An optimized protocol for the enrichment of small vesicles from murine and non-human primate heart tissue
DOI: https://doi.org/10.47184/tev.2022.01.03 Over the last few years, the interest in extracellular vesicles (EVs) function has exponentially grown. However, methods for isolating these small vesicles from tissue are still not trivial. Few protocols that allow EV isolation from whole tissue samples, including the heart, are available and they are based on organ perfusion unsing Langendorff method. In this work, aiming at analysing in vivo biology of small EVs, we implemented a simple method to obtain enrichment of these vesicles from murine heart tissue. We tested a titration of Liberase for tissue digestion, which was subjected to differential ultracentrifugation combined with iodixanol cushion and presented the step-by-step procedure of this protocol. Validation was done with Nanoparticle Tracking Analysis, transmission Electron Microscope and Western Blot analysis of EV markers and organelle contaminants. Furthermore, we tested the suitability of the protocol for isolating EVs from heart tissue obtained from a pre-clinical translational non-human primate animal model. Therefore, this protocol should be suitable for isolating vesicle from human heart tissue. Additionally, this method could potentially be applied beyond heart tissue
Tissue, heart, EVs purification, cushioning, murine, non-human primates, EVs quality
Introduction
EVs are a heterogeneous group of bilayer membrane nanoparticles that play roles in diverse intercellular communication in short and long-range distances. Due to their prominence in cellular communication, the study of EVs has recently been gaining great attention from scientists. In the cardiovascular system, physiological and pathological adaptations require a coordinated cell-to-cell communication mediated by cytokines, peptides, hormones and, as recently recognized, also by EVs. Within the EVs group, we can highlight exosomes, which can transport RNA species, lipids, and proteins. These nanoparticles are sensitive to various stress stimuli mediating locally and remotely signaling transduction. Furthermore, they are intensively studied as diagnostic markers as well as cell-free strategies for regenerative medicine [1, 2]. EVs were reported to protect the heart against myocardial infarction and stimulate angiogenesis [3]. Different subtypes of EVs are classified based on physical and/or biochemical properties. In general, based on physical characteristics such as size, EV subtypes can be classified as "small" or "medium/large". Small EVs size ranges between 100 and 200 nm, whereas medium/large EVs size is > 200 nm [9]. Small EVs are released by exocytosis of multivesicular bodies, while whole large EVs and apoptotic vesicles are shed from the plasma membrane. Apoptotic vesicles are in the range of > 1 mm in diameter [4]. EVs, including exosomes (or cardiosomes), were described to be released from all major heart cell types in vitro, suggesting that they play an important role in the cardiovascular system. However, the problem represented by the isolation of these small particles present in the interstitium of cellular tissues in vivo [5], is preventing scientists from advancing more rapidly along this area. Specifically, EVs isolated from heart tissue remained challenging due to the susceptibility of heart cells to hypoxia, including cardiomyocytes, which can cause changes in ultrastructure and secretory activities [6].
The majority of available protocols and data are focused on EVs isolated from cell culture conditioned media or body fluid [4, 5], however, transferability of EV secretion studies in vitro to a disease-dependent condition in vivo is limited [7]. At present, it is still challenging to isolate EVs from tissue homogenates and no standardized protocol has yet been develop that can be considered as a gold standard [4]. Differential centrifugation combined with several steps of further separation including density gradient (sucrose or preferably iodixanol) is recommended for purification of EVs along with co-pelleted protein complexes and lipoproteins [8]. EVs can be isolated from hearts via Langendorff perfusion however, the buffers used can cause exosome-sized and calcium-phosphate nanoparticle formation, due to the spontaneous formation in Ca+2- containing bicarbonate buffer. These features, can directly interfere with some analysis such as nanoparticle tracking analysis [4, 9].
In this study, we aimed to obtain interstitial EVs contained in the heart in vivo and reflecting the physiology of the murine heart with an easy-following protocol with gentle digestion, as an alternative to Langendorff perfusion. Standard methods of EV characterization including, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and detection of positive and negative markers for EVs were used to characterize the enriched EVs. EVs were reported to be produced by almost all organisms and cell types studied; however, research has mainly focused on EVs of human or mouse origin, leaving other species less investigated [10]. In this article, we showed the suitability of the protocol for isolating EVs from heart samples of non-human primates (Callithrix jacchus, common marmoset); a pre-clinical animal model providing the optimal compromise for human related therapeutic studies due to the high homology with the human genome [11]. Currently, only physical characterization of Callithrix jacchus derived cardiac EVs can be performed due to the lack of suitable specie-specific antibodies, which remain to be established.
Methods and Materials
Solution preparation
Solutions are provided in Table 1, 2 and 3.
