differential ultracentrifugation results in co-isolation of protein and other contaminants and incomplete separation of vesicles from lipoproteins. combining ultracentrifugation with micro-filtration or a gradient can improve purity. single step isolation of extracellular vesicles by size-exclusion chromatography has been demonstrated to provide greater efficiency for recovering intact vesicles over centrifugation, although a size-based technique alone will not be able to distinguish exosomes from other vesicle types. to isolate a pure population of exosomes a combination of techniques is necessary, based on both physical (e.g. size, density) and biochemical parameters (e.g. presence/absence of certain proteins involved in their biogenesis). the use of reference materials such as trackable recombinant ev will assist in mitigating technical variation introduced during sample preparation and analysis. novel selective isolation methodology has been using a combination of immunoaffinity chromatography and asymmetric-flow field-flow fractionation to reduce the contamination from lipoproteins and other proteins when isolating from blood plasma. often, functional as well as antigenic assays are applied to derive useful information from multiple exosomes. well-known examples of assays to detect proteins in total populations of exosomes are mass spectrometry and western blot. however, a limitation of these methods is that contaminants may be present that affect the information obtained from such assays. preferably, information is derived from single exosomes. relevant properties of exosomes to detect include size, density, morphology, composition, and zeta potential. detection since the diameter of exosomes is typically below 100 nm and because they have a low refractive index, exosomes are below the detection range of many currently used techniques. a number of miniaturized systems, exploiting nanotechnology and microfluidics, have been developed to expedite exosome analyses. these new systems include a micronmr device, a nanoplasmonic chip, and a magneto-electrochemical sensor for protein profiling; and an integrated fluidic cartridge for rna detection. flow cytometry is an optical method to detect exosomes in suspension. nevertheless, the applicability of flow cytometry to detect single exosomes is still inadequate due to limited sensitivity and potential measurement artifacts such as swarm detection. other methods to detect single exosomes are atomic force microscopy, nanoparticle tracking analysis, raman microspectroscopy, tunable resistive pulse sensing, and transmission electron microscopy. bioinformatics analysis exosomes contain rna, proteins, lipids and metabolites that is reflective of the cell type of origin. as exosomes contain numerous proteins, rna and lipids, large scale analysis including proteomics and transcriptomics is often performed. currently, to analyse these data, non-commercial tools such as funrich can be used to identify over-represented groups of molecules. with the advent of next generation sequencing technologies, the research on exosomes have been accelerated in not only cancer but various diseases. recently, bioinformatics based analysis of rna-seq data of exosomes extracted from trypanosoma cruzi has showed the association of these extracellular vesicles with various important gene products that strengthens the probability of finding biomarkers for chagas disease. therapeutics and carriers of drugs increasingly, exosomes are being recognized as potential therapeutics as they have the ability to elicit potent cellular responses in vitro and in vivo. exosomes mediate regenerative outcomes in injury and disease that recapitulate observed bioactivity of stem cell populations. mesenchymal stem cell exosomes were found to activate several signaling pathways important in wound healing (akt, erk, and stat3), bone fracture repair and participates in the regulation of immune-mediated responses and inflammatory diseases. they induce the expression of a number of growth factors (hepatocyte growth factor (hgf), insulin-like growth factor-1 (igf1), nerve growth factor (ngf), and stromal-derived growth factor-1 (sdf1)). exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling, exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (mir-126, mir-130a, mir-132) and anti-inflammatory (mir124a, mir-125b) micrornas, and a microrna regulating collagen deposition (mir-21). researchers have also found that exosomes released from oral keratinocytes can accelerate wound healing, even when human exosomes were applied to rat wounds. exosomes can be considered a promising carrier for effective delivery of small interfering rna due to their existence in body's endogenous system and high tolerance. patient-derived exosomes have been employed as a novel cancer immunotherapy in several clinical trials. exosomes offer distinct advantages that uniquely position them as highly effective drug carriers. composed of cellular membranes with multiple adhesive proteins on their surface, exosomes are known to specialize in cell–cell communications and provide an exclusive approach for the delivery of various therapeutic agents to target cells. for example, researchers used exosomes as a vehicle for the delivery of cancer drug paclitaxel. they placed the drug inside exosomes derived from white blood cells, which were then injected into mice with drug-resistant lung cancer. importantly, incorporation of paclitaxel into exosomes increased cytotoxicity more than 50 times as a result of nearly complete co-localization of airway-delivered exosomes with lung cancer cells. unapproved marketing different forms of unproven exosomes are being marketed in the u.s. for a wide variety of health conditions by clinic firms, without authorization from the fda. often, these firms also sell non-fda-approved stem cell injections as well. in late 2019, the fda issued an advisory warning about noncompliant marketing of exosomes and injuries to patients in nebraska related to injections of exosomes. the agency also indicated that exosomes are officially drug products requiring pre-market approval. in 2020, the fda cautioned several firms about marketing or use of exosomes for covid-19 and other health conditions.