Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current Status and Prospects

Abstract

Exosomes have come a long way since they were first described in 1981 by Trams et al. as small lipid bilayer-enclosed vesicles of endocytic origin. Their ability to alter cell bioactivity combined with their advancing popularity as disease biomarkers and therapeutic delivery systems has compelled major Government institutions and regulatory authorities to invest further in this ever-growing field of research. Being relatively new, exosome research is besieged by challenges including but not limited to inefficient separation methods and preservation techniques, difficulties in characterization, and lack of standardized protocols. However, as excitement and research on exosomes increase, their relevance and capacity to elicit a distinct biological response is reinforced. Therefore, it is pertinent to further explore their potential as cell-free therapeutics. This review focuses on current difficulties and subsequent strategies to refine existing methodologies for efficient clinical translation of exosomes in a streamlined and cost-effective manner. The chapter is briefly divided into subsections, each relevant for sequential therapeutic development such as their classification, isolation, scaling up, storage, characterizations, regulatory requirements, therapeutic developments, and perspectives. Apart from literature search, we have endeavored to bring in our own experience in this field including some recent clinical developments.

1. Introduction

This chapter reiterates the central dogma that mesenchymal stem cells (MSCs) ameliorate disease not just by virtue of their differentiation and self-renewal abilities, but in a paracrine manner, by secreting anti-inflammatory, immunomodulatory, and regenerative factors. Among these paracrine mediators, nanosized extracellular vesicles “exosomes” have generated supreme interest, owing to reports of their standalone therapeutic effect. Stem cell exosomes can circumvent the safety risks associated with the administration of cell therapy.

2. History and evolution of exosomes

A ground-breaking study by Chargaff and West in 1946 [1] for the first time detailed the phenomenon of plasma membrane fragments being shed off viable cells and forming “high particle weight lipoproteins.” It was not until two decades later, when the vesicular particles isolated from body fluids were given some attention. Initially disregarded as artifacts of the separation technique [2], these were later believed to be associated with viruses [3]. Wolf and Prince then identified the usefulness of these serum-isolated extracellular vesicles and termed them “phospholipid-rich platelet dust” that could essentially be separated out by ultracentrifugation [4]. It was only in 1975, when particles of 30 to 60 nm diameter, containing an electron-dense core enveloped by a membrane, were recognized as microvesicles, and were firmly established as “breakdown products of normal cellular components,” thus freeing them from any association with viruses’ [5]. In 1981, Trams and co-workers coined the term “exosomes” for microvesicles harvested from tissue culture supernatant [6]. The exact physiological function of exosomes remained unknown, but reports of specific plasma membrane domains within sparked interest. In 1983, the phenomenon of formation and release of cellular vesicles by exocytosis was outlined by the works of Stahl and Johnstone, respectively [7, 8]. These reports individually identified exosomes as 50-nm spheres displaying receptors on their external surface and originating from a “non-lysosomal endocytic compartment.” By mid to late 80s, the term exosome had caught on [9], and “exosome secretion pathway” was acknowledged for the existence of a novel intracellular trafficking pathway via shedding of cell membrane [10, 11]. In 1991, Johnstone identified cellular stress as the primary factor to aid internalization and shedding of archaic components of the plasma membrane in the form of exosomes, the mechanism of which was not yet known [12]. However, it was Johnstone’s pivotal paper in 2005 [13], which underlined the biological significance of exosomes, establishing for the first time a fate for them that was beyond cellular waste. Following that, the field saw an exponential increase in exosome research including scale-up processes, use of direct modifications, and genetic engineering, paving the way for regenerative therapy. Figure 1, adapted from a landmark article in the field, depicts a timeline on exosome [14].

Figure 1.

Evolution and timeline of research conducted on exosomes. A brief timeline of when EVs were discovered, and coining of the term “exosomes.”

3. Classification of exosomes

An inherent size overlap between distinct EV subtypes poses a challenge for characterization solely based on size [15]; however, the presence of markers associated with EV origin can further assist classification. Based on size, either small or large EV categories comprise of five major populations: exomeres, exosomes, migrasomes, apoptotic bodies, and large oncosomes [16]. The largest among these are apoptotic bodies, with a diameter > 800 nm (can go up to 5 μM), and consist of plasma membrane and cytoplasmic components of post-apoptotic (dying) cells. Smaller than these are microvesicles, or ectosomes ranging in size from 100 nm to 1 μm. These originate from an irregular blebbing of the plasma membrane. The smallest EVs, exosomes generate from multi-vesicular endosomes, and contain proteins, lipids, and nucleic acids [17, 18]. Their size is much under debate, with smallest exosomes at 30 nm and largest anywhere between 150 and 200 nm. Since exosomes are secreted only upon environmental and physiological cues like cellular stress, their selectively integrated cargo carries specific instructions for modulating target cells. Hence, this EV subclass is often referred to as “signalosomes.” Interestingly, microvesicles and exosomes are both released by non-apoptotic cells (Table 1).

4. Exosome morphology

Dehydration during sample preparation in conventional electron microscopic techniques forces exosomes to reveal a cup-shaped structure, and they appear as flattened spheres [19, 20]. Cryo-electron microscopy helps exosomes remain fully hydrated and enables exosomes to retain a proper spherical morphology, thus it is a superior technique [21].

5. Biogenesis, release, and uptake of exosomes

Exosome biogenesis and their secretion involve a complex molecular pathway and exchange of material which is tightly regulated by each source cell [22, 23]. Cells secrete exosomes at different rates, depending on their type, metabolism, and other factors. The first step in this multistep pathway is invagination of the plasma membrane via endocytosis [24], forming endosomes, which mature to late endosome, also known as Multivesicular Bodies (MVBs), containing a population of ILVs (Intraluminal Endosomal Vesicles) [24]. The final fate of MVBs is to either i) undergo degradation via lysosomes or ii) fuse with plasma membrane and release the ILVs as exosomes into extracellular space [25, 26, 27]. Most crucial step is the process of channeling and deposition of a specific subset of proteins (including tetraspanins and some endosomal proteins), lipids (ceramide), and other macromolecules into the ILVs, for which the Endosomal Sorting Complex Required for Transport (ESCRT) pathway is recruited [28, 29, 30]. The ESCRT pathway is an intricate, Adenosine Tri Phosphate (ATP)-dependent process involving the use of four complexes—ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III along with associated proteins Tsg101 and Alix among others [31, 32, 33]. Alternatively, the sorting of exosomal content and biogenesis may occur via an ESCRT-independent pathway [34, 35] that surpasses ceramide-mediated membrane budding [15]. Trafficking of exosomes to plasma membrane and their subsequent release involve binding to tether proteins mediated by Rab GTPases [36], followed by the fusion complex SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that brings membranes into close proximity [37], while sphingomyelinase mediates release [29]. The possible outcomes after exosome release are as follows:

1. capture by neighboring cells

2. reabsorption by the parent cell

3. relocation and uptake by a remote cell

4. entry into circulation via body fluids

Target cells uptake exosomes through (i) endocytosis mediated by clathrin, claveolin, or lipid rafts; (ii) direct plasma membrane fusion; or (iii) receptor-ligand interaction on the cells surface [38], as shown in Figure 2. Once internalized by the target cell, exosomes will fuse with an endocytic vesicle, releasing RNA and proteins in the cytosol. All cell types including stem cells secrete exosomes, found in various body fluids such as saliva, tears, plasma, serum, cerebrospinal fluid, bronchial fluid, synovial fluid, amniotic fluid, breast milk, urine, semen, lymph, bile, gastric acids [39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49]. However, this endosomal pathway for exosome biogenesis is the one factor that distinguishes exosomes from other extracellular vesicles (EVs) [50, 51]. Contrastingly, both Apoptotic bodies and Microvesicles are formed via outward blebbing of plasma membrane [15, 52, 53, 54].

Figure 2.

MSC exosome biogenesis, release, and uptake. Pathway for stem cell exosome biogenesis, release, and three mechanisms for subsequent uptake by recipient cells (1). Direct fusion with target cell (2). Endocytosis (3). Binding to cell surface receptor on target cell and internalization.

6. Exosome composition

Stem cell exosomes partly replicate the content of their cells of origin [26, 55]. This discovery, coupled with the revelation that they represent a very specific subcellular compartment, the components of which are selectively sequestered, led to the hypothesis that exosomes are more than just cell debris. Stem cell exosomes comprise a specific milieu of cytoplasmic and membrane proteins including receptors, enzymes, transcription factors, extracellular matrix proteins, lipids, and nucleic acids all of which target molecular pathways and are biologically active in recipient cells [56, 57]. Proteomic databases have now made available the protein composition of exosomes [58, 59]. Irrespective of their origin, all exosomes share few proteins, for instance a specific subset of endosomal, plasma membrane, and cytosolic proteins including cell adhesion molecules (CAMs), integrins, tetraspanins (CD9, CD63, and CD81), heat shock proteins (Hsp60, Hsp70, and Hsp90), biogenesis-related proteins (ALIX and TSG101), and Major Histocompatibility Complex—MHC-I/II proteins). Other transfer and fusion proteins such as Flotillins, Annexins, Heat Shock Proteins, Rab2, Rab7 may be up- or down-regulated depending on the tissue of origin [17, 60], which also include MVB Biogenesis proteins, prostaglandins, platelet-derived growth factor, latherin, transmembrane proteins, lysosome-associated membrane protein-2B, and other phospholipases [61, 62, 63]. The lipid bilayer of exosomes (Figure 3) with a characteristic thickness of 5 nm [64] is enriched in cholesterol, sphingomyelin and other sphingolipids, ceramide, phosphoglycerides like phosphatidyl serine, and diacylglycerol, which are usually conserved and specific to the parent cell [65]. Since lipids are a key component forming and protecting the exosome structure, lipid content is conserved, and variations are only observed among different cell types [66]. Lastly, another essential cargo that is conserved is nucleic acids, which are relatively distinct from the cytosolic pool of the parent cell. These include single- and double-stranded deoxyribonucleic acids (ssDNA and dsDNA), mitochondrial (mtDNA), and coding and non-coding ribonucleic acid (RNA) such as mRNA and microRNAs [67]. Cholesterol and sphingomyelin along with GPI-anchored proteins and Flotillin are also enriched in “lipid-rafts,” implying a role for exosomes in transport [68]. Exosomes are a source of pro-inflammatory cytokines—Interleukins such as IL-1β, IL-6, and IL-8, monocyte chemoattractant protein 1 (MCP-1), Tumor Necrosis Factor-alpha (TNF-α) [69, 70]. Figure 3 is a visualization of the identity and function of well-established components that make up the stem cell exosome structure, which consists of a lipid bilayer backbone, along with protein corona/glycocalyx attached to the surface (based on initial findings) [71].

Figure 3.

Mesenchymal stem cell exosome structure – A representation. Exosome structure comprises of a cargo-bearing cytosol enveloped by a bilayer membrane carrying receptors, transmembrane proteins, integrins, lipid rafts, lipid molecules, and a range of surface markers, along with associated protein corona.

7. Exosome function

The process of exosome biogenesis aids packaging and transfer of information in the form of lipids, nucleic acids, and proteins from the parent cell into the recipient [70]. Consequently, the precise biological functions of exosomes are dependent on their composition, and the reason stem cell exosomes receive tremendous attention is their biological fingerprint and functionality mirroring that of their parental cells [72]. Stem cell exosomes have generated a lot of scientific interests owing to their primary role as message and cellular cargo transporters in intercellular communication as well as involvement in processes such as coagulation, antigen presentation, immune modulation and inflammation, regeneration, cell differentiation, waste management, proliferation and apoptosis, tumor growth, and metastasis [73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85]. Owing to their specific lipid content, they can alter lipid composition, specifically cholesterol and sphingomyelin in target cells, thereby regulating target cell homeostasis [86]. Exosomes, owing to their unique composition, provide innovative opportunities as biomarkers for diagnosis and in treatments. Currently, many clinical trials have been registered for exosome-related studies [87].

8. Isolation methods

Stem cell exosomes are multi-component vesicles with heterogeneity among populations, which necessitates the development of an effective isolation method. Many isolation techniques have been developed, which enable high yield, variable purity, and quality of isolated exosomes. The relative merits and demerits of each of these procedures are summarized in Table 2.

8.1 Differential ultracentrifugation

Based on small size and low density, stem cell exosomes can easily be separated by ultracentrifugation that works on stepwise speed increments (300 x g to 2000 x g to 10,000 x g to 100,000 x g) or alternating between high and low speed (100,000 x g to 200,000 x g), ensuring that different sized particles are separated at different times. Despite its many shortcomings, ultracentrifugation is considered the gold standard for exosome isolation and accounts for 56% of all exosome isolation techniques used in research [93]. Since this process does not separate different sized EVs, the final product is more heterogeneous and may not be suitable for governing bodies [94].

8.2 Density gradient ultracentrifugation

There are two types of preconstructed density gradient media—moving-zone gradients and isopycnic gradients. Moving-zone gradients allow EVs of distinct sizes, but same density to separate out together. Once the sample is layered from top into a tube containing progressively increasing density from top to bottom, it is subjected to multiple rounds of ultracentrifugation allowing the exosomes to individually move toward the bottom, based upon their sedimentation rate. Moving gradient ultracentrifugation employs sucrose, deuterium oxide, or iodixanol-based gradients [95, 96]. When isopycnic density gradients (like caesium chloride) are employed, separation occurs because of differences in sedimentation rates/densities between exosomes and other impurities [97]. Exosomes concentrate at the density region of 1.10 and 1.21 g/cm³, and pure exosome pellets are obtained by ultracentrifugation at 110,00 x g to 120,000 x g [98, 99, 100]. As inferred in Table 1, this may not be ideal, especially since the density separations of microvesicles as well as apoptotic bodies are remarkably close to exosomes.

Type of EV	Mechanism of Biogenesis	Size	TYPE
Exomeres	Currently unknown	<50 nm	SMALL EVs
Exosomes	Inward budding of plasma membrane (PM) - endocytosis	30–150 nm
Ectosomes or Microvesicles	Direct shedding into extracellular matrix (ECM) via outward budding of PM	100 nm – 1 μm	LARGE EVs
Migrasomes	Generated during cell migration, secreted into ECM	500–3000 nm
Apoptotic Bodies	Outward blebbing of apoptotic cell membrane	800 nm– 5 μm
Large Oncosomes	Released from membrane blebs of amoeboid tumor cells	1–10 μm

Table 1.

EV classification.

Exosomes are the only EV class that originate through multivesicular body formation via endocytosis. Endosome-derived EVs are generally referred to as ‘exosomes’ throughout this chapter and may be interchangeably used with the term ‘EVs’.

Isolation Method	Basis of separation	Potential advantages	Potential limitations
Differential Ultracentrifugation	Density and size	High purity; Affordability over time; Ease of use; Minimal technical expertise; Reduced contamination risk; Large yields	Low recovery rate; Poor reproducibility; Time consuming (>4 hrs); Labour-intensive; Expensive set-up; Aggregation of exosomes; Presence of impurities (other EVs); Unsuitable for clinical use
Density Gradient Centrifugation	Size, mass and density	Standard protocol; High purity; High capacity	Narrow loading zone; Low Yield; Contamination due to size overlap; Time consuming (>4 hrs)
Size Exclusion Chromatography (SEC)	Size	High yield; Intact proteomic identity; Preserves biophysical properties; Improved distribution of exosomes injected in vivo; Lower aggregation; Intact vesicle structure; Good reproducibility; Enables quantitative detection	Long run time; Requires specialized equipment; Does not separate protein aggregates; Not applicable for high throughput; No scale-up
Ultrafiltration	Size; (For Example, TFF)	High purity; Applications in protein characterization studies; Rapid; No specific equipment; High exosomal RNA yield [88]; Easy scale-up	Low recovery; Trapping of exosomes on membrane filter; Shear-induced exosomal damage; Reduced lifetime of membrane due to clogging; No scale-up
Asymmetric Flow Field-Flow Fractionation	Size	Rapid isolation; High purity; No centrifugal force; No shear stress	Equipment cost
Immunoaffinity; Capture-based techniques	Molecular recognition	High affinity; High specificity of isolation; High yield (10–15 times) [89]. Easy to use and rapid; No specialized equipment; Cost effective at large scale	Low stability of antibodies; High reagent cost; Cumbersome; Low yield; Not applicable to all cell types; Potential for false readouts
Polymer based Precipitation	Polymer-based precipitation	High recovery; High yield; Few steps and rapid; Ease of use; particles within size range clinical application; Easy scale-up	Low purity of exosomes; No method for removing the polymer; Impaired downstream analysis
Microfluidic separation	Density, size, affinity	High yield; Portable technology; Cost effective; Rapid; High purity of exosomes	Low sample capacity,; Complex set-up [90, 91, 92]

Table 2.

The merits and demerits of stem cell exosome isolation methods.

Table lists and summarizes advantages and limitations of techniques regularly employed for isolation and enrichment of exosomes.

8.3 Size exclusion chromatography (SEC)

SepharoseCL-2B, Sepharose CL-4B, Sephacryl S-100 columns, or SEC matrices exploit the property of smaller vesicles having longer diffusion paths in the paths between porous gels, leading to their retention in the columns. Since exosomes have large hydrodynamic radii, these are excluded from entering the pores, resulting in longer retention times within the column [101, 102, 103]. Widely used in many areas of biology, size exclusion chromatography (SEC) can be used to isolate EVs based on the molecular size, for example, QEV (izon), EC SEC columns (Stem cell). This method is used in collaboration with filtration or multiple columns and is reported to alter the characteristics of the EVs to a lesser extent (than other methods like Precipitation) [104].

8.4 Ultrafiltration

One of the most popular size-based separation methods is where particles in suspension are separated on basis of their size/molecular weight. Exosomes are isolated using membrane filters of specific molecular weight, using molecular weight cut-offs (MWCO). A great example is TFF (Tangential Flow Filtration), a method that filters EV sizes 100 kDa and above, using tangential flow of the fluid across the filter surface, avoiding filter cakes and clogging of the pores. This is a preferred method compared to UC. Most commercial large-scale EV purification use either 100 kDa or 300 kDa molecular weight cut-off filters. Because TFF has a history of use in biopharma, it can be easily translated into large-scale downstream processing. Companies such as Pall and Sartorius have developed TFF systems that can be modified to work with their 3D bioreactor systems making EV scale-up easier.

8.5 Asymmetric-flow field-flow fractionation (AF4)

AF4 employs a porous rectangular channel, which is subjected to parabolic flow around its axis, and sample retention and diffusivity are controlled by a cross-flow [93].

8.6 Immunoaffinity-capture

Precise isolation, based on antigen molecules highly concentrated on exosomal surface, targeted by specific fluorescently labeled antibodies immobilized on a polystyrene substrate is purified either using a microplate (ELISA) or using submicron-size magnetic beads [89]. Coupling with mass spectrometry significantly enhances the capacity and is referred to as “Mass Spectrometric immunoassay.”

8.7 Polymer-based precipitation

Hydrophilic polymers like polyethylene glycol (PEG) are employed to tie up water molecules and thereby force less soluble exosomes from stem cell-secreted culture superfluates. The resulting exosome-precipitate is separated by low-speed centrifugation or ultrafiltration [105, 106].

To meet the demands of the ever-growing field of exosome therapeutics, there is a desperate need for a robust, highly reproducible and high-throughput isolation method that is still under development.

9. Exosome (EXO) characterization

9.1 Characterization based on physicochemical properties

Depending on their physiological origin, exosomes differ in the composition (quantity and quality) of their bioactive cargo capable of modulating and reprogramming recipient cells. While this property has conferred therapeutic prowess to exosomes, it also proves an impediment to the accurate assessment of their potency and efficacy. Consequently, a crucial question for clinical development of exosome therapeutics is to unravel the precise molecular composition and specific characteristics of exosomes. Unlike isolation, there is no available gold standard technique for either quantification or potency assessment of exosomes, owing mainly to inconsistencies in isolation methods and batch-to-batch variations. There are multiple guidelines available from ISEV (International Society for Extracellular Vesicles) to accurately characterize exosomes.

Table 3 represents a comprehensive list of the different physicochemical properties such as size, shape, surface charge, density, and porosity assessed by various Exosome Characterization methods.

Physicochemical Property	Exosome Parameter	Unit	Techniques Used
Size and Concentration	Particle Number Particle Size Particle/protein ratio	Particles/mL nm particles/mg	Nanoparticle Tracking Analysis (NTA), Tunable Resistive Pulse Sensing (TRPS), Quantitative Electron Microscopy (qEM) NTA, Dynamic Light Scattering (DLS), TRPS TRPS, calculated
Source Cells	1) Particle number	Cell number	Cell Counters, Flow Cytometer
Morphology	Structure and Size, Vesicular Diameter		Atomic Force Microscopy, Cryo-Transmission Electron Microscopy (TEM), TEM (side effects – dehydration of Exos)
Composition	Protein Content Lipid Content RNA Content Cytokine Content	mg/mL %, Absolute levels/protein content Yield (pg/uL) Size Distribution Relative measurement based on qualitative differences	micro-BCA/Bradford Assay, ELISA, MS LC-ESI-MS/MS RNA Sequencing, qPCR, Bioanalyzer Cytokine Array, Luminex/ProCartaPlex Assay
Identity	Transmembrane Proteins (Tetraspanins)	CD9/63/81, CD44, CD29, CD37, CD53, CD82	ELISA(concentration of marker), EXO Flow Cytometry using bead capture, NANO FLOW CYTOMETRY
	Transmembrane Integrins	MFG-E8, α3, α4
	Antigen Presentation	MHC Class I, MHC Class II
	Source Cell Markers	MSC: CD105, CD73, CD90, CD44 (Positive Surface Markers); CD34, CD45, HLA-DR (Negative Surface Markers)
	Membrane Trafficking	Annexins: I, II, IV, V, VI, VII, XI; Rab 2, Rab 5c, Rab 10, Rab 7; Clathrin
	ESCRT Proteins	Alix, Tsg 101
	Heat Shock Proteins	Hsc70, HSP70, HSP90
	Cytoskeletal Proteins	Tubulins: α1, α2, α6, β5, β3; Actin, Cofilin 1, Moesin
	Enzymes	GAPDH, Pyruvate Kinase
Identity	Signal transduction	Syntenin-1	ELISA(concentration of marker), Flow Cytometry (% expression)
	Lipid Rafts (Cytosolic recovered in Exos)	Flotillin-1, Flotillin-2
	Lipoproteins	ApoA1
	Miscellaneous	Lactadherin, Lamp2
	Secretory Pathway	Negative Markers: Calnexin, GRP94
Quality	Intact membrane	Calcein Staining, Cell Trace Violet Staining	Flow Cytometry, Fluorescent Microscopy

Table 3.

Exosome characterization methods.

Table summarizes different physicochemical properties of stem cell exosomes that can be characterized by different methods as listed.

9.1.1 Nano flow cytometry (nFCM)

nFCM is a high-resolution flow cytometric approach that allows generic quantitative and qualitative analysis of individual EVs as well as sorting of EV subsets (including exosomes), based on antibody and/or fluorescence staining. Nano Flow requires elaborate staining protocols that efficiently eliminate confounding variables such as high background noise caused by buffers, unbound fluorophore-conjugated antibodies, unincorporated dyes, protein aggregates, and other submicrometer-sized particles that can interfere with the EV measurements [107, 108, 109].

This is perhaps the only technique that enables analysis of exosome particles in low abundance (for instance, disease-related exosomes purified from clinical samples).

nFCM is the future of quantitative and standardized measurement of therapeutically significant nanoparticles, especially EVs. In our study, we optimized conditions for antibody-labeled, precipitation-enriched exosomes to be analyzed by nFCM based on exosome-specific markers.

9.1.2 Cytokine Array

We used a cytokine array kit to determine the expression of 36 different cytokine-related proteins in our EV/EXO preparations, using the Human Cytokine Array Kit (Proteome Profiler; R&D Systems) according to the manufacturer’s instructions. This is essentially a membrane-based sandwich immunoassay, where a biotinylated antibody-stained exosome sample is incubated with an array membrane that is spotted with capture antibodies to the Exosomal target proteins and visualized using chemiluminescence. This process is semi-quantitative in that the signal produced is proportional to the amount of bound analyte. In our study, we discovered at least 106 different cytokines and growth factors bound to the Exosome membrane.

Figure 4 enlists the methods used to characterize Exosomes based on their physical properties, concentration, cargo, and function.

Figure 4.

Stem cell exosome quality control: Methods for multi-parameter characterization. Exosomes can be characterized based on multiple parameters that include qualitative and quantitative analyses such as their morphology, physical characteristics, size, concentration, cargo content, intact-ness, origin as well as their function.

9.2 Characterization based on exosome potency

Exo potency is assessed by a matrix of functional assays that are performed under a tightly regulated validation process. Some of the in vitro potency assays that exploit the immunomodulatory properties of exosomes including immune cell signaling, wound closure, and angiogenesis are as listed below:

9.2.1 In vitro potency assays

9.2.1.1 Angiogenic evaluation of exosomes

Tube formation assay/vascular-like Network Formation Assay

To assay the angiogenic potential of MSC Exos, human endothelial cells are treated with exosome solution in variable ratios (1:10, 1:00, 1:1000) for 12 h in a 96 well plate, on Matrigel or Geltrex basement membrane matrices that enable vascular network formation. This assay works around the principle that MSC Exos significantly enhance formation of tube-like structures, thereby promoting angiogenesis in human endothelial cells. Validity of the assay can be reinstated by using non-supplemented cell growth media as a negative control and complete media as positive control. Imaging of capillary network is usually acquired with regular light microscopy; total tube length is measured using the Angiogenesis Analyzer plugin of ImageJ software and plotted for different Exo concentrations [110]. MSC Exos are internalized by many cells, including human Endothelial cells, and this assay proves the potency of Exos to promote angiogenesis in vitro.

9.2.1.2 Vascular endothelial growth factor (VEGF) immunoassay

VEGF plays a crucial role in angiogenesis and immunomodulation. This assay investigates the proteolytic stability of VEGF in Exo preparations, by mixing them in 1:1 ratio with Trypsin and analyzing via a membrane-based immunoassay [111]. This assay runs on the principle that the presence of MSC Exos protect secreted factors like VEGF from protease-mediated degradation. Functional testing of the angiogenesis assay is done by blocking VEGF with an anti-VEGF antibody along with protease treatment of Exos, which will inhibit tube formation in vascular endothelial cells. This is manifested in significantly reduced vascular network formation.

9.2.1.3 Immunomodulatory assessment of exosomes

9.2.1.3.1 T cell proliferation assay

MSC Exos inhibit T cell proliferation in vitro, leading to impaired T cell function, and 300,000 CFSE-labeled PBMCs from donors are stimulated with 5ug/mL PHA (Phytohemagglutinin) in a CD3-coated flat bottom 96 well-plate to induce mitogenesis at 96 h (Day4), after which Exos are introduced in varying doses. Flow cytometry determines the percentage of proliferating T cells by measuring percentage of viable CD3 positive cells, which also depict lower CFSE staining compared to non-PHA stimulated cells. No Exos Negative control is used to express maximum T cell proliferation. Maximum proliferation is carried out using Flow Cytometry. Production of CD3-stimulated T cells significantly decreases when treated with exosomes in vitro. Exos significantly inhibit T cell proliferation in a dose-dependent manner.

9.2.1.3.2 IL-10 release assay

Another in vitro potency assay for MSC exosomes is based on the release of IL-10 from PBMCs following incubation with exosomes. Post-incubation with Exos at 37C for 16-18 h, PBMCs are stimulated with LPS (Lipopolysaccharide) for 5 h. The Supernatant is assayed for IL-10 using ELISA and raw absorbance values are converted to concentration. Higher IL-10 secretion indicates high exosome potency of exosomes [112].

9.2.1.3.3 Macrophage polarization assay

This assay exploits the phenomenon that macrophages maintain a proinflammatory (classically activated M1) phenotype during active infections, and then switch back to a normal, anti-inflammatory M2 phenotype. Here, macrophages are incubated for 3 hours in the presence of PKH7-stained Exos and the principle of this assay is that the majority of the macrophages (>70%) should efficiently internalize Exos in their cytoplasm. This will significantly increase cell proliferation of the Exo-recipient macrophages, which is measured by Flow Cytometry using BrDU incorporation, as compared to Exo-untreated cells. This phenotype modulation of Macrophages from M1 to M2 can be quantified by assessing relative intensities of pro-inflammatory markers (like Ly6C, CD11b, CD40, and CD86) which are downregulated, while anti-inflammatory markers such as CD36, CD51, CD206 are upregulated [113].

Another form of potency assessment is to check the capacity of MSC Exos to suppress mRNA induction of Tumor Necrosis Factor Alpha (TNF-α) in M1 macrophages generated by LPS and IFN-γ (Interferon gamma) stimulation, in the presence or absence of MSC Exos for 24 h. After 24 hours, TNF-α mRNA levels are quantified by RT-qPCR. The functional end point of this assay is half minimal effective concentration or 50% decrease in levels of TNF-α, relative to control (EC₅₀) during the 24 hours [114].

Exosomes play a role in macrophage phenotype modulation by triggering their proliferation and polarization to decrease inflammation.

9.2.1.3.4 Exosome uptake by peripheral blood Mononucleocytes (PBMCs)

Exosome uptake by cells gives us an inkling about their ability to alter signaling in the recipient immune cells and subsequently their potency toward immunomodulation. In this simplistic assay, PBMCs are incubated for different time intervals (maximum being 48 hours) with pre-stained Exos in a ratio of Exo: PBMC = 5:1. The percentage and intensity of Exo uptake is quantified using either Flow Cytometry or fixing and visualizing with laser scanning confocal microscopy.

9.2.1.4 Wound healing scratch assay

This assay works on the simple principle that Exosomes activate human fibroblast cells to proliferate and migrate to the site of injury. Varying concentrations of Exosomes are added to wells containing Fibroblast cells, with manual scratch where cells lift off. After 24 hours, gap closure is measured using Image J Analysis software and visualization of gap closure under light microscope.

9.2.1.5 Multidrug resistance protein 1 (MRP1) assay

This assay investigates the ability of MSC Exos to downregulate the expression of MRP1 in a dose-dependent manner.

All in vitro assays should be performed in triplicates, with at least two independent experiments. It would be interesting to compare potency of freshly isolated stem cells Exos with frozen Exos and lyophilized Exos stored at different time intervals (3 months, 6 months) to obtain important insights into their stability over an extended period. It would also be interesting to see the differences in potency of Exos produced under hypoxic conditions as compared to normoxia-produced Exos.

9.2.2 In vivo potency assays

Biological in vivo potency assays need to be disease-specific, fit-for-purpose and should employ relevant functional end points. These cover multiple aspects of applicability, from administration route to dosing, and provide information about therapeutic effects and toxicity. Moreover, only an in vivo system will be able to precisely map exosome distribution and localization post-administration, circulation time, half-life, and target organs.

Most in vivo assays are extensions of their in vitro counterparts, including in vivo angiogenic assay (myocardial infarction model), in vivo macrophage modulation (M1-M2) in skeletal muscle injury model, in vivo wound healing assays.

10. Exosome preservation and storage

Stem cell-derived exosomes are being developed for variety of applications from primary diagnostics and drug delivery (engineered exosomes). Traditional storage method, cryopreservation has been found to cause decrease in exosome concentration and quality over extended period and is cost intensive. Herein, we discuss current developments in preservation of exosomes through techniques such as cryopreservation, freeze drying, and spray drying [115, 116, 117].

Cryopreservation involves freezing exosomes at temperatures much below the optimum required temperature for enzymatic and biochemical activity of constituent biomolecules. Common challenge is formation of ice crystals, which affects the functional and morphological properties of exosomes [118]. To overcome this, various cryoprotectants (CPAs) are used which controls the kinetics of ice formation and concentration of solute pockets, prevent aggregation of particles, and maintain osmotic balance internally and externally [119, 120]. Penetrating CPAs are permeable to the exosome membrane due to their low molecular weight (e.g., dimethyl sulfoxide, ethylene glycol, and glycerol), while non-penetrating CPAs form a glassy matrix or coating externally due to their high molecular weight (e.g., trehalose, sucrose, mannitol, and other sugars) [119, 121, 122]. Studies have shown that cocktails of these cryoprotectants aide in extending the shelf life of the exosomes at low temperatures [116]. Chung et al. [123] in their patent (US2020/0230174A1) used carboxylated Poly-lysine as cryoprotectant to avoid using DMSO. Critical parameter that needs to be optimized is the concentration of CPAs as higher concentration can have toxicity effects, while lower concentrations can cause cryoinjury [124].

Freeze-drying or lyophilization involves freezing exosome solutions followed by sublimation of ice to vapor phase under vacuum forming a dry powder/precipitate thereby maintaining biological properties [125]. Challenges with this method involve uncontrolled ice formation and stresses caused due to drying that affects the exosome stability and membrane integrity. Lyoprotectants such as trehalose, sucrose, buffers, or cocktails can be used to extend the product life [126, 127]. Driscoll et al. [128] compared sucrose, trehalose, and mannitol as lyoprotectants and found trehalose to be most economical and effective lyoprotectant in conjugation with manifold-based lyophilization. Kim et al. and Lim et al. in their patents present a technique to lyophilize exosomes in the presence of one or more lyoprotectants [129, 130]. Since lyophilized products involve storage temperatures between 4°C and room temperature as compared to cryopreservation at freezing conditions, this technique can increase dry state stability, reduce cold chain transportation, and can be used by dissolution in water.

Spray drying is a continuous drying process where wet solution is first atomized, followed by contacting with dry warm air, which leads to instant vaporization of moisture producing dry powder [131]. This technique reduces the risks associated with freezing and dehydration stresses. As this is a continuous operation, spray drying can be economical as well as scalable for large-scale manufacturing. Some critical parameters that affect the shelf life and quality of dry powder are initial feed concentration rate, atomization pressure, and outlet temperature. Behfar et al. [132] have patented a system of exosomes encapsulated in alginate by spray drying. A cyclone separator/electrostatic precipitator can be used for better retention of dry powder. However, experiments are required to be carried out to evaluate if the final product meets required critical quality attributes.

Other than preservation techniques, storage conditions such as pH, temperature, and number of freeze thaw cycles also affect the relative particle concentration, protein content, particle diameter, shelf life, and cellular uptake of exosomes [133, 134].

In summary, trehalose, a FDA approved non-reducing disaccharide sugar, presents as the most suitable option for cryopreservation and lyophilization by stabilizing the lipid membrane preventing exosome aggregation [135].

11. Exosome engineering

11.1 Targeted delivery of pay loads

Native exosomes present therapeutic properties and cargo similar to parent cell type thereby limiting the cargo quality and delivery of exosomes to targeted tissues or cells. Engineered exosomes with targeted delivery overcome these limitations by enhancing exosome efficacy as well as avoiding possible adverse reactions. Some of the methods to modify or load cargo onto native exosomes are as follows: (a) surface modification or passive cargo loading where the exosomes are modified chemically or physically directly to deliver desired cargo and (b) genetic engineering or active cargo loading where the parent cell type is genetically modified to secrete exosomes with specific cargo [136, 137, 138, 139, 140]. Some drawbacks to these modifications are compromised exosomal structure and possible induction of immunogenic effects as compared to native exosomes, Challenges linked to engineered exosomes are scalability, production cost, trained professionals, and downstream processing. Hence, there is a need to conduct further studies on these shortcomings at the same time maintaining exosome therapeutic value in conjugation with delivery systems.

11.2 Hydrogel and exosome engineering for tissue regeneration and enhanced secretion

Hydrogels are 3D network of physically or chemically crosslinked polymeric materials with a high affinity for water. Moreover, hydrogels have tissue-mimicking properties such as porous hydrophilic structure, biocompatibility, tunable stiffness, response to external stimuli (pH, temperature, enzymes, cells, etc.), and controlled degradability; thus, hydrogels present a suitable choice for targeted drug delivery system and tissue engineering.

Due to their porous structure and biocompatibility, exosomes are readily encapsulated in hydrogel matrix, offering a sustained and controlled bulk exosome delivery system for therapeutics. For instance, ADSC exosomes encapsulated in GelMA promote tendon regeneration [141], UMSC exosomes laden low-stiffness HA-MA hydrogels stimulate nervous tissue regeneration [142], UMSC-exosomes encapsulated in a hydrogel wound dressing encourage diabetic wound healing [143], and MSC-Exosomes in a sprayable fibrin heart patch regulate myocardial infarction [144]. Hydrogels can be printed with exosomes providing off-the-shelf solution to customized tissue constructs [145, 146, 147].

As hydrogels represent a native ECM such as environment and control over their functional chemistries, they can affect exosome secretion activity of hMSCs. Some examples are hydrogel composition and stiffness enhancing MSC secretome and exosome secretion profile [148, 149, 150]. Chen et al. [151] have developed a 3D bioprinting method, which augmented exosomes secretion compared to plastic cell culture.

Thus, hydrogels present an exciting drug delivery system and improve exosome secretion profile for native and engineered exosomes.

12. Exosomes: clinical and regulatory guidance

Thirty-seven clinical trials in Phase 1–4 are currently listed at clinicaltrilas.gov using the term extracellular vesicle (EV) or exosomes. The most abundant are in lung and respiratory diseases followed by graft-versus-host disease (www.clincaltrilas.gov). The rise in respiratory and lung diseases is a direct result of the current pandemic (Coronavirus-19), which has a fast track to authorization due to its disease class. Most jurisdictions look to the Food and Drug Administration (FDA) in the United States for guidance. The current guidance for industry is the same as the current guideline for mesenchymal stem cells, which requires a demonstration of safety and efficacy across multiple clinical trials and shows product purity and potency. Consumer alerts have been issued by authorities due to unregulated stem cells and exosomes. The International Society for Extracellular Vesicles (ISEV) and the European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD) have formulated certain guidelines to foster their clinical use [152]. However, three is no harmonized regulatory framework around exosomes at the international level yet but some isolated approvals provided on case-to-case basis. The current status of ongoing trails in MSC derived exosomes can be seen in Table 4 [87, 153].

Title	Condition	Locations
Effect of UMSCs Derived Exosomes on Dry Eye in Patients With cGVHD	Dry Eye	Zhongshan Ophthalmic Center,; Guangzhou, Guangdong, China
Effect of Microvesicles and Exosomes Therapy on β-cell Mass in Type I Diabetes Mellitus (T1DM)	Diabetes Mellitus Type 1	Sahel Teaching Hospital, Sahel, Cairo, Egypt
Evaluation of Adipose Derived Stem Cells Exo.in Treatment of Periodontitis	Periodontitis	Beni-Suef University, Banī Suwayf, Egypt
Exosome of Mesenchymal Stem Cells for Multiple Organ Dysfunction Syndrome After Surgical repair of Acute Type A aortic Dissection	Multiple Organ Failure	Fujian Medical University, Fujian, China
MSC-Exos Promote Healing of MHs	Macular Holes	Tianjin Medical University Hospital, Tianjin, Chin
MSC EVs in Dystrophic Epidermolysis Bullosa	Dystrophic Epidermolysis Bullosa	Aegle therapeutics, Miami, Florida, USA
The Use of Exosomes In Craniofacial Neuralgia	Neuralgia	Neurological Associates of West LA,; Santa Monica, California, USA
Focused Ultrasound and Exosomes to Treat Depression, Anxiety, and Dementias	Neurodegenerative Diseases	Neurodegenerative Diseases
The Safety and the Efficacy Evaluation of Allogenic Adipose MSC-Exos in Patients With Alzheimer’s Disease	Alzheimer Disease	Ruijin Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China
Allogenic Mesenchymal Stem Cell Derived Exosome in Patients With Acute Ischemic Stroke	Cerebrovascular Disorders	Saeed Oraei Yazdani, Tehran, Iran
iExosomes in Treating Participants With Metastatic Pancreas Cancer With KrasG12D Mutation	Stage IV Pancreatic Cancer, Pancreatic Ductal Adenocarcinoma, Metastatic Pancreatic Adenocarcinoma	M D Anderson Cancer Center,; Houston, Texas, United States
A Pilot Clinical Study on Inhalation of Mesenchymal Stem Cells Exosomes Treating Severe Novel Coronavirus Pneumonia	COVID-19	Ruijin Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China
A Tolerance Clinical Study on Aerosol Inhalation of Mesenchymal Stem Cells Exosomes In Healthy Volunteers	Healthy	Ruijin Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China
Evaluation of Safety and Efficiency of Method of Exosome Inhalation in SARS-CoV-2 Associated Pneumonia.	COVID-19	Medical Centre Dynasty, Samara, Russian Federation
Organicell Flow for Patients With COVID-19	COVID-19	Landmark Hospital, Naples, Florida, United States
Safety and Efficiency of Method of Exosome Inhalation in COVID-19 Associated Pneumonia (COVID-19EXO2)	COVID-19	Medical Centre Dynasty, Samara, Russian Federation

Table 4.

Current clinical trials using MSC exosomes.

13. Exosomes: scale-up production

13.1 Upstream processing

In order to meet demand of aforementioned EVs in clinical trials and success thereof, upscale technologies must be employed [154, 155]. One of the major challenges to cellular and non-cellular biologics is upstream scaling of manufacture to bioreactor culturing. Generally, manufacture relies on the development from T flasks to multi-layer stacks; however, this method of culturing is restrictive in terms of surface-to-volume ratio. From 2D culture, the system can be scaled into roller bottles or spinner flasks. From there, lab-scale technologies can be employed which can then be transferred or scaled into high-capacity bioreactor systems. Manufacturing current Good Manufacturing Practice (cGMP) EVs to a commercial scale which are not only (i) reproduceable but (ii) cost effective remains a somewhat arduous task. Upscaling manufacture is a necessary step on route to commercialization; however, large investment is required to ensure a smooth translation from bench to bedside and gain successful regulatory acceptance. With upscaling comes less batch testing, less lot release criteria, less labor, less facility time, less consumables and reagents costs, and perhaps most importantly less impact of variation. However, there are also higher risk considerations including higher costs of failure, larger equipment costs and depreciation, more upfront research and product development, and undesired product changes [156]. Although much can be learnt from stainless steel bioreactors that are currently in use in other fields (Monoclonal antibody and vaccine production) at a scale of 20,000 Liters (L), cell and EV therapy scale-up solutions need to generate high volumes in single-use sterile bioreactor systems (SUBs). SUBs allow for less qualification and validation due to pre-sterilization and minimal contact. Capacity of SUBs currently stands at 6000 L (Wuxi Biologics, China); however, downstream systems for purification and sensors are not necessarily compatible with the SUBs and product requirements.

13.1.1 Single-use technology

With an ever-changing landscape in bioreactor technology, it is important the technology of choice has longevity. A change of method nearing Phase 3 or commercialization could be devastating. System suitability relies on many factors including cell type, downstream processing, and carrier suitability. For EVs to reflect the expression pattern from the parent cells, it is a challenge with most historical characterization being completed on cells from 2D culture systems and most cell types being extremely sensitive to hydrodynamic conditions [157]. Hydrodynamic conditions within a bioreactor will significantly impact the biological performance of the cells and/or secreted molecules like EVs. This makes the choice of SUB of upmost importance [158]. Bioreactors enable the user to control gas, temperature, pH, and feed addition; however, these factors are reliant on which type of reactor is employed [159]. The main types of reactors used for EV manufacturing include those in which employ microcarriers or macrocarriers that are utilized in the following: 1. continuous stirred tank bioreactors, 2. hollow fiber reactors, 3. packed bed bioreactors, and 4. wave reactors [155, 160, 161]. By far the most widely used bioreactor type is the stirred tank reactor due to its high flexibility and low-operating costs, Tables 1 and 2 outline a range of SUBs currently available and some of their limitations and advantages for EV production The Xcellerex (GE Healthcare), Allegro (Pall), and BIOSTAT (Sartorius) offer manufacturing platforms of SUBs with designs that closely match traditional reactors (Table 5) [162].

Type	Commercial examples	Advantages	Limitations
Wave (Rocking bed)	GE healthcare, Finesse (ThermoFisher),	Versatile	Not easily scaled, low cell recovery (beaching)
Stirred tank	Xcellerex (GE healthcare/Cytiva), Finesse (ThermoFisher), Mobius 2000 (Millipore), BIOSTAT (Sartorius), BioBLU (Eppendorf)	Functional at large volumes>50 L, most abundant	Stirred tank reactors have high shear force which can affect cell characteristics
Perfusion/hollow fiber bioreactor	Fibercell (FiberCell systems), Quantum ell expansion	Minimal shear stress, isolation of EVs and cells easy	Very low throughput
Fixed bed	iCellis (Pall)	Scalable from lab, small footprint with large surface area	Difficult to remove cells, easy for EVs

Table 5.

Examples of SUBs commercially available.

13.1.2 Substrate technology

Microcarriers and macrocarriers are generally used in SUB systems as the 3D surface the cells can attach and grow. Microcarriers come in many forms and can be characterized based on matrix, coating or size. This includes glass, diethylaminoethyl (DEAE)-dextran, acrylamide, polystyrene, collagen, and alginate [163]. To increase cell attachment, they are either coated (collagen) or non-coated (charged). Regulatory bodies require the culture system to be animal origin free for human use to avoid xenogeneic reactions that limits the choice. In the case of xeno-free microcarriers, some examples include Hillex and Star Plus (Sartorius, USA) as well as other dissolvable carriers like Synthemax (Corning, USA). One advantage of not being required to harvest the adherent cells as with EV production is the challenge of enzymatic detachment. Using microcarriers in stirred tank or wave reactors can cause issues including aggregation, engulfment, and beaching where the microcarriers and cells are stuck together or in the case of the wave bag reactor are caught in the corners of the bag. For EV manufacture using adherent cells, it is therefore preferred to use macrocarriers, generally made as disks (fibra-cel, Eppendorf) or strips from polytetrafluoroethylene (PTFE).

13.2 Downstream processing

Selective purification is necessary to isolate exosomes from other EV subsets as well as from the heterogeneous “soup” that is the stem cell secretome. This secretome contains a myriad of analytes, including cytokines, chemokines, enzymes, growth factors, Extracellular Matrix (ECM) proteins, and factors involved in ECM remodeling, different types of Extracellular Vesicles including Exosomes, microvesicles, apoptotic bodies, and others. The downstream processing of a mixed biological like the secretome requires careful precision. From regulatory standpoint purity, potency, safety, and efficacy of derived exosomes are of upmost importance for clinical relevance [164]. A pure product without contaminants from culture media and cells is critically important. Methods for EV isolation are broad and rely on alternative characteristics of the EVs for purification; however, challenges remain on which on scale vs. purity. Many methods are starting to become available to researchers and manufacturing organizations, for example, immunocapture of CD81-, CD63-, and CD9-positive molecules and microfluidics and the methods described below include Ultracentrifugation (UC), Precipitation, Size exclusion chromatography (SEC), and Tangential Filtration Flow (TFF) are most commonly used in larger-scale downstream processing. Currently, there are no well-defined methods for exosome isolation in high-efficiency and high-throughput, and the recommendation is a combination of the below methods.

14. Perspectives

Secretion of extracellular vesicle (EVs) is a universal phenomenon and hence must have biological implications. They have come a long way since first discovered in 70’s by Peter Wolf [4]. Fast forward to today, there are extensive publications and clinical trial data to promote their role in diagnostics and therapeutics, including in drug delivery. However, the jury is still out there on EVs to fulfill these roles and gain prime time.

Among the two broader categories of EVs: ectosomes and exosomes, the latter are inward budding and hence endosomal origin with a size range of ∼40 to 160 nm in diameter. Endosomes undergo a process of systematic invagination of the plasma membrane with the formation of multivesicular bodies that intersect with other intracellular vesicles (phagosomes) and organelles, contributing to diversity of exosome composition before they are released back to intercellular space. These subset of EVs as exosomes have attracted attention for their critical role in cell-to-cell communication and hence therapeutic values. In particular, the mesenchymal stem cells (MSCs)-derived exosomes seem to have strong anti-inflammatory and immunomodulatory roles and hence have been studied extensively.

The role of exosomes in intracellular and intercellular communication is quite apparent from many critical studies thus far. The developmental pathways for exosomes are highly regulated that are being unraveled at the molecular level; however, how they impact cells is still elusive. Nevertheless, recent studies including pre-clinical and clinical appraisals have demonstrated their role in mitigating symptoms of various diseases as delineated in this chapter. Given the cellular therapy still facing huge challenges regarding cell differentiation, maturation, and integration, opportunity with exosomes to develop non-cellular active pharmaceutical ingredients (API)-based allogeneic therapy may be more appeasing to the regulatory authorities.

This chapter has endeavored to address questions systematically by first reviewing the developments in the field and then putting perspectives on feasibility of their roles and the technical and regulatory hurdles involved. The scientific rigor behind exosomal research puts a demand on regulatory bodies to develop appropriate framework for promoting their developmental pathways toward human medicine.

15. Challenges

The current drug developmental paradigm critically requires defining the active ingredient in the product to its finest detail to eliminate heterogeneity and batch-to-batch variations before defining its pharmacology and pharmacokinetic profiling for final approval after clinical trials. This is followed up by rigor of their scale-up production under GMP from established master cell bank (MCB) with quality controlled (QC) protocols, cryopreservation if any, and shelf life for potency. This is a very arduous, time-consuming, and expensive journey in drug development and takes about 5–10 years with a projected cost of 3–4 billion USD.

15.1 Regulatory challenges

Purification of exosomes by various methods described in this chapter gives rise to a heterogeneous EV population consisting of 40–160 nm diameter vesicles, putatively classified into many subcategories based on size, Exo A, B, C; content, Exo 1, Exo 2, Exo 3; function, Exo α, Exo β, Exo γ; and source, Exo I, Exo II, Exo III [28]. However, as per societal guidelines, all EVs qualify as one based on their characterization. The major question is whether such heterogeneity matters, particularly when blood and blood-derived products are approved for therapeutic use. In fact, the pleotropic effects of exosomes are beneficial in some cases that include blood transfusion. Therefore, the current classification of exosomes API (active pharmaceutical ingredient) needs due diligence and redefining by the regulatory authorities so that it is at par with blood and blood products.

There are no harmonized protocols available as yet that endow regulatory approvals on exosome production for clinical development. Approvals that have been granted are on a case basis depending upon clinical needs, but no main stream approvals yet.

15.2 Technical challenges

Characterization: The current societal guidelines for characterizing exosomes include the following:

For size and number, Nanoparticle Tracking Analysis (NTA) or Dynamic Light Scattering (DLS) are both complementary techniques that offer different insights. DLS will generally measure a wider size range than NTA, but NTA offers greater resolution than DLS. There are inherent difficulties in both these techniques with wide variations within each sample and aggregation of exosomes affecting measurements. Stabilizing exosomes with some cryoprotectants such as trehalose can offer some respite. Alternatively, quantification using the Exo-Flow-ONE staining kit may be more accurate.

Exosome-specific surface markers such as CD9, 63, 81, 107, Alix, are generally assessed by using Western blot; however, the procedure is cumbersome and not efficient for a large number of samples. Additionally, nano-flowcytometry that is more efficient is not widely used because of the cost involved and lack of harmonized published protocols. Nevertheless, some recent publications including our own endeavour showed success with this technique [162, 163, 164, 165, 166]. Alternately super-resolution microscopy topology could be employed though it is neither cost effective nor efficient.

Proteomics and micro-RNA profiling are generally outsourced for bulk analyses that are more cost effective but relevance of such comprehensive data for drug development is far from clear, though may be relevant for diagnostic purposes.

Exosome potency assays include inhibiting T cell proliferation, macrophage phagocytosis, fibroblast activity, vascular-like network formation assay on Matrigel [167] and scratch wound healing assays [168] that are very useful and can be optimized quickly in the lab. Emerging in vivo potency assays such as EV-mediated wound healing [166, 169] (assess the biological response of exosomes in a disease model [170]. Critically understanding the mechanism of how exosomes elicit a response will assist in regulatory approval.

Scale-up production involves establishing a stem cell biobank made up of MSCs extracted from biologically available sources such as umbilical cord, bone, tooth, fat; however, these MSCs have finite life and reach senescence after 6–7 passages. To circumvent this problem, immortalized cell lines were used in the past that caused regulatory and safety issues. Current approach is therefore to reprogram pluripotent stem cells like iPSC (induced pluripotent stem cell) from human blood or skin tissue and differentiate these into MSCs so that there is an unending source of materials in the biobank. These iMSC derived from iPSCs continue to proliferate beyond passage 10–12 and do not senesce easily, likewise eMSC derived from hESC is other source of long-life stock from which MSCs can be derived easily. Producing these sources of iPSC and MSCs under GMP is very expensive proposition. We have recently moved from planar culture to 3D culture using bioreactors for improved exosome production. We observed an improvement in both quality and quantity of EVs produced. Incorporating such devices within aseptic environment is leading the way for transition to clinical trials and then clinics.

While cryopreservation of cell lines is a standardized procedure, storing exosomes is still an evolving field. Notionally, these can be stored in saline at -80°C with good keeping quality for years; however, there are inherent problem with exosomes aggregation and/or lysis happening during storage. Lyophilization is a quick and efficient way to store exosomes at room temperature for clinical purposes. Cryoprotectants like trehalose help in protecting against aggregation as discussed in this chapter.

Targeted delivery of exosome payloads by genetic engineering increases efficiency and efficacy. Considerable progress has been made toward understanding the logistics for exosome delivery. The controlled release of exosomes at the site of injury is by using various biodegradable gels, and extracellular matrices are on trials as in our labs. Particularly in this regard is 3D printing of exosomes onto bandages as therapeutics is of great relevance in this field.

16. Conclusions and perspectives

In conclusion, exosomes field has emerged as a critical area of therapeutic development as a third pillar of medicine with proof of principle and good science behind it. However, bringing it to fruition requires liaising with the regulatory authorities to harmonize framework around quality control protocols, which will further facilitate clinical trials and more importantly bring focus and excitement for continued funding in this field.

Conflict of interest

The authors declare no conflict of interest.