Extracellular Vesicles: Applications to Regenerative Medicine, Therapeutics and Diagnostics

358 Pages·2021·16.286 MB·English

by  Wojciech Chrzanowski, Chwee Teck Lim, Sally Yunsun Kim

Extracellular Vesicles
Applications to Regenerative Medicine, Therapeutics and Diagnostics
Biomaterials Science Series
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Extracellular Vesicles
Applications to Regenerative Medicine,
Therapeutics and Diagnostics
Edited by
Wojciech Chrzanowski
University­of­Sydney,­Australia
Email:­[email protected]
Chwee Teck Lim
National­University­of­Singapore,­Singapore
Email:­[email protected]
and
Sally Yunsun Kim
Imperial­College­London,­UK
Email:­[email protected]
Biomaterials Science Series No. 9
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Electronic ISSN: 2397- 141X
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Preface
Extracellular vesicles (EVs) are membrane- enclosed structures secreted ubiq-
uitously by cells. They are thought to mediate communication between cells.
Understanding this cellular communication offers potential for new diagnos-
tics, drug/small molecule delivery vehicles and therapeutics for a variety of
medical conditions, including incurable ones such as chronic lung diseases/
injury, Parkinson's disease and multiple sclerosis.
EVs have also been lauded as the new generation of medicine. In stark con-
trast to conventional pharmacological interventions, most of which are based
on a single drug with a limited number of actions, EVs contain a multitude of
naturally produced molecules that can act synergistically to activate multiple
cell types and accelerate tissue repair. It is envisaged that in the future, EVs
containing particular combinations of these naturally produced molecules
will be able to be ‘farmed’ from cultures of specific cell types under specific
conditions, and then delivered to selected tissues or organs for specific ther-
apeutic purposes; e.g. to repair damaged lung tissue due to smoke inhalation
injury or to repair myelination around nerve cells to cure multiple sclerosis.
Since the composition of EVs mirrors that of the cell from which they are
secreted, EVs can be the ideal biomarkers for disease diagnosis, especially for
early detection. Additionally, because of their composition, namely what is in
and on EVs, they have inherent suitability to target specific cells and tissues
in the body, and hence are ideal vehicles for drug delivery. The key benefit is
that EVs can precisely deliver their payloads without activating the innate or
acquired immune system.
While EVs present us with the possibility to revolutionise medicine – with
astonishingly real possibilities for success – they are very heterogenous, have
not been fully characterised, are difficult to isolate and the regulatory path-
way for this new class of biologics is still not well defined.

Biomaterials Science Series No. 9
Extracellular Vesicles: Applications to Regenerative Medicine, Therapeutics and Diagnostics
Edited by Wojciech Chrzanowski, Chwee Teck Lim and Sally Yunsun Kim
© The Royal Society of Chemistry 2022
Published by the Royal Society of Chemistry, www.rsc.org
v
vi Preface
In this book, we present state- of- the- art EV science with a specific focus on
the basic biology of EV biogenesis and production, isolation, physicochemi-
cal characterisation and their role in regenerative medicine for the treatment
of major medical conditions. We also present key aspects related to the stan-
dardisation and commercialisation landscape of EVs.
We hope that this book will help to advance the fundamental science of
EVs and contribute to the new developments to overcome current major lim-
itations in isolation, identification and characterisation, as well as applica-
tion and commercialisation of EVs. We believe that EVs will soon become
mainstream medicine.
Wojciech Chrzanowski
University of Sydney
Chwee Teck Lim
National University of Singapore
Sally Yunsun Kim
Imperial College London
Contents
Chapter 1 Therapeutic Potential of Mesenchymal Stromal
Cell- derived Small Extracellular Vesicles 1
Fabiola Nardi Bauer and Bernd Giebel
1.1 Concepts in Regenerative Medicine 1
1.2 MSCs Act in a Paracrine Rather than Cellular Manner 3
1.3 MSC- EVs Mediate Their Therapeutic Effects via
Extracellular Vesicles 4
1.4 MSC- sEVs Improve Disease Symptoms in
Many Different Disease Models 6
1.5 MSC- sEVs Have Successfully Been Applied to Patients 7
1.6 Immunomodulatory Activities of MSC- EV Products 8
1.7 Other Activities Mediated by MSC- EV Products 9
1.8 Heterogeneity of MSC- sEV Preparations 9
1.9 Take- home Message 11
References 11
Chapter 2 Separation and Purification of Extracellular and Bio-fl uid
Vesicles – State of the Art: Past, Present, and Future of
Extracellular Vesicle Separation and Purification 22
Vijaya Sunkara, Hyun- Kyung Woo and Yoon- Kyoung Cho
2.1 Introduction 22
2.2 EV Separation Methods 24
2.2.1 Density- based Separation 24
2.2.2 Size- based Separation 27
2.2.3 Affinity (AFF) 28
2.2.4 Precipitation (PPT) 29

Biomaterials Science Series No. 9
Extracellular Vesicles: Applications to Regenerative Medicine, Therapeutics and Diagnostics
Edited by Wojciech Chrzanowski, Chwee Teck Lim and Sally Yunsun Kim
© The Royal Society of Chemistry 2022
Published by the Royal Society of Chemistry, www.rsc.org
vii
viii Contents
2.3 Comparative Evaluation of EV Separation
Methods 29
2.4 Combination of Methods for EV Separation 33
2.5 Emerging Technologies 34
2.6 Conclusions and Future Perspectives 38
References 40
Chapter 3 Physicochemical Characterisation of Extracellular
Vesicles 45
Su Hyeon Park, Thanh Huyen Phan, Jeung Eun Kim
and Wojciech Chrzanowski
3.1 Introduction 45
3.1.1 Perceived Significance of Single
Particle Analysis 46
3.2 Characterisation Methods 47
3.2.1 Characterisation of the Size, Size
Distribution and Concentration of EVs 47
3.2.2 Morphological Descriptive Analysis 57
3.3 Summary 69
3.3.1 Advantages and Disadvantages of
Each Technique 69
3.3.2 Types of Analysis Conducted With
Each Technique 70
References 71
Chapter 4 Biological Characterization of Extracellular Vesicles –
Methodologies to Characterize Molecular Composition
of Extracellular Vesicles – Advances Towards Single
Vesicle Characterization 76
Kirsi Rilla
4.1 Introduction 76
4.2 The Demand for Single Vesicle Characterization
Methods 78
4.3 Imaging Methods 79
4.4 Light Microscopy 80
4.4.1 Widefield Fluorescence Microscopy 80
4.4.2 Confocal Microscopy 80
4.4.3 Super- resolution Techniques 81
4.4.4 Fluorescence Correlation Spectroscopy (FCS) 82
4.5 Electron Microscopy 82
4.5.1 Transmission Electron Microscopy (TEM) 82
4.5.2 Cryo- electron Microscopy 82
4.5.3 Scanning Electron Microscopy (SEM) 83
4.5.4 Immunoelectron Microscopy (iEM) 83
4.5.5 Correlative Imaging Techniques 83
Contents ix
4.5.6 Flow Cytometry 84
4.5.7 Label- free Techniques 85
4.6 Detection of Specific Molecules in EVs by Labeling
Methods for Light Microscopy 85
4.6.1 Labeling Methods for EVs 85
4.6.2 Lipid Stains in EV Detection 86
4.6.3 Proteins 86
4.6.4 Live Cell (EV) Imaging by Light Microscopy 87
4.6.5 Analysis of Carbohydrates 89
4.7 Future Challenges and Opportunities 89
References 90
Chapter 5 Extracellular Vesicles in Bone Tissue Engineering 95
Owen G. Davies
5.1 Introduction 95
5.1.1 A Vesicle- based Solution 97
5.1.2 Advantages of Using EVs 98
5.1.3 The Role of EVs in Bone Formation 114
5.2 EVs in Bone Tissue Engineering 115
5.2.1 Priming EVs for Bone Tissue Engineering 117
5.2.2 Scaffold- based Approaches 120
5.2.3 EV Modification Strategies 124
5.2.4 EVs for the Delivery of Biotherapeutic
Molecules 125
5.3 Concluding Remarks 126
References 127
Chapter 6 Mesenchymal Stromal Cell (MSC)- derived Small
Extracellular Vesicles as Next- generation
Therapeutics for Cartilage Regeneration 138
Kristeen Ye Wen Teo, Shipin Zhang and Wei Seong Toh
6.1 Introduction 138
6.2 Stem Cells 139
6.3 Mesenchymal Stromal/Stem Cells (MSCs) 140
6.4 MSC Therapies for Cartilage Injury and
Osteoarthritis 141
6.5 Paracrine Mechanisms of MSCs in Cartilage
Repair 143
6.6 Extracellular Vesicles 144
6.7 Mesenchymal Stem Cell- derived Small Extracellular
Vesicles (MSC- sEVs) 145
6.8 MSC- sEV Therapies for Cartilage Injury and
Osteoarthritis 147
6.9 MSC- sEVs as Next- generation Therapeutics for
Cartilage Repair 150