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Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at −80 °C.
Like other biological fluids, i.e., breast milk, plasma or urine, bile contains exosomes, lipid rich vesicles1–4. Exosomes can induce or alter biological functions in recipient cells5, 6, a form of cell-cell communication that might be part of their normal function4, 7–9. Exosomes can contain miRNA species which can provide a valuable source of biomarkers for diagnosis10. miRNAs profiles have gained substantial attention in recent years as diagnostic and prognostic markers for a variety of diseases11–14.
miRNA itself can be found in biological fluids, possibly released by dead cells and the amount of shed cells can be greatly influenced by an underlying disease. The miRNA profile of exosomes does not necessarily reflect the miRNA profile of the originating cell6, 10, 15–17, yet exosomes may carry miRNA signatures that might be characteristic for the parental cell like a tumor cell. Tumor-derived exosomes have been identified in the plasma of patients with lung adenocarcinoma, prostate cancer and other tumors8, 16, 18.
The goal of this method is the reliable and robust isolation of exosomes from human bile specimens, largely independent of their prior handling procedures. It was developed to utilize bile as a reliable source of miRNA for the potential diagnosis of diseases of the bile duct such as cholangiocarcinoma19. It consist of several steps of centrifugation with increasing speeds to isolate exosomes and might be applicable to other biological fluids.
Obtaining the bile from patients by endoscopic retrograde cholangiopancreatography (ERCP) requires the approval of a human subjects study protocol by the Institutional Review Board. All work presented here was approved by the Johns Hopkins University Institutional Review Board.
Note: Isolation of miRNA from bile exosomes is carried out using a modified miRNA isolation based on a commercially available kit, but any other RNA isolation method can be used or adapted.
Since exosomes are too small to be detected by regular microscopy or flow cytometry, electron microscopy or nanoparticle optical analysis has to be performed. The nanoparticle optical analysis has the advantage over electron microscopy that it is also quantitative and provides size distribution and concentration. The instrument introduces a finely focused laser beam through a glass prism into the sample. The Brownian motion of the isolated vesicles is captured via an EMCCD high sensitivity camera and tracked on a frame-by-frame basis. The nanoparticle tracking analysis (NTA) software measures this Brownian movement frame to frame to calculate the size, mode and distribution of the bile exosome preparations. Figure 1 shows the result of a typical NTA analysis of exosome isolated from a human bile sample.
Alternatively, electron microscopy can be utilized to confirm the size of the isolated particles, albeit without quantification. Figure 2A shows the typical result of transmission electron microscopy for exosome isolated from human bile.
For further verification of the exosomal nature of the isolated particles, Western blots probing for the presence of proteins like Tsg101 or tetraspanin CD63, shown in Figure 2B, have to be used. Exosome-specific proteins do not exist per se, but exosomes are enriched in tetraspanins, especially CD9, CD63, CD81 and CD82 with CD63 and CD81 referred to as classical exosome markers, and proteins involved in exosome formation like TSG101 and Alix21. Other proteins like heat shock protein cognate 70 (Hsc70) and 73 (Hsc73), as well as major histocompatibility complex (MHC) class II molecules can also be found enriched in exosomes with some like Hsc73 and the peripheral membrane-associated protein milk fat globule - epidermal growth factor -factor 8 (Mfge8) being quite specific for exosomes secreted from dendritic cells21.
Figure 3 shows the typical real-time amplification plots of a variety of miRNA species extracted from exosomes of human bile. Since exosomes, unlike cells, lack reliable standards like 18S or 28S rRNA or housekeeping genes like GAPDH or β-actin for normalization, the spiking with a synthetic miRNA is important. The synthetic miRNA should be derived from a different species like cel-miR 39 from Caenorhabditis elegans to enable one to normalize the expression levels effectively.
For reproducibility it is critical that the bile is processed as soon as possible and not frozen prior to processing as these conditions lead to degradation of miRNAs present in bile. On the other hand, once isolated, exosomes are very stable and quite resistant as storage at RT for 48 hr or up to three freeze-thaw cycles cause negligible effects on the levels of at least two species of miRNA, although the stability for the miRNA of interest has to be determined empirically.
To reliably utilize exosomes isolated from bile, it is important to employ consistent high quality isolation methods to obtain high quality samples in return. The methodology defined in this paper is a well-established way to isolate exosomes and miRNA from human bile. It highlights several crucial steps in the characterization of the isolated exosomes which at a minimum should comprise electron microscopy or nanoparticle optical analysis and Western blots.
The most crucial step in the isolation of exosomes, at least when it comes to miRNA stability, is to process fresh bile as soon as possible. Prolonged storage of whole bile at RT or even a single freeze-thaw cycle can significantly reduce the miRNA content while in contrast the isolated exosomes are very stable even when stored at RT or undergoing several freeze-thaw cycles. As long as the samples in question have been stored at −80 °C, successful isolation of exosomes and miRNA from bile can be performed with great reproducibility to identify miRNA signatures in the samples. It is recommended to initially start with fresh bile to ensure proper miRNA integrity. This is an important consideration when attempting to build up a collection of bile samples over time as would be the case for patient samples. It enables one to process samples that have been collected over months or even years to be processed and evaluated at the same time.
This greatly enhances the use of bile for diagnostic purposes, either to look for miRNA signatures that confirm the presence of a disease state like cancer or the response of a disease to therapeutic interventions. In fact, results from clinical samples have been used to establish miRNA signatures as biomarkers for cholangiocarcinoma19.
The first centrifugation step removes intact cells that are present in bile. In the second, higher speed centrifugation cell debris, apoptotic bodies and other larger organelles are pelleted. To ensure that only particles smaller than 200 nm are collected in the ultracentrifugation step, the filtration step with a low protein binding filter is important. Some protocols omit this step, but we think it is necessary. After the ultracentrifugation at 120,000 × g the pellet obtained contains fairly pure exosomes that can either be processed immediately or after resuspension in PBS be stored at −80 °C. A limitation of this method is that the pellet obtained is only highly enriched in exosomes but this is true for other enrichment methods such as size exclusion and polymeric precipitation. Further processing might entail another purification step by sucrose gradient or binding to antibody-coated magnetic beads. If the source of the exosomes (like for example plasma) contains precipitates as a result of clotting, this extra purification might be necessary as these particulates can often have the size of exosomes. In particular, the immuno-purification leads to a more exosome marker enriched preparation. The disadvantage is that this further reduces the amount of exosomes and/or miRNA present, and for most purposes does not justify the time and resource consuming process. If, however, Western blot analysis detects the presence of microsome contamination through an endoplasmic reticulum protein such as calnexin, further purification, in particular immuno-isolation based, is advised. In our experience this is rarely the case for bile, if one uses other sources of biological fluids an immune-based purification step would be recommended.
The use of differential ultracentrifugation to isolate and enrich exosomes from bile fluid is a relatively fast and cost-effective method. While it requires the use of an ultracentrifuge, preferably with a swing bucket rotor, these devices are commonly found in many institutions. Further purification via density centrifugation are possible with the same equipment and the tubes are reusable several times after cleaning and sterilization. While polymer-based precipitation requires only the use of microcentrifuges or standard centrifuges to pellet the precipitates at speed of 10,000 × g or less, it generally co-isolates non-vesicular contaminants including lipoproteins. While lipoproteins are not generally present in bile, the cost of the often proprietary and patented polymers make the isolation expensive in comparison. The polymers also are sometimes incompatible with downstream applications such as mass spectrometry, but when downstream analysis is compatible with the polymer (RNA or protein isolation), the method is easy, fast and does not require specific equipment.
Size exclusion chromatography has the downside of long running times and the requirement of special equipment, but allows the precise separation of large and small particles. Immunoaffinity purification yields the highest purity of exosomal vesicles and even allows the isolation of specific exosomal fractions such as epithelial derived, but this comes at the cost of total yield. Furthermore, it is limited to small samples volumes requiring a first enrichment step if a large volume is to be processed and the isolated exosomes may lose functional activity or exhibit altered biological function due to the bound antibodies.
It is important to consider the downstream application(s) and availability of specialized equipment when it comes to the isolation of exosomes. The characterization of the isolated/enriched particles is important regardless of the isolation method used. Ultracentrifugation has so far been a “gold standard” used by many laboratories due to its robust and fast (and cost-effective if an ultracentrifuge is available) enrichment of exosomal particles.
This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).
The video component of this article can be found at http://www.jove.com/video/54036/
None of the authors have competing financial interests.