores suggest better scalability of the method.
•
Specialized Equipment: Higher scores imply higher requirements for specialized
equipment.
•
Commercial Availability: Higher scores indicate wider availability of commercial
kits.
•
Automatization: Higher scores suggest better suitability for automation.
•
Cost of Consumables: Lower scores indicate lower consumable costs per
sample.
20XX
9
Conclusions and perspectives
•
Role of EVs in Intercellular Communication: As potent mediators of intercellular communication
by transferring proteins, lipids, and nucleic acids.
•
Emerging Clinical Applications of EVs: Exploring EV-based therapies and disease-specific
diagnostic or prognostic tools, emphasizing the need for reliable automated production
platforms and highly purified EV fractions for biomarker discovery.
•
Advantages of SEC-Based Isolation: SEC is user-friendly, less time-consuming, and maintains
EV properties effectively, making it suitable for research and clinical applications.
•
Preservation of EV Functionality: Isolation method can influence the functional activity of EVs,
with SEC-isolated EVs maintaining genuine functions compared to other methods, such as
precipitation-based isolation.
•
Potential EV-Based Clinical Applications: Cell-Free Therapies and Biomarkers for Disease
Diagnosis and Prognosis.
SEC, with its technical advantages and scalability, holds significant potential for advancing EVbased research and clinical applications, providing a versatile and effective method for EV
isolation in diverse laboratory settings.
Presentation title
20XX
10
• BIOCHEMICAL TECHNOLOGY,
•
• DEPARTMENT OF CHEMICAL ENGINEERING,
Thank you
Presentation title
• BANGLADESH UNIVERSITY OF ENGINEERING TECHNOLOGY,
DHAKA , BANGLADESH
20XX
11