Liver-derived extracellular vesicles: A cell by cell overview to isolation and characterization practices
Introduction
Extracellular vesicles (EVs) is a collective term referring to a diverse group of small membrane vesicles virtually released by all cell types, and which are generally being categorized according to their biogenesis: [1,2] apoptotic bodies are blebs of the dying cell membrane and have the broadest size range; microvesicles, sometimes referred to as microparticles or ectosomes, stem from the outward budding of the cellular membrane. Exosomes, which tend to be the smallest subpopulation, are released into the extracellular space after multivesicular bodies (MVB) fuse with the cell membrane (Fig. 1). It is still not really possible to isolate one subpopulation from the others, and while it is believed that they may display biomolecules that are enriched to different extents, their overlapping composition, density and size, as well as the absence of subtype-specific markers still make for a considerable challenge. [3,4] The recent discovery of their role in intercellular communication captivated the attention of a growing number of scientists anticipating the enormous potential of EVs in the fields of diagnostics and drug delivery. [5,6] For some pathological dispositions EVs can be applied as liquid biopsies, and that has sparked a lot of interest from a diagnostic perspective. EVs are enriched in selected biomolecules, they are intrinsically equipped to protect their cargo from degradation, and while their complexity offers many characterization opportunities (see Fig. 1), they are still simpler to analyse than total blood or serum samples. The interest in EVs as drug delivery systems stems from their potential advantages over synthetic carriers: they are bioavailable, biocompatible, resistant to RNAases and proteases (high physicochemical stability), capable of long-distance communication and they are intrinsically able to interact with cells even across species. [[7], [8], [9]].
The increasing scientific interest in EVs [10] has led to the establishment of dedicated, open access databases such as Vesiclepedia, [11] EVpedia [12] and EV-TRACK, [13] which are being regularly updated. The majority of the information is concerning proteins, whereas EV-TRACK sets itself apart by focusing on method transparency.
As reviewed elsewhere, [14,15] there are significant challenges (small yields, co-purification of contaminants, etc.) in finding the most efficient protocols for the isolation and sufficient characterization of EVs. We will discuss them in an effort to encourage sharing the current knowledge of the more practical scientific trends in those areas, starting with the present review on liver-derived EVs.
The liver is a large and complex organ responsible for a variety of essential physiological tasks including protein synthesis, lipid storage regulation, xenobiotic detoxification, and offering support to both immunological activity and food digestion. [16] It is difficult to understate its importance. When organ function is compromised, hepatic diseases are directly responsible for as many as 2 million deaths per year: liver cirrhosis alone kills 1.16 million people every year, and hepatocellular carcinoma accounts for the death of 788,000 more, meaning that combined they cause 3.5% of all yearly deaths in the world. [17,18] The global health burden of liver associated conditions is not sufficiently addressed as of yet. [19] With its unique ensemble of diverse cells (see Fig. 2), the liver offers the opportunity to study intra- and inter-cellular communication. Elucidating the role of EV-mediated hepatic cellular crosstalk has gained the attention of many researchers, who have been able to review its critical role in both health and disease, pointing to differences in the set of EVs that are released, especially in the case of tumors. [[20], [21], [22], [23], [24], [25]].
The present review shifts the perspective into practice: we aim to critically consider the methods used to purify and to biochemically analyse EVs from specific cells, offering a user/reference guide to current practices (see Table 1). The most common and (when applicable) the most original/promising protocols will be examined in order to provide a discussion frame. Diving into the different methodological approaches, we will also highlight limitations and possible inconsistencies. Finally, the (interspecies) method transferability and the translational applicability of these practices will be examined. We will look within the individual cell specific sections if the same or similar methods could be transferred to different in vitro systems (rodent cells, co-cultures), but we will also explore the practical strategies for EV-isolation and characterization that were applied ex vivo and in the clinic from patients with liver-related conditions (Section 8). It is worth noting, that we are only covering method employment and it is not our role to comment on the quality of the results gained from them. As to the nomenclature, we strived to keep it as it was applied in the referenced manuscripts, only changing it to “EVs” if we needed to paraphrase, since it is the preferred generic term. [1,2].
Section snippets
EVs from hepatocytes
Hepatocytes are the most abundant cells in the liver, comprising about 70–80% of its mass, they are dedicated to protein synthesis (serum albumin, transferrin and other glycoproteins), while also being the main site for glycolysis and only site for bile salts production in the body. [[222], [223], [224]] Another essential task relying on hepatocytes is detoxification: they metabolize both exogenous (e.g., drugs, toxins) and endogenous (e.g., steroids) compounds. [225,226] More notable still is
EVs from hepatic stellate cells
Hepatic stellate cells reside in the space of Disse (Fig. 2), between hepatocytes and endothelial cells, and are mainly responsible for storing vitamin A in cytoplasmic lipid droplets. [238] Upon liver injury, however, they undergo transdifferentiation into a myofibroblast-like state, i.e., they become activated, progressively lose their lipid droplets and start promoting fibrogenesis, most notably by deposition of excessive and collagen rich extracellular matrix. [239,240] When the injuries
EVs from cholangiocytes
Cholangiocytes are epithelial cells lining the bile ducts (Fig. 2), which can differ in size and morphology just as the bile duct tree itself does. Their main physiological role is the modification of the bile coming from the liver while it is being transported along the biliary ducts into the intestine. [[247], [248], [249], [250]] Pathologies directly associated with cholangiocytes include primary biliary cholangitis, and primary sclerosing cholangitis, for the latter of which liver
EVs from liver sinusoidal endothelial cells
Liver sinusoidal endothelial cells (LSECs) form the fenestrated endothelial layer at the interface between cells in the blood and the HSCs in the space of Disse. LSECs act as efficient pinocytotic scavengers for particles that are smaller than 0.2 μm, making them the central players in clearing the body of blood-borne viruses. [253,254] When working with EVs from LSECs, researchers were interested in infections caused by hepatitis B and C viruses (HBV and HCV).
EVs from Kupffer cells
The resident macrophages in the liver, found in the hepatic sinusoid, are known as Kupffer cells. [258] Since the liver is frequently in contact with exogenous material, suppressing “unwanted” immune responses is essential. Kupffer cells have been shown to provide anti-inflammatory signals that allow homeostatic immunological tolerance under healthy conditions. As part of the innate immune system, these macrophages phagocyte invading pathogens and play a critical role for the initiation of
EVs from liver stem cells
Several studies have suggested the presence of liver-resident stem cells, which along with hepatocytes contribute to liver regeneration, but it remains a controversial topic, as reviewed elsewhere. [264] The identification of oval cells and their role in liver regeneration contributes to our current understanding of the process, [265] more confidently so after establishing their precursor role to hepatocytes, [266] and their localization in the canals of Hering [267] between bile capillaries
EVs from clinical settings
There has been extensive research delving into the diagnostic potential of EVs in the context of liver-associated conditions, albeit mostly looking into circulating vesicles, both in human patients [[100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142],
Final general remarks
Strategies for liver-derived EV isolation and characterization are as varied as the research groups tackling the challenge. While the main advantages and disadvantages for each approach have been presented, we would like to highlight some of the key aspects that emerged.
Conclusions and perspectives
We reviewed the current methodological practices for the isolation and analysis of liver derived EVs, making a cell type specific user guide. Liver-derived EVs are gaining attention as a research topic, and there are already a few research papers addressing EVs from every liver resident cell type. Much work has already been published, but a rigorous standardization is needed. We moreover highlighted common causes for concern and critically reviewed room for improvement: the bigger issue that
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
G.F. is a holder of a NanoMatFutur grant from the Federal Ministry of Research and Education (13XP5029A). The Phospholipids Research Center is gratefully acknowledged for its support.
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