The isolation and identification of exosome subsets in human blood plasma and serum
Introduction
Exosomes are nanosized cell-derived vesicles which can predominantly be retrieved from many extracellular body fluids (ECF), including human blood plasma, serum, and urine (Edgar, 2016).
Their main roles consist of facilitating cell to cell communication, protein transportation, and antigen presentation. This involves the transportation of the molecules encapsulated in the exosomal lipid bilayer membrane including; nucleic acids, lipids and proteins from their unique cells of origin to their recipients. Furthermore, this intercellular communication impacts physiological processes and recent research from Andaloussi et al. exploits the potential of extracellular vesicles (EV) including exosomes in inducing both local and systemic changes in the body, thus having the capability to promote disease (Andaloussi, Breakefield and Wood, 2013).
These nanovesicles, which are the smallest among secreted EV (approximately 30-150nm in diameter) have introduced the potential of exosome-based biomarkers in the monitoring, prognosis, and diagnosis of cancer, neurodegenerative disease and CVD and has successfully been used in the detection of ovarian, lung and pancreatic cancers (Inamdir and Rege, 2017). This combined with their quick and easy access in body fluids, along with their ability to cross biological barriers and reflect the progression and stages of disease opens many exciting opportunities for their use in both laboratory research and clinical medicine (P. Li et al., 2017).
Further advantages of exosome research include their fairly non-invasive retrieval over long periods of time which favors/allows continuous monitoring. Moreover, exosomes are fairly abundant in extracellular fluids as well as durable, allowing them to maintain their integrity through several freeze and thaw cycles which aids in their potential use as biomarkers.
Upcoming research sheds light on the possibility of exosomes as a drug delivery system. This exploits the long half-life in circulation, low immunogenicity and ability to cross the blood-brain barrier and biological barriers properties of exosomes which could aid in the treatment of various cancers in the future (Y. Li et al., 2017).
There are currently various methods of separating exosomes based on their sizes and content. Recent research by Li et al. discovered the two main exosome subpopulations, Exo-L (90-120nm) and Exo-S (60-80nm) in human ECF (Li, Yao and Kaslan, 2017).
The main issue impeding the advancement of exosome research is the lack of standardized methods for exosome retrieval. Each method has its advantages and disadvantages for exosome isolation and have been discovered to retrieve vesicles of various populations and characteristics which only reduces their purity and usability when isolating from complex biological fluids (Lobb et al., 2015).
Although the debate for exosome subpopulation isolation is ongoing, the current gold standard method of recovering exosomes from samples is ultracentrifugation. However, this technique has been suggested/reported to be expensive, time-consuming, produce exosomes of reduced quality and yield and dependant on the skill of the operator which reduces reproducibility. Further studies also indicate that the high centrifugal forced used in this method (approx. 100,000-200,000 x g) promotes exosomal fusion, coagulation and can damage structures of exosomes, thus impacting analysis of populations (Wu et al., 2017). Isolation of exosomes is a complex task due to the lack of definition of exosome subpopulations, hence can cause an overlap in their size and density. In addition, the high costs involved, and the time required to enrich and purify EV in large volumes only adds to the problem (Yanez-Mo et al., 2015)
The aim of this project is to determine whether Size Exclusion Chromatography (SEC) will permit the separation of the two exosome fractions from human plasma and serum. Both of these are derived from full blood but undergo different biochemical processes. Blood serum has a similar composition to plasma but is from blood that has coagulated with fibrin clots and excludes clotting factors and blood cells using centrifugation. Plasma is a blood medium which consists of red and white blood cells along with various other components including albumin and globulin (He et al., 2011).
Moreover, after assessing the exosome yield from SEC, we will compare this with the yields obtained from the Exo-spin and PEG methods of isolation.
Polyethylene glycol (PEG) is a form of polymer facilitated precipitation which uses a neutral pH to isolate exosomes. Research suggests the technique may lead to the contamination of the populations with non-exosomal materials. Moreover, despite the widespread use of the technique and reagents, minimal research has been released on the quality and quantity of harvested exosomes in comparison to other methods of isolation (Rider, Hurwitz and Meckes, 2016). Hence this method will be compared with SEC which focuses on separating macromolecules and exosomes using minimal shear force resulting in reduced vesicular damage and deformation (Munagala et al., 2016).
Although exosomes are highly abundant in a wide range of biological fluids, the challenge lies with overcoming the limited methods of isolating purified exosome populations of a high yield. This is partly due to their biophysical properties overlapping with other secreted cellular products. Various commercial kits for exosome harvesting are popular in today's research, however, reports of their low purity and high costs restrict their utility. Nevertheless, we will investigate the method of Exo-spin kits and see how it compares to other common methods of isolation.
Additionally, the exosomes isolated using these three techniques will be suspended in phosphate buffer solution and analyzed for particle number and size distribution using Nano-tracking analysis. This has demonstrated to be a reproducible and highly sensitive method to visualize and analyze the number and size of exosomes using light and particle movement (Mehdiani et al., 2015).
The method of western blot will then be used to characterize the populations further, in terms of their protein content and specific biomarkers and the presence of contaminating lipoprotein particles in the exosome fractions will be assessed using an LDL and HDL quantification kit.
Methods
Plasma preparation
Plasma and serum samples were obtained from the blood bank and then centrifuged, stored in aliquots of 250ml and frozen at -80°C until needed/required.
Exo-spin isolation:
Prior to the Exo-spin isolation technique being undertaken, the blood serum/plasma samples were defrosted and spun at 20,000 x g for 10 minutes in order to remove any cell and cell debris. The supernatant was then transferred to a second microtube and spun again at 20,000 x g for 30 mins to remove any remaining cell debris. 400μL was measured from the 250ml aliquots and ½ of this volume of buffer A added, in this case, 200μL. Buffer A appears as a thick, viscous texture to aid in binding to water molecules and separate the exosomes during centrifugation. The sample was then incubated at 4 °C for 5 minutes. Research suggests that the incubation time may be increased up to 60 minutes in order to enhance the exosome yield, however, in this study the incubation time was kept at 5 minutes. The samples were centrifuged using Labogene at 20,000 x g for 30 minutes, ensuring prior to each spin that the centrifuge is correctly balanced.
A pipette was then used to carefully remove and discard the supernatant, leaving the exosome containing pellet remaining at the base of the tube undamaged. The exosome containing pellet was then resuspended in 100 μL of Phosphate Buffer Solution (PBS) which aids in gently dissolving the exosomes without causing their lysis. When discarding the supernatant, it was important to immediately add the PBS in order to prevent the exosome containing pellet to dry off and become damaged.
Next, a vortex machine was used for 15-30 minutes until the sticky pellet dissolved into a yellow solution.
The spin column was prepared in order to purify the exosomes. After removing the screw cap and outlet plug, the spin column was placed into a waste collection tube and spun briefly at 50 x g for 1 minute in order to draw out any PBS solution stored in the column. The eluate was discarded. 200μL of PBS was added to the top of the column and centrifuged again at 50 x g for 2 minutes. If any PBS remained at the top of the column filter after centrifugation it was carefully removed using a micropipette and discarded with the remaining eluate. Next, 100μL of the exosome pellet solution was pipetted to the top of the column and centrifuged for 2 minutes at the same speed of 50 x g and the eluate discarded.
The waste collection tube was replaced with a collection tube and 200 μL of PBS was pipetted to the top of the column and centrifuged again at the same speed for another 2 minutes. This allowed the exosome sample to be pushed through the column and the eluate containing the purified exosomes.
If the solution did not completely pass through the spin column during the centrifugation then the tube was recentrifuged for a further 2 minutes at 150 x g. This allows the sample to fully pass through the spin column and gain a high exosome yield.
This Exo-spin method was repeated to obtain 4 serum samples and 4 blood plasma samples.
The purified exosome samples were each labeled and stored at -80°C and the columns placed at 4°C in the fridge.
Nanosight Analysis
Once the exosome samples were extracted using the three methods (Exo-spin, SEC and PEG), it was necessary to measure the size and concentration/amount of exosomes per each sample. This was conducted using Nanosight analysis/Nanotracking analysis (NTA) machine which takes advantage of light and particle movement to visualize and analyze exosomes in a highly sensitive manner (Mehdiani et al., 2015)
Each sample prior to analysis was defrosted and brought to room temperature. Each 200 μL was divided in half, resulting in two 100 μL samples. One half to be used for the nanosight analysis and the second half to be used in the Western Blot procedure. Approx. 50ml of PBS was used to prime the machine prior to analysis and the nanosight was adjusted. The field of view was checked using a camera, this ensured no… and prevented contamination.?
Then dilutions were made using the exosome sample and PBS solution. This is demonstrated in the table below. It is important to vortex each sample before and after the addition of PBS to ensure proper mixing.?
The diluted sample was loaded onto the machine and the field of view was checked again, this time at the highest camera brightness setting. The focus adjustment was used to find a balance between the particles, this was vital as our sample was polydisperse which meant it included various different samples of exosomes. The camera was then turned low until the dullest particle could be seen and the SOP tab was used to create and export 4 videos each lasting 30 seconds for our samples. The 30 second videos are long enough to capture the particle movement and not too long that the particles agglutinate to the side of the walls. The detection threshold was adjusted between each sample which allowed for a more accurate exosome measurement by avoiding the measurement of background particles in the sample.?. The ideal number of particles per frame allowed the most accurate size and concentration of exosomes were approximately 20 to 60. If the number of particles per frame exceeds this by a great amount, then the sample can be diluted further.
Before analyzing the next sample, the machine is flushed with PBS and the field of view is checked to be clear. If after measuring a sample, the field of view is not clear then flushing with deionized water and PBS again may solve this. A background PBS check can also be conducted. This is completed by measuring the particles in the PBS sample being used and subtracting the number of particles measured in the PBS from the number of particles in the plasma or serum sample being measured. This would take into account any particles in the PBS which could affect or alter results and acts as a control.
(add each method, sample and dilutions table)