Migrasomes, Matrix-bound Nanovesicles, and More: Messengers in the Matrix

Abstract

Extracellular vesicles (EVs) and particles (EPs) are diverse micro- and nanoparticles that circulate in bodily fluids and can attach to, or be deposited onto, the extracellular matrix (ECM) and other surfaces. To date, the nomenclature and classification of matrix-bound or matrix-associated EVs and EPs (MEVPs) has been unclear, largely due to a lack of consensus guidelines and a relatively miniscule amount of received attention in comparison to EVs found in fluids. Recently, there has been a growing appreciation for several subtypes of MEVPs and their roles in applications ranging from wound healing to metastasis. However, progress in these fields has largely been achieved in silos, with minimal consideration for overlap or complementary function between different MEVPs. In this article, we briefly describe this growing field with a focus on several MEVP subtypes and the lack of consensus, then discuss challenges and opportunities in improving MEVP isolation and characterization. Importantly, proteomic analyses of these unique MEVPs will be crucial in promoting rigor, reproducibility, and understanding in this exciting new field.

Introduction

The extracellular matrix (ECM) is the immobilized, noncellular network that plays a critical role in providing structural, homeostatic, and communication support to eukaryotic and prokaryotic cells. It is highly tissue-specific as a result of several unique constituents which alter its biochemical and biophysical properties [1]. For example, growth factors and chemokines attached to the basal lamina contribute to tissue repair and regeneration, while overly abundant collagen and hyaluronic acid increase both ECM stiffness in the tumor microenvironment and proliferation of cancer cells [1,2].

Notably, several types of extracellular vesicles (EVs) and particles (EPs) have been found to be deposited onto or attached to the ECM, and may likewise fall into this group of unique ECM constituents. Their important roles in repair and disease have already been demonstrated, and further exploration is crucial as this may have large implications in the development of new therapeutics and diagnostics [3–7].

However, unlike for EVs and EPs found in fluids, referred to as fluid EVs, there is limited consensus and defined guidelines for those bound to the ECM [8]. While several studies have detailed various types of ECM-bound EVs and EPs, only a few specialized comparisons have been made. For example, the interaction between the ECM and EVs has been examined both broadly and in relation to bones and joints [7,9]. These reviews highlight specific subsets of ECM-associated EVs, and we aim to build on them by incorporating other ECM-associated EVs and EPs to further clarify the field.

To do so, we propose the term matrix-bound or matrix-associated EVs and EPs (MEVPs). We define MEVP to describe EVs and EPs that are typically formed and found in or on surfaces, such as the ECM, tissues, culture surfaces, or synthetic biomaterials. A key difference between MEVPs and fluid EVs is their isolation method. Typically, fluid EVs are isolated from cell culture medium, blood, and urine using centrifugation, ultrafiltration, size exclusion chromatography, and immunoaffinity precipitation [8,10]. MEVPs, however, typically require an initial step of detachment from the surface via enzymatic digestion, which is followed by a combination of centrifugation, density gradient centrifugation, and filtration. It is important to note that the goal of this paper is not to suggest MEVP as a permanent term, but rather encourage the conversation to build a more cohesive foundation for this growing field.

In this paper, we describe the biogenesis pathway, biological relevance, isolation technique, and protein markers for several MEVP subtypes, including matrix vesicles, matrix-bound nanovesicles, migrasomes, retractosomes, filopodia-derived vesicles, blebbisomes, protrusion-derived vesicles, matrimeres, and non-animal EVs. We identify potential overlaps between MEVP subtypes and address prevalent challenges in their study, as well as stress the unique importance of proteomics in advancing the MEVP field [4,11–19].

Section 1: MEVPs

Section 1.1: Matrix vesicles

Matrix vesicles (MVs) are considered to be the earliest observed MEVP as they were first imaged in 1967 and later classified in 1969 [20,21]. MVs are small EVs that range in diameter from 20–200 nm. Their biogenesis mechanism entails budding from microvilli-like protrusions of mineral forming cells, such as chondrocytes and osteoblasts. After budding, MVs are released into the ECM where they serve as mineralization nucleation sites and promote hydroxyapatite crystal formation in calcified cartilage, bone, and dentin [12,22–25].

The MV isolation process typically includes enzymatic digestion of mineralized tissue, centrifugation, and filtration [24]. Tissue sources include in vitro models, where mineralization is induced in cell culture and the deposited matrix is collected, or in vivo models, commonly the bones of chicken embryos [24,26]. After collection, the mineralized tissue is enzymatically digested with collagenase, followed by sequential centrifugation steps (500 ×g, 2500 ×g, 10000 ×g) and filtering (0.22 μm) to remove large ECM debris. MVs are collected with a final centrifugation step (100,000 ×g) [24].

Mass spectrometry-based proteomics (MSP) analysis showed an overlap with proteins associated with the cytoskeleton, P_i and Ca²⁺ ion homeostasis (TNAP and AnxA1), and adhesion proteins (various integrins) [22]. However, it has been noted that the MV proteomic profile varies based on the sample and isolation technique [23,24].

Along with facilitating the mineral formation process by regulating P_i and Ca²⁺ ion homeostasis and modifying and interacting with the surrounding ECM, MVs have been shown to have a pro-inflammatory effect on macrophages [23,24]. Interestingly, MVs isolated from centrifuged conditioned medium of an osteocyte cell line (MLO-Y4) functionalized to scaffolds demonstrated increased osteogenic differentiation and mineralization of osteoblast precursor cells, indicating MV’s utility in bone repair applications [5].

Section 1.2: Matrix-bound nanovesicles

Matrix-bound nanovesicles (MBVs) were first identified in 2016 as small EVs that are embedded in the ECM of soft tissues, such as the heart and urinary-bladder, and range in diameter from 80–150 nm [11,24,27]. Although the effects of MBVs have been studied, their biogenesis mechanism and role in matrix formation remain poorly understood.

MBVs can be isolated from either experimentally derived or commercially available animal or human tissues, or from 2D and 3D in vitro cell culture [11,24,28]. Out of many isolation methods, the optimal method to isolate MBVs of high yield and bioactivity is collagen or liberase enzymes in combination with size exclusion chromatography. In more detail, porcine urinary bladder ECM can be disrupted with enzymes to release MBVs, followed by centrifugation and filtration steps identical to MV isolation. Finally, MBVs are collected using size exclusion chromatography [29]. Of note, to isolate MBVs the ECM is often decellularized with harsh chemicals and must be broken apart with enzymes, leading to a potential loss or alteration of EV markers and components of the MBVs. The impact of decellularization methods on MBVs has yet to be explored in detail, but enzymes such as proteinase K compared to collagenase, and ultrafiltration compared to ultracentrifugation, all lead to changes in MBV purity and protein content [29].

Although MBVs have a similar isolation method to MVs, extensive proteomics analysis has shown significant differences between the two MEVP subtypes. Specifically, western blot analysis identified TNAP as a protein unique to MVs that is not present in MBVs [24]. Additionally, MBVs are distinct from other small EVs as western blot analysis has shown no expression of common small EV markers such as CD63, CD81, CD9, Hsp70, HRS, and HSC70 [11,28]. Interestingly, when comparing the MSP analysis of MBVs isolated from 2D versus 3D cell culture of mesenchymal stromal cells (MSC), there was a 51.6% proteomic profile overlap between the two groups, the enrichment of protein targeting and localization pathways in 3D-derived MBVs, and the abundance of proteins associated with extracellular environment organization and immune-related pathways in both groups [30]. This highlights the importance of accounting for both the ECM source and isolation method when comparing proteomic profiles of MEVP subtypes.

MBVs have been shown to play a role in immunomodulation, fibroblast proliferation, angiogenesis and wound healing, which largely overlaps with many of the ECM functions [6,11,28,30,31]. Specifically, MBVs derived from decellularized and liberase-digested urinary-bladder ECM have also demonstrated promising roles in wound repair, by modulating the immune system to alleviate bone resorption in particulate-induced periprosthetic osteolysis mice models. 2 × 10¹¹ MBVs per 20 g mouse were necessary to produce this effect [32]. Additionally, MBVs isolated from this same ECM can be rapidly taken up by various cell types including fibroblasts, stem cells, and cancer cells, are not cytotoxic, and do not suppress the immune response like other drugs such as cyclophosphamide [33].

Section 1.3: Migrasomes

Migrasomes were first discovered in 2015 as large EVs that have a biogenesis mechanism associated with cell migration and that range in diameter from 0.5–3 μm. As a cell migrates, retraction fibers (RFs) are left behind and migrasomes form on the RF tips and intersections, containing up to 300 intraluminal vesicles [13]. While the migrasome is attached to RFs, it is classified as an organelle since it is the primary site for cytokine secretion, and when it detaches from RFs, it is classified as an EV since it mediates long distance intercellular communication [14]. After migrasomes detach, they are either engulfed by surrounding cells, rupture to release their intraluminal vesicles, or remain adhered to cell or ECM surfaces to leave a “breadcrumb trail” of spatial and biochemical information [13,34].

Migrasomes can either be isolated from cultured cells or from cell-free serum samples. Tissue culture cells are grown on 30–80 fibronectin (FN) coated dishes for 12 hours, followed by collection by trypsinization, whereas 50 mL of serum is used for isolating them from blood. At this point, samples from either source follow the same isolation protocol of sequential centrifugation steps (1000 x×g, 4000 x×g, 20000 ×g) to remove cells and debris, as well as to collect a crude large EV pellet. This pellet is fractionated via density gradient centrifugation and each fraction is concentrated with a final centrifugation step (20,000 ×g). Migrasomes are collected from fractions 4–6 (from the top) for tissue culture samples and from fractions 6–8 (from the top) for serum samples, assuming 9 equal volume fractions [35].

A key step in migrasome biogenesis is the concentration of TSPAN4 and integrins at points along the RF to create migrasome formation sites and anchor the migrasome to the ECM [3,36]. The migrasome acts as the tethering point to the ECM, although there is a point of contention in literature whether RFs “sway around” or are matrix-adherent [36,37]. While TSPAN4 and various integrins can be used as migrasome markers, they are somewhat nonspecific due to their presence on other small EVs [35]. MSP analysis revealed four proteins unique to migrasomes: NDST1, PIGK, CPQ, and EOGT. Briefly, the workflow to establish these makers included tandem mass tag labeling followed by quantitative MSP on two separate groups, migrasomes and small EVs, finding a 27% overlap in protein expression. Proteins that were only expressed in migrasomes were screened using western blot analysis and fluorescence live cell imaging to identify the four unique migrasome markers [35]. A separate macrophage-focused experiment conducted on mouse and human macrophage cell lines in vitro and a zebrafish model in vivo revealed pTRAP SCIMP, a signaling adaptor for immune receptors, to be a unique marker for macrophage-derived RFs and migrasomes [37].

In a physiological context, migrasomes have been found to conduct lateral transfer of mRNA between cells, to maintain mitochondrial homeostasis by storing damaged mitochondria, and to deliver cargoes to spatially defined locations [3]. Of note, the crucial role of migrasomes in organ morphogenesis has been observed in vivo. In this experiment, zebrafish grastrula expressing Tspan4a-GFP were recorded via live-cell imaging and migrasomes were shown to concentrate in a cavity underneath the embryonic shield to ensure proper positioning of dorsal forerunner cells [38]. As migrasomes have been found to be associated with various disease states, such as cancer, cardiovascular conditions, immune regulation, proliferative vitreoretinopathy, and cerebral amyloid angiopathy, there is potential for migrasomes to serve as powerful diagnostic markers and therapeutic targets [3,39]. Interestingly, nanoliposomes that prevent migrasome formation by tumor-associated osteoclasts have recently been developed to prevent early bone metastasis, further showing the therapeutic potential of migrasomes [40].

Section 1.4: Retractosomes

Retractosomes were identified in 2022 as migration-dependent small EVs that originate from cell-detached RFs. Following migrasome formation, cells continue to migrate while distal RFs undergo ‘pearling’ to form a beaded-chain of vesicles, which further break up into individual vesicles, each ranging in diameter from 50–250 nm [15,37]. TSPAN4 is crucial to retractosome biogenesis as it is initially evenly distributed along the RF and then is concentrated into small puncta prior to RF breakage. However, unlike migrasome biogenesis, this process is not cholesterol-dependent. Disagreement exists in literature on the nomenclature of the RF breakdown products. These vesicles are commonly referred to as retractosomes, however a recent study highlighted that this term may not fully describe the heterogeneity of both the RF vesicles and fragments [15,37]. For consistency, we will refer to these vesicles as ‘retractosomes’.

Retractosomes can be isolated from tissue culture cells, in a method similar to migrasome isolation. The trypsinization of cells is followed by centrifugation (1000 ×g, 4000 x×g), filtration of the supernatant (0.22 μm), and two final centrifugation steps (18000 ×xg) to collect the crude retractosome pellet [15]. MSP and western blot analysis revealed the presence of migrasome protein markers PIGK, EOGT, and PCCA in retractosomes, although EOGT and PCCA were less enriched in retractosomes than in migrasomes. Additionally, this analysis showed a high similarity of the retractosome and migrasome protein profiles, but low similarity to the small EV profile. Although retractosomes have been observed in vivo, their biological role is yet to be determined [15].

Section 1.5: Filopodia-derived vesicles

Filopodia-derived vesicles (FDVs) were first described in 2021 and are considered to be large EVs that range in diameter from 150–400 nm. They are derived from filopodia, which are thin, finger-like protrusions that extend from the leading edge of a migrating cell. FDVs are produced via the scission of filopodia, a process that is promoted by the I-BAR MIM protein [16,41]. Although this protein is expressed in FDVs, no unique markers have been identified, and immunoaffinity capture showed that CD63, a common small EV or exosomal marker, was absent [16].

FDVs are isolated through the collection of cell culture media and two rounds of centrifugation (3000 ×g, 16500 ×g) [16]. The biological relevance of FDVs has not been determined, but as filopodia are involved in processes such as cell migration, interaction with the ECM, wound healing, and even cancer progression, this may imply the potential physiological role of FDVs [16,41,42].

Interestingly, filopodial-tip vesicles (FTVs) were first described in 2023 as large EVs that specifically form from macrophage filopodia. FTVs have a diameter ranging from 500 to 1500 nm and contain numerous internal-vesicles that are enriched in chemokine IL11. Along with in vitro observation, FTVs have been observed in vivo, ingrained in tissues of diabetic nephropathy patients and diabetic mice [43].

FTVs exhibit numerous similarities to migrasomes, including their isolation process and expression of TSPAN4 and integrin α5. However, no unique marker for FTVs has been described [43]. The biological relevance of FTVs has been explored through an in vivo study where FTVs derived from both M1 and M2 macrophages were injected into diabetic mice. The M1 group exhibited an increase in collagen deposition while the M2 group showed the opposite. Therefore, FTVs may be key in regulating not only fibrogenic response, but also the communication between macrophages and fibroblasts [43]. Importantly, since FDVs and FTVs are understudied in comparison to migrasomes, further characterization and proteomic analysis is necessary to better understand their biological functions and clarify the differentiation between each other and migrasomes.

Section 1.6: Blebbisomes

Blebbisomes are large EVs that have an average diameter of 5–20 μm and exhibit constant membrane blebbing, hence their name. Blebbisomes lack a nuclear membrane but contain various cellular organelles such as functional mitochondria, endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes. Proteomic analysis reflects the blebbisome composition as a large number of mitochondrial (VDAC2 and VDAC1), cytoskeleton (myosin Iia, alpha tubulin, actinin-4 and beta actin), ribosomal (RPS8 and EEF2), endoplasmic reticulum (calreticulin), and Golgi (TGN protein 2) proteins are expressed.

Similar to previous MEVPs, blebbisomes can be isolated from cell cultures. After trypsinization of the cells, the cell solution is centrifuged (1000 ×g) and the supernatant is filtered (10 and 5 μm), followed by two final centrifugation steps (2000 ×g) to produce a blebbisome pellet [18].

Additionally, blebbisomes have the ability to secrete and internalize other EVs, produce actin protrusions, and undergo apoptosis. Blebbisome formation occurs in a single retraction event where a portion of a migrating cell remains attached to the substrate, a membrane nanotube connects to and eventually breaks off of the blebbisome, which releases the blebbisome into the surroundings where it can remain for days. Interestingly, both blebbisome and migrasome biogenesis depend on retraction events, however simultaneous formation of these two MEVPs has not been observed [18]. Blebbisomes have been observed in vitro in both cancer and healthy cells, and in vivo in the bone marrow of healthy mice. Notably, blebbisomes derived from cancer cells expressed immune evasion and inhibitory checkpoint proteins, meaning that they could play a role in cancer progression.

Section 1.7: Protrusion-derived vesicles

Although protrusion-derived vesicles (PDVs) are not a specific subtype of MEVP, it is important to include PDVs in this conversation as it similarly groups several types of MEVPs together. The term PDV was introduced in 2021 after a key observation. Cells have various protrusions, which are actin fibers covered by a membrane, including filopodia, RFs, tunneling nanotubes, microvilli, and blebs [17,44]. Actin filaments are assembled into these protrusions at will and can facilitate cell adhesion, secretion, communication, interaction with the ECM, and in the case of cancer cells, invasion [17,41]. These protrusions can also release EVs, referred to as PDVs, via outward blebbing, shedding, pearling, or scission [17]. The release of PDVs is efficient as shedding from a protrusion is energetically favorable and increases spreading into the surrounding environment [17]. PDVs are hypothesized to play a facilitative role in the same processes as protrusions.

Although all protrusions depend on the polymerization of actin, it cannot be used as a PDV marker as it is not expressed on all PDVs [17]. However, CD133 (Prominin-1) has recently emerged as a potential marker for PDVs. Interestingly, CD133-positive EVs have been found to contribute to cancer metastasis and immune evasion [45].

As PDVs are a broad category, many of the MEVPs listed above fall into this group, including MVs, migrasomes, retractosomes, FDVs, FTVs, and blebbisomes. This overlap in classification may be beneficial as this could provide guidance to determine unclear biogenesis mechanisms, such as those of MBVs. It is important to note that there are subtypes of PDVs, such as secreted midbody remnants (sMB-Rs), that do not classify as MEVPs as they are typically shed from protrusions into the central lumen rather than into the ECM [46].

However, it is clear that the nomenclature used to describe cell protrusions has some of the most obvious inconsistencies, as protrusions can be referred to differently based on the cell type and environment studied [17]. Additionally, protrusions can shed vesicles into the lumen or ECM [17]. These inconsistencies may translate to MEVPs, stressing the importance of further proteomic analysis to define distinct markers for each MEVP subtype as well as provide further insights into the protein cargo and resulting effect on cells.

Section 1.8: Matrimeres

Matrimeres are non-vesicular EPs that are found in the ECM and are made up of at least one matrix protein and one DNA molecule. Their diameter ranges from 40–105 nm, a value that is likely dependent on the length of the incorporated DNA strand. Although only FN matrimeres have been studied, they can be formed using other ECM proteins. Even though the matrimere biogenesis pathway is unknown, they can be reconstituted in an acidic environment (pH 5.5), suggesting a potential dependence on secretory autophagy or lysosomal exocytosis [4].

Matrimeres can be isolated from cultured MSCs and blood plasma. To collect blood plasma, 5% EDTA solution is added to a 600 uL blood sample to serve as an anticoagulant, followed by two rounds of centrifugation (3000 ×g). The MSC cell culture media or plasma collection is followed by two rounds of centrifugation (2000 ×g, 10000 x×g), density gradient centrifugation with a 30% sucrose solution (100000 ×g), and additional centrifugation (100000 x×g) to produce a crude matrimere pellet. Immunoaffinity selection is used to isolate FN matrimeres, and both NTA and TEM are used to perform characterization. This isolation process results in 4 × 10⁶ matrimeres per μL of plasma [4].

MSP analysis of both plasma and MSC secreted FN matrimeres was completed to show an abundance of FN1, Alb, and Ighm [4]. In a biological context, matrimeres have been shown to expedite tissue repair and regeneration by restoring endothelial barrier integrity via matrix protein deposition. Specifically, MSC-secreted matrimeres, delivered via the intratracheal route, reversed lung edema in mice. 3 × 10⁸ matrimeres per 20 g mouse were necessary to produce this effect. Interestingly, reconstituted matrimeres remain in the lung tissue of mice for up to 9 days after delivery, with no trace in other organs, which holds great promise for matrimeres in a therapeutic context [4].

Section 1.9: Non-animal MEVPs

As EV production is conserved across kingdoms, EVs can be isolated from plants and microorganisms, such as fungi, bacteria, and parasites. Likewise, MEVPs can be isolated from microorganism-produced biofilms and plant tissue. For example, isolation of plant-EVs has recently been optimized in Morinda officinalis via enzymatic digestion of roots. This procedure potentially releases EVs from the plant tissue itself and produces a large EV yield with a smaller diameter and different lipid, RNA, and protein content, in comparison to the standard isolation from pulp liquid [19].

Beyond plants, biofilms of fungal and bacterial species contain distinctly different EVs than their planktonic counterparts. For example, the fungal Candida albicans-produced biofilm-EVs contain up to 45% of the proteins found in the biofilm matrix, including the ESCRT subunits, Hse1 and Vps27 [47]. These biofilm-EVs play a role in matrix biogenesis and function, and could be integral to biofilm production [48]. Of note, these biofilm-EVs were isolated from the conditioned medium surrounding the fungi, not the matrix itself, so it is unclear how fungal biofilm-residing EVs may impact biofilm formation.

However, biofilm-EVs have been isolated from the biofilm itself in both gram-positive and gram-negative bacteria. Gram-negative Pseudomonas aeruginosa biofilm-EVs were isolated by vortexing or homogenizing biofilms before centrifugation to isolate entrapped EVs. These biofilm-EVs had different protein and physical characteristics than planktonic EVs, and 30% of the proteins identified in the biofilm were also present in biofilm-EVs [49]. Furthermore, biofilm-EVs were found to accumulate within biofilms, to serve as signal “depositories” and to be released into the surrounding liquid [50]. Less commonly studied, gram-positive biofilm-EVs have been isolated from a few different strains and were similar in size to planktonic EVs, but still lack significant characterization of cargo differences and functions [51,52]. The exploration of non-animal MEVPs is still a largely unexplored field, and potential insights into the relationship between microorganismal biofilm-EVs and the biofilm may unlock an understanding of the role of MEVPs in tissues.

Section 1.10: MEVPs summary

In this section, we highlighted the biogenesis pathways, isolation techniques, protein markers, and biological relevance of numerous MEVP subtypes. All described MEVPs and their potential biogenesis mechanisms have been depicted in Figure 1. A commonality between these different MEVPs is that they are typically formed and found in or on surfaces, and also have overlapping biological roles with ECM functionality. However, no common protein marker has been found or verified for all MEVPs. Although many of the described MEVPs are classified as PDVs, meaning that they all potentially share CD133 as a marker, this would not include matrimeres. We have summarized the included MEVP proteomic studies in Table 1, detailing the proteins associated with each MEVP. While unique markers have been identified for migrasomes, further proteomic studies would be beneficial to identify uniquely enriched proteins for all other MEVPs, and general proteins associated with their cargo and external corona.

Figure 1.

Schematic of (left) potential biogenesis mechanisms and (right) reported MEVP subtypes. Potential MEVP biogenesis mechanisms include scission, pearling, shedding, budding, and exocytosis, which are not necessarily distinct from fluid EV biogenesis. Subtypes of MEVPs currently reported in literature include migrasomes and migrasome-derived intraluminal vesicles and retractosomes, matrix-bound nanovesicles, matrimeres, blebbisomes, matrix vesicles, and filopodia-derived vesicles. Non-animal MEVPs are less studied and are not included in the schematic, but are briefly described in the main text. Adapted from [17].

Table 1.

Known proteins enriched in established MEVP subtypes

MEVP Subtype	Sample of Origin	Isolation Technique	Characterization Technique	Protein Markers	Source	Notes
Matrix-bound nanovesicles	C57BL/6 mouse skeletal muscle	enzymatic digestion of ECM, centrifugation, and filtration	western blotting and RNA sequencing	Anxa5	[24]
	human MSCs in 2D cell culture	enzymatic digestion of ECM, differential centrifugation, and filtration	LC-MS/MS	COL6A1, COL6A2, COL6A3, COL12A1, FN1, TNC, TGFBI, ANXA2, MVP, STOM, MME	[30]
	human MSCs spheroid culture	enzymatic digestion of ECM, differential centrifugation, and filtration	LC-MS/MS	COL6A1, COL6A2, COL6A3, ITGA1, ITGA2, ITGB1, LRP1, ANXA6, FN1, MVP, STOM, MME, ITGA1, ITGA2, ITGA3, ITGA5, ITGAV, ITGB1, ITGB3, ITGB5
Matrix vesicles	murine pre-odontoblast cells (ECM of 17IIA11 cells)	enzymatic digestion of ECM, centrifugation, and filtration	western blot analysis	TNAP, Anxa5	[24]
	Human osteosarcoma Saos-2 cells (ATCC HTB-85)	enzymatic digestion of ECM, centrifugation, ultracentrifugation, and density gradient centrifugation	LC–ESI-MS/MS	TNAP, NPP1, Na+/K+-ATPase, plasma membrane Ca2+ ATPase, PHOSPHO1, AnxA1, AnxA2, AnxA4, AnxA5, …	[23]	Table 3 includes overlapping proteins from this study and three other MV proteome studies
	chicken embryo leg bone	enzyme digestion and differential centrifugation	LC-MS-MS/MS	collagen type X, core and link proteoglycan proteins, TNAP, AnxA2, AnxA5, AnxA6, LDH, actin, carbonic anhydrase type II, PHOSPHO1, Na1/K1 ATPase, 50-nucleotidase, …	[77]	Table 1 in the reference lists 126 gene products
	murine calvaria-derived osteoblast cells (MC3T3-E1)	enzyme digestion and ultracentrifugation	nanoRPLC-MS/MS	type IV collagenase (MMP-2), aminopeptidase A and N (zinc metalloproteases), Neprilysin (CD1), emilin-1, alpha-actinins, thrombospondin-2, vascular cell adhesion protein 1 (V-CAM 1), and periostin (osteoblast-specific factor 2)…	[78]	Supplemental Table 2 in the reference lists partial list of MV proteins
Migrasomes	rat kidney cells (NRK)	density gradient centrifugation	MS	TSPAN4	[13]
	murine fibroblast cells (L929) and dental pulp stem cells (DPSCs)	differential centrifugation and total membrane protein isolation	MS, SIM and EM, western blot	Myosin-5a, Rab8a, Rab11, VAMP2, VAMP3	[14]
	human gastric cancer cells (MGC-803)	density gradient centrifugation	MS	integrin alpha-5 beta-1 (with fibronectin pairing)	[36]	Other integrins were identified for different ECM proteins
	rat kidney cells (NRK)	density gradient centrifugation	MS and western blotting	NDST1, PIGK, CPQ, EOGT, TSPAN4, and integrin alpha5	[35]	TSPAN4 and integrin alpha5 are also present in other small EVs
	human serum	density gradient centrifugation	western blotting	NDST1, PIGK, CPQ, EOGT, TSPAN4, and integrin alpha5
	murine macrophage cells (RAW264.7) and mouse bone-marrow derived macrophages (BMMs)	-	immunofluorescence microscopy and live-cell fluorescence microscopy	pTRAP SCIMP	[37]	Study specific to macrophages
	zebrafish (in vivo)	-	confocal fluorescence microscopy	pTRAP SCIMP
Intraluminal vesicles	murine fibroblast cells (L929)	-	SIM and EM	Rab8a, Rab5, Rab10, Rab11, VAMP2, VAMP7, Myosin-5a	[14]
	dental pulp stem cells (DPSCs)	-		Rab8a, Rab11, VAMP2, VAMP3
Retractosomes	murine fibroblast cells (L929)	centrifugation and filtration	MS and western blot analysis	PigK, Eogt, and PCCA	[15]	PCCA and Eogt are less enriched in retractosomes than migrasomes
	murine macrophage cells (RAW264.7) and mouse bone-marrow derived macrophages (BMMs)	-	immunofluorescence microscopy and live-cell fluorescence microscopy	pTRAP SCIMPs	[37]	Study specific to macrophages. BMM = mouse bone-marrow derived macrophages.
	zebrafish (in vivo)	-	confocal fluorescence microscopy	pTRAP SCIMP
Filopodia-derived vesicles	human embryonic kidney cells (HEK293)	centrifugation	LC-MS/MS	IRS4 and GTPase Rac1	[16]	Contents of FDVs, not surface markers
Filopodia-tip vesicles	murine macrophage cells (RAW264.7)	density gradient centrifugation	western blot analysis and immunofluorescence staining	IL11, TSPAN4 integrin alpha5	[43]

It is important to note that some EVs and EPs can move dynamically from being defined as fluid EVs to MEVPs. For example, migrasomes are typically formed and reside on the ECM, yet may later detach and enter the bloodstream or other biofluids [13]. Meanwhile exosomes are typically released into surrounding fluids but may become embedded within or on the ECM [53,54]. While this complicates the field and may confound certain experimental workflows, it may be a unique opportunity to exploit this transitory ability of EVs and EPs to move from fluid to matrix-bound. Our goal is not to establish a prescriptive definition but rather to begin a discussion with the wider community of how this unique subset of EVPs should be classified.

Section 2: Challenges in MEVP production, isolation, and characterization

One significant challenge in the study of MEVPs is producing them at scale from tissue explants, in vitro cultures, or even biofluids. For example, initial proteomic studies of migrasomes required the culture of 30–80 FN-coated tissue-culture polystyrene flasks followed by enzymatic release from the surface along with cultured cells, then differential centrifugation and density gradient ultracentrifugation [35]. A similar approach has also been used for the isolation of MBVs, which can be extracted using a variety of enzymes (collagenase, proteinase K, liberase) and non-enzymatic (potassium chloride) treatments and separation methods (ultracentrifugation, ultrafiltration, density barrier, size exclusion chromatography). However, the optimal established method to yield MBVs with highest quantity, bioactivity, and purity is a combination of liberase/collagenase and size exclusion chromatography [29]. While several experimental factors, such as optimized ECM coatings, micropatterned growth surface topographies, or 3D cultures could likely increase these yields, the production of sufficient MEVPs for most researchers are, at least to some degree, limited by the cell density achievable in monolayer cultures [30,55].

Additionally, enzymes such as trypsin have been shown to contribute to non-vesicular proteins during EV isolation, and cell detachment using EDTA can cause a rapid release of exosomes. The large volumes of trypsin, or other enzymes used for MEVP cleavage from the substrate, may damage MEVPs or remove valuable biomolecular content from their protein corona while also requiring rapid neutralization [56,57]. In general, the corona can be significantly altered based on the EV source and isolation steps. Specifically, it has been shown that high-salt washing of EVs can alter the protein corona and expose EV surface molecules, ultimately changing EV function [58,59]. While it may be helpful in the long term for defining MEVPs, particularly in the context of their interactions with the ECM, the understanding of the EV corona is in its infancy and thus it is too early to make any prescriptive rules. However, given the likelihood that MEVP-ECM interactions are driven or mediated by the corona, we thought it was necessary to at least mention its relevance to encourage researchers in this space to characterize the corona when possible. For example, researchers attempting to recapitulate MEVPs in vitro can perform patterning and internalization studies with native vs “corona-shaved” MEVPs to determine differences in patterning and/or functional effects on cells [59,60].

MEVPs isolated from biofluids or tissue explants are also limited by the volume of biofluid or mass of tissue available. For example, the first demonstration of migrasome isolation from blood required roughly 50 ml of serum to be withdrawn from blood samples [35]. While some collected tissues, such as discarded organs, may enable downstream orthogonal characterization techniques, other tissues which are considered precious or provide low MEVP yield will require highly efficient isolation strategies. It is important to note the heterogeneity of in vivo sources. For example, ECM matrix scaffolds, prepared from decellularized tissue, proved to have different remodeling effects based on the age of the source tissue [61]. This could imply further differences in MEVP characteristics and protein expression.

While improving isolation efficiency is necessary for tissue-derived MEVPs, yields from in vitro cultures have multiple limitations and may also be improved in several ways. It is well-known that in vitro growth conditions can affect the amount, type, and cargo of EVs produced [62]. In particular, flat tissue-culture polystyrene is routinely used, a material that lacks many microenvironmental cues, which affects both cell behavior and EV production. Ultimately, these produced EVs are significantly different from those derived from in vivo conditions [30].

However, conventional cultures do offer unique advantages, particularly their accessibility, scalability, and amenability to common imaging and high throughput systems. To harness these advantages, there are several ways to address the limitations of in vitro growth conditions. Culture surfaces can be coated with ECM proteins or tissue-derived ECM materials to increase biomimicry [35]. Additionally, semi-3D cultures can be used. For instance, both commercially available and custom-built bioreactor systems have demonstrated immense promise in improving the yield of fluid EVs in conditioned media by greatly increasing cell density, in combination with other factors such as growth surface topography and unique fluidic interactions [63–68]. However, these bioreactor systems are opaque, difficult to monitor, entail semiweekly maintenance, and will require further development if they are to be adopted for scalable MEVP production. These limitations can be addressed by monitoring bioreactor parameters (media pH, glucose levels, color, etc.) and potentially exploring sustained nutrient release mechanisms, such as micro- or nanocarriers [69]. Additionally, a comparative analysis will be necessary for each MEVP subtype to demonstrate clear differences, if any, between conventional and more advanced culture systems.

Regardless of the production method used, MEVPs must generally be fractionated from a heterogeneous pool of other EVs using one or more conventional EV isolation techniques [8]. However, as MEVPs are unique, in that if they are harvested directly from synthetic growth surfaces, it may be possible to isolate them based on their unique biogenesis mechanism. This is in contrast to the difficulty of harvesting specific subtypes of fluid EVs with high specificity, such as exosomes.

For some MEVPs, specific physical properties such as density, have been exploited to improve isolation specificity. However, analogous to the fractionation of fluid EVs, the field of MEVPs should remain free of prescriptive isolation strategies or suggestions, and instead rely on the researcher to use them depending on the starting material and the downstream application. As shown in Figure 2, the conventional EV isolation strategies that can be applied to MEVP fractionation include differential ultracentrifugation, density gradient ultracentrifugation, size exclusion chromatography, ultrafiltration, polymer solution-based precipitation, tangential flow filtration, and immunoaffinity-based capture. Many of these techniques have not been reported for specific application to diverse subtypes of MEVPs, and immunoaffinity-based capture in particular may require extensive proteomic analyses to establish reliable markers for highly specific and efficient capture.

Figure 2.

Illustration of potential MEVP sources, production methods, and isolation approaches. (A) MEVP sources include tissues, biofluids, ECM material, tissue culture flasks, and bioreactor flasks. (B) Following initial collection, which may include various forms of enzymatic digestion, cells and large debris are depleted typically using low-speed centrifugation. Several established isolation methods can be applied to MEVP isolation independently or in combination, including (C) ultracentrifugation, (D) density gradient ultracentrifugation, (E) size exclusion chromatography, (F) dead-end ultrafiltration, (G) tangential flow filtration, (H) PEG-based precipitation, and (I) immunocapture. Figure created in biorender.com.

Similar to fluid EVs, MEVPs can be characterized using a variety of well-established techniques and there are several unique opportunities to develop characterization strategies specific to MEVPs in situ. Based on the strategies outlined in the MISEV 2023 guidelines, one should consider three orthogonal characterization techniques when possible to validate the presence of MEVPs, including one for sizing/counting (NTA, RPS), one for morphological analysis (TEM/SEM/super-resolution microscopy), and one for validating some known MEVP marker (immunoblotting, proteomics) [8]. However, given that most established workflows for these techniques were not uniquely designed for MEVPs, researchers should also strongly consider characterization techniques that can be employed prior to detaching MEVPs from their original surface. We expect that scanning electron microscopy (SEM) will become a standard characterization technique in this field and it has been used to identify migrasomes and tissue-derived MBVs [13,70]. Additionally, single MEVP fluorescence may become valuable in situ given the recent growth of super-resolution single EV and EP analysis techniques, and to the best of our knowledge, atomic force microscopy has not yet been explored for detailed MEVP analysis.

Section 3: MEVP Proteomics

Proteomic analysis offers the unique opportunity to characterize the contents and surface proteins of MEVPs as well as deconvolute potential heterogeneity between subtypes. MSP is a widely used tool in EV research, enabling the identification, characterization, and understanding of the biological roles of EVs by analyzing the mass-to-charge ratio of molecules. Not only does MSP enable the identification, characterization, and understanding of the biological roles of MEVPs, but its high throughput and specificity could allow for the identification of unique protein markers of different subtypes and serve as an information-rich benchmark to optimize EV fractionation in the future [71].

However, the application of MSP in EV proteomics faces several challenges. One such challenge is the complexity of biological fluid and culture media samples, which contain non-vesicular proteins and contaminants that can interfere with the detection of low-abundance proteins within EVs [71]. As previously mentioned, the results of MSP can also vary based on the isolation technique used, which introduces additional complexity to the analysis. Furthermore, for low-abundance EV samples, the sensitivity of MSP for detecting small amounts of proteins is limited, especially in the absence of large sample quantities or protein amplification steps [72].

MSP can also be employed for targeted analyses to assess contamination and EV purity, as well as to quantify protein abundance [73]. It is important to note that the EV isolation strategy preceding targeted MSP will affect the proteomic yield, purity, and representation of EV subtypes [74]. Non-targeted analyses, on the other hand, can provide a comprehensive protein composition and enable biomarker discovery [75]. Due to the complexity of samples and need for high throughput, MSP for EVs is typically performed in a bottom-up approach, where proteins are broken down into peptides, and are then separated using liquid chromatography prior to MSP analysis. Although top-down analysis has been employed for the characterization of protein cargo of small EVs, the limitation in maximum protein size and required extraction of intact proteins proves to be unfavorable [76].

Currently, MSP is widely used for characterizing EVs in fluids, including both biological fluids and culture media. However, it is less commonly applied to MEVPs, primarily due to the differences in isolation techniques. Proteomics studies on EVs in matrix-bound environments face the additional challenge of removing ECM proteins or other matrix components that may interfere with MSP analysis [72]. Clearly, the field of MEVPs has relied heavily on MSP and other protein validation techniques to improve production, isolation, and characterization workflows and will continue to do so as it progresses. Known proteins associated with reported MEVP subtypes are listed in Table 1.

Conclusions and Future Outlook

Based on the continued growth of EV and EP research more broadly, we believe that there has never been a better time to shine light on these unique ECM-associated messengers. However, as with many emerging fields, the diversity of nomenclature, experimental approaches, and characterization standards across studies has created challenges in establishing a cohesive understanding of MEVP biogenesis and function. Current MEVP researchers should work collectively to ensure that the subfield of MEVPs does not suffer from potential rigor and reproducibility issues. Significant overlaps in certain biogenesis mechanisms, isolation protocols, and proteomes indicate that while MEVPs are currently investigated in silos, there are also many similarities. For example, it is unclear how FDVs and FTPs differ from migrasomes in biogenesis and function. To increase distinction between subtypes, more detailed investigations of MEVP biogenesis from different cell types and their relationship with the ECM are needed to better understand their biological function. Additionally, identification of specific protein markers of MEVP subtypes could help us to determine their localization in the ECM, transfer and uptake by cells, and general biogenesis.

The scalable production and isolation of MEVPs using conventional cultures poses several challenges, however there is potential for optimization of production by using established bioreactor systems and other types of 3D cultures. Regarding isolation, enzymatic digestion has been widely used in plant-EVs, MBVs, and MVs, yet the effect of enzymatic digestion on MEVP contents remains unclear. Additionally, there is uncertainty surrounding tissue-derived EVs and their potential to be either matrix-associated or fluid-derived. This will be clarified via the combination of proteomic analyses, including spatial proteomics to study MEVPs in a native state, and other characterization techniques. To develop efficient MEVP classification and improve production and isolation workflows, proteomic databases should begin to distinguish between fluid EVs and MEVPs. Although this paper emphasizes the potential of proteomics in advancing the MEVP field, it is crucial to explore other characterization techniques in parallel. For example, mass spectrometry-based lipidomics and RNA-sequencing have been used to show distinct differences between MBVs and fluid EVs [79].

In conclusion, now is the time for the EV and EP research community to take action in the development of the MEVP field, ensuring responsible growth through rigor and reproducibility. This could involve the consensus on nomenclature, reporting standards, and experimental guidelines. Unlocking the full potential of MEVPs will be made possible by improving our understanding of their role in ECM development and maintenance, cellular communication, and disease and repair mechanisms, which will ultimately lead to improved diagnostic and therapeutic applications.

Abbreviations:

ECM

extracellular matrix

EV

extracellular vesicle

EP

extracellular particle

MEVP

matrix-bound or matrix-associated EV and EP

MV

matrix vesicle

MSP

mass spectrometry-based proteomics

MBV

matrix-bound nanovesicle

MSC

mesenchymal stromal cell

RF

retraction fiber

FN

fibronectin

FDV

filopodia-derived vesicle

FTV

filopodial-tip vesicle

PDV

protrusion-derived vesicle

sMB-Rs

secreted midbody remnants

NTA

nanoparticle tracking analysis

RPS

resistive pulse sensing

TEM

transmission electron microscopy

SEM

scanning electron microscopy