The overall goal of this method is to isolate exosomes from human bile for further analyses including microRNA profiling. This method can help answer key questions in the liver disease field such as detection of a particular bile marker for cholangiocarcinomas or other biliary diseases. The main advantage of this technique is that this is robust and repeatable.
To perform endoscopic retrograde cholangiopancreatography, or ERCP, begin by placing the patient in a supine position. After intubating and passing a duodenoscope through the mouth of the patient, insert it into the second part of the duodenum and identify the major papilla. Selectively cannulate the common bile duct by inserting a triple lumen sphincterotome preloaded with a 0.35-inch or a 0.9-millimeter hydrophilic guidewire.
Under fluoroscopic guidance, advance the guidewire to the left hepatic duct then advance the sphincterotome five centimeters into the bile duct to acquire a stable position. After removing the guidewire, attach a 10-millimeter syringe to the guidewire port through the lower lock and use 10 milliliters of negative pressure to aspirate the bile. Remove the syringe containing the bile and after reinserting the guidewire, inject contrast agent through the injection port to opacify the biliary tree.
Then, transfer the collected bile into a 15-milliliter tube on ice and if storing for a later time, centrifuge the sample at 500 times g and four degrees Celsius for 10 minutes before freezing at minus 80 degrees Celsius. To immediately proceed with exosome isolation, transfer 400 microliters of bile into a microcentrifuge tube and centrifuge at 300 times g and four degrees Celsius for 10 minutes to collect cells and cell debris. To further clear the supernatant, transfer the solution to fresh microcentrifuge tubes and centrifuge the samples at 16, 500 times g and four degrees Celsius for 20 minutes.
Then in a biosafety cabinet, collect the supernatant into a syringe and filter it through a 0.2-micrometer polyethersulfone membrane filter to remove any remaining particles larger than 200 nanometers. Transfer the filtered supernatant to ultracentrifuge tubes and use PBS to bring the tubes up to at least two-thirds full to prevent the tubes from collapsing during centrifugation. Pellet the exosomes through ultracentrifugation at 120, 000 times g and four degrees Celsius for 70 minutes.
A small, yellow pellet will contain the exosomes. Discard the supernatant by carefully decanting and disinfect the waste by adding bleach to a final concentration of 10%volume per volume. Then let it stand for 20 minutes.
For downstream experiments, either process the pellets in a small volume of appropriate solution or re-suspend them in 50 to 100 microliters of PBS and store at minus 80 degrees Celsius for future use. After preparing grids of contrast-stained extracellular vesicles, or EVs, according to the text protocol, acquire images with an electron microscope using an acceleration voltage of 80 kilovolts starting at magnifications of 20, 000 X, and increasing to 100, 000 X, when determining the size of the particles. Exosomes are too small to be detected by regular microscopy or flow cytometry;however, this figure shows the results of a typical nanoparticle tracking analysis, or NTA, of exosomes that measure the concentration and size distribution, which averaged 97 nanometers in this isolated human bile sample.
Electron microscopy can also be used to confirm the size of the isolated particles in human bile and this figure shows a typical transmission EM result. These western blots were probed for proteins enriched in exosomes such as the tetraspanin CD63 and Tsg101. This real-time amplification plot shows a variety of microRNA species extracted from exosomes of human bile.
Since exosomes lack reliable standards like 18S or 28S ribosomal RNA or housekeeping genes, the spiking of a synthetic microRNA is important for normalization. the exos isolation technique can be done in three hours if it is performed properly. After watching this video, you should have a good understanding of how to obtain and process bile from patients or animals to isolate and analyze extracellular vesicles.
While recanting this procedure, it is important to remember to centrifuge the sample at 500 G and four degrees before freezing at minus 80, if you can't proceed with ultracentrifugation. Following this procedure, other methods like microRNA studies can be performed in order to answer additional questions like microRNA profiles. Don't forget that working with bile can be extremely hazardous since it could contain hepatitis, and precautions such as wearing gloves and safety glasses should always be taken while performing this procedure.
After its development, the technique paved the way for research in the field of cholangiocarcinoma research to explore extracellular vesicles as potential biomarker in bile from patients with biliary problems such as obstruction of PSA.
