Effective methods for isolation and purification of extracellular vesicles from plants

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.     

Identification and characterization of hADSC-derived exosome proteins from different isolation methods

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.     

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.     

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge.ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells.MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM).ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield.ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.     

An improved method for preparing rat small intestine microsomal fractions for studying drug metabolism.

Epithelial cells from isolated rat small intestine were harvested by vibration of the everted intestine in 0.14 m NaCl containing 5 mm EDTA. These cells, which were largely free of mucus contamination, were homogenized in hypotonic (74 mm) sucrose using a Potter-Elvehjem homogeniser. After successively sedimenting a “brush border plus nuclei” and a “mitochondrial” fraction, microsomes were prepared from the postmitochondrial supernatant by ultracentrifugation or by precipitation at pH 5.0. The isolation and fractionation procedure was validated by the distribution of marker enzymes and by light microscopy and found to be largely uncontaminated by other subcellular components or by haemoglobin. The usefulness of this preparation was demonstrated by determining drug-metabolising enzyme activity and by substrate- and metabolite-binding spectra to cytochrome P-450. A comparison of precipitated “acid” and “normal” intestinal microsomes indicated similar apparent K_m and V_max values for a number of drug-metabolising enzymes. The content of components of the microsomal electron transport system were also similar in both preparations while the “acid” microsomes contained approximately 50% more protein.     

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates

Background

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial.

Principal Finding

In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7–10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30–43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37°C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4°C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates.

Conclusion

These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.     

Cell wall glycoproteins and polysaccharides of mature runner beans

A novel use of chlorite-HOAc treatment (delignification procedure) for the isolation of hydroxyproline (HP) rich “glycoproteins” from the depectinated cell wall material of mature runner beans is described. This procedure can be used for the isolation of wall proteins even from heavily lignified tissues. Its main disadvantage is that some of the constituent amino acids are either destroyed or modified; the nature of these changes was studied using gelatine, lysozyme and “cytoplasmic proteins” of mature beans. The main amino acids to be affected were tyrosine, cystine, methionine and lysine. The chlorite-HOAc solubilized proteins were separated by PhOH-H₂O fractionation into two distinct “glycoprotein fractions”. The major fraction (isolated from the aqueous layer) contained most of the HP of the solubilized proteins. The sugars obtained on hydrolysis of both “glycoproteins” were galactose, arabinose, glucose, xylose, rhamnose and uronic acid. Most of the proteins remaining in the holocellulose could readily be extracted with cold alkali and were relatively poor in HP.     

Low level electricity increases the secretion of extracellular vesicles from cultured cells

Exosomes, a type of extracellular vesicles, can be collected from the conditioned medium of cultured cells, and are expected to be used in disease therapy and drug delivery systems. However, since the yield of exosomes from conditioned medium is generally low, investigations to develop new methods to increase exosome secretion and to elucidate the secretion mechanism have been performed. Our previous studies demonstrated that activation of intracellular signaling including Rho GTPase and subsequent endocytosis of extraneous molecules in cells could be induced by low level electricity (0.3–0.5 mA/cm²). Since exosomes are produced in the process of endocytosis and secreted by exocytosis via certain signaling pathways, we hypothesized that low level electric treatment (ET) would increase exosome secretion from cultured cells via intracellular signaling activation. In the present study, the influence of ET (0.34 mA/cm²) on extracellular vesicle (EV) secretion from cultured cells was examined by using murine melanoma and murine fibroblast cells. The results showed that the number of EV particles collected by ultracentrifugation was remarkably increased by ET in both cell lines without cellular toxicity or changes in the particle distribution. Also, protein amounts of the collected EVs were significantly increased in both cells by ET without alteration of expression of representative exosome marker proteins. Moreover, in both cells, the ratio of particle numbers to protein amount was not significantly changed by ET. Rho GTPase inhibition significantly suppressed ET-mediated increase of EV secretion in murine melanoma, indicating that Rho GTPase activation could be involved in ET-mediated EV secretion in the cell. Additionally, there were almost no differences in uptake of each EV into each donor cell regardless of whether the cells had been exposed to ET for EV collection. Taken together, these results suggest that ET could increase EV secretion from both cancer and normal cells without apparent changes in EV quality.     

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.     

Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signaling mediators of cellular responses. However, despite representing a promising alternative to cell-based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred-tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred-tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC-derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential.     

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.     

One hundred years of zygotic embryo culture investigations

Summary Isolation of zygotic embryos from seeds and their culture in a defined medium, initiated by Hanning in 1904, has proved to be a promising method to study the factors that control growth and differentiation of embryos. Using this technique, several investigations have focused on the carbohydrate and nitrogen nutrition during germination of cultured seed embryos and on the effects of plant hormones on their morphogenesis. Culture of immature embryos leads to their germination into weak seeldings, skipping the later stages of embryogenesis, by a process known as precocious germination. Progressively smaller embryos have been cultured by supplementation of the medium with coconut milk or hormonal additives or by osmotic adjustment of the medium by high concentrations of sucrose or mannitol. Although methods have not been developed for large-scale isolation and culture of zygotes, zygotes of maize isolated from embryo sacs and those obtained by in vitro fertilization have been grown in culture into full-term embryos. Embryo culture techniques are widely used to rescue embryos from seed of wide crosses which usually abort and to overcome dormancy of recalcitrant seeds.     

Extraction of recombinant protein from Escherichia coli by using a novel cell autolysis activity of VanX

Escherichia coli is a versatile, low-cost, and popular host for expressing recombinant proteins. However, extracting recombinant proteins from E. coli requires cell wall breakage, which is both time- and effort-consuming. Here we report a novel cell breakage method based on our recent finding that VanX, which is a d-Ala-d-Ala dipeptidase encoded in a vancomycin-resistant VanA gene cluster, exhibits a strong cell lysis activity when expressed in isolation in E. coli. In our strategy, we coexpress VanX with the target protein, causing cell autolysis and release of the cellular content into the culture medium. We demonstrated this strategy for two model proteins, a green fluorescent protein variant (GFPuv) and Gaussia luciferase, and optimized the autolysis conditions and coexpression vectors. The fluorescence activity of GFPuv collected from the medium was identical to that of GFPuv purified by conventional methods. Cell breakage by VanX-mediated autolysis is very simple to implement and will efficiently complement traditional methods.     

Molecular composition and origin of substrate-attached material from normal and virus-transformed cells

The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein C_o (the LETS glycoprotein); protein C_a (a myosin-like protein); protein C_b (MW 85,000); protein C₁ (MW 56,000, which is apparently not tubulin); protein C₂ (actin); proteins C₃–C₅ (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins C_c, C_d, C_e, and C_f. The LETS glycoprotein (C_o) and C_d appear in newly-synthesized SAM (which is probably enriched in “footpad” material – “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material – remnants of footpads left behind as cells move across the substrate). C_o and C_d turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.     

Isolation and characterization of RNA-containing exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen, eradication of established tumors in mice, inhibition and activation of natural killer cells, promotion of differentiation into T regulatory cells, stimulation of T cell proliferation and induction of T cell apoptosis. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.     

Toward Standardization of Mesenchymal Stromal Cell‐Derived Extracellular Vesicles for Therapeutic Use: A Call for Action

Extracellular vesicles (EVs), which include a variety of nano‐sized membrane‐encapsulated particles, are released to the extracellular microenvironment by the vast majority of cells and carry lipids, proteins, mRNA, and miRNA or non‐coding RNA. Increasing evidence suggests the great versatility and potential of EV‐based applications in humans. In this issue, van Balkom et al. explore and compare the reported proteomic signature of mesenchymal stromal cell (MSC)‐derived small EVs. In particular, their paper offers a valuable approach and point of view on MSC‐EV manufacturing and therapeutic potential. Briefly, van Balkom et al. aimed to identify a common protein signature that may be useful in ensuring the homogeneity of therapeutic MSC‐EVs. In addition to excessive variability in EV‐producing cell sources and culture conditions, the harvesting time for the EV‐containing conditioned medium, and EV isolation procedure, the authors found a specific protein signature from the publicly available MSC‐EVs proteome. In light of their findings and those from the plentiful studies published in this continuously growing area of research, potential focus areas and issues are outlined for the more rational design and optimization of MSC‐EV production and potency for therapeutics.     

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation

Enterovirus A71 (EV‐A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV‐A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV‐A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV‐A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV‐A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV‐A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID₅₀) values calculated in viral proliferations (ranged from 10^7.6 to 10^7.8); the TCID₅₀ being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV‐A71Jinan1002 amplifies more efficiently in MA104, Hep‐2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV‐A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV‐A71.     

Commonly used methods for extracellular vesicles’ enrichment: Implications in downstream analyses and use

Extracellular vesicles (EVs) are becoming promising tools for clinical application, either as sources of disease-specific molecular signatures for the unraveling of disease pathophysiology and establishment of novel biomarkers, or as platforms for cell-free nanotherapy. Yet, an unsolved issue is to define standardized techniques for EV isolation allowing data comparison across laboratories worldwide. Considering the difficulties to find this necessary consensus, it has to be stressed out that the outcome of the downstream analysis might be deeply biased by the isolation method, among other variables. Thus, it is crucial that the researcher is aware of the strengths and weaknesses of each method keeping their intended use in mind, and to sufficiently report the methodology details for the results to be comparable and solid. This review aims to present the most widely used EV isolation methods, from the initial differential ultracentrifugation (dUC) to newest approaches.