The storage stability of extra cellular vesicles is an important parameter for their prospective therapeutic use. Our protocol provides a readily applicable tool for evaluating how storage effects the vesicles. The main advantage of our technique is that by introducing glucuronidase as an exogenous marker, extracellular vesicles derived from different parent cells an be easily compared regarding their storability.
Asymmetric flow field flow fractionation, or AF4, is a mild alternative to size exclusion chromatography purification for very sensitive compounds. Because the compounds encounter less sheer stress in the channel. As glucuronidase is a sensitive enzyme, the quality of the resides benefits from thorough planning and carrying out the steps for purification and analysis back to back.
After culturing the cell line of interest according to standard conditions for those cells, replace the supernatant with serum free or extra cellular vesicle depleted medium and return the cells to the cell culture incubator for an additional 24 to 72 hours. At the end of the incubation, collect the super natant from each flask and centrifuge the samples to sediment the cells. Carefully collect the cell conditioned medium without disturbing the pellet.
And centrifuge the medium again to remove cell debris and large aggregates. Then carefully transfer the supernatants into ultracentrifuge tubes. If using a fixed angle rotor, mark the orientation of the tubes in the centrifuge to facilitate the retrieval of the extracellular vesicle pellet after the centrifugation.
At the end of the ultracentrifugation, use a serological pipette to carefully discard the supernatant without disturbing the extracellular vesicle pellet. At 200 microliters of 0.2 micrometer pore filtered PBS to the residual supernatant of the first ultracentrifuge tube. After resuspending the pellet, transfer the resulting extracellular vesicle suspension to the next tube for resuspension of the subsequent pellet until all of the extracellular vesicle pellets have been resuspended.
Add beta glucuronidase to the final resuspended pellet solution to a final concentration of 1.5 milligrams per milliliter. And saponin to a final concentration of 0.1 milligrams per milliliter. Then mix well by vortexing for three seconds and place the reaction at room temperature for 10 minutes with intermittent gentle flicking.
Once the glucuronidase has been encapsulated, be sure to perform all of the subsequent purification vesicle evaluation steps on the same day to obtain unbiased storage stability results. Have a size exclusion chromatography column equilibrated with at least two column volumes of PBS ready to purify the pellet immediately after the saponin incubation. To purify, add up to 400 microliters of extracellular vesicle suspension to the column.
Then collect one milliliter fractions of the eluate. To conform the separation of the extracellular vesicles from the contaminating proteins and free glucuronidase, measure the protein concentration by bicinchoninic acid assay according to the manufacturers instructions. Using parameters optimized for the respective instrument and vesicle type, use a nano particle tracking analysis instrument to measure the extracellular vesicle size and concentration.
To measure the glucuronidase activity, add 25 microliters of freshly prepared fluorescin di-beta-D-glucuronide to 125 microliters of the purified extracellular vesicles and add the sample to one well of a black 96-well plate. Measure the time zero fluorescin production on a plate reader at a 480 nanometer excitation and 516 nanometer emission. Cover the plate tightly with transparent plastic foil to reduce evaporation.
Then incubate the plate protected from light for 18 hours at 37 degrees Celsius. Measure the 18 hour fluorescin production on the plate reader as just demonstrated. For extracellular vesicle lyophilization, add four milligrams per milliliter of trehalose to the purified vesicles.
And freeze the vesicles at minus 80 degrees Celsius for at least one hour. To lyophilize the samples, set the shelf temperature of a lyophilizer to 15 degrees Celsius and the pressure to 0.180 millibars. Dry the vesicles under these conditions for 46 hours.
At the end of the main drying step, set the shelf temperature to 25 degrees Celsius and the pressure to 0035 millibars and dry the samples for two hours under these conditions. Then store the lyophilized samples at four degrees Celsius. To rehydrate the samples after storage, add a volume of water equal to the volume of the extracellular vesicle suspension prior to the lyophilization.
To assess the enzyme activity after storage, load an AF4 instrument with freshly prepared 0.1 micrometer filtered PBS for the mobile phase and insert a 0.1 micrometer filter membrane into the peak inlet filter after the pump to reduce particle noise from the solvent. After disassembling, clean the small channel for AF4 with millEq water. Hydrate a new regenerated cellulose membrane with a molecular weight cut off of 30 kilodaltons and place the membrane on top of the fret with the shiny side up.
Place a wide 350 micrometer spacer below the top half of the channel and assemble the channel parts. Using a torque screwdriver, tighten the screws first to a torque of five Newton meters, then to a torque of seven Newton meters. Tightening the screws around the block in a crisscross pattern.
Connect the channels inlet, outlet, and cross flow port to the eclipse. Then from at least three old samples to equilibrate the membrane. Program a fractionation run with the detector flow and focus flow set to one milliliter per minute and an inject flow of 0.2 milliliters per minute.
After a one minute pre-focus step, inject the sample over a period of 10 minutes in the focus and inject step, before decreasing the cross flow from two milliliters per minute to 0.1 milliliters per minute over the course of eight minutes. Finish the fractionation run with an elution step without cross flow for 10 minutes and add a washing step of elution and injection, followed by an elution. Then equilibrate the channel for the next run with an initial cross flow of two milliliters per minute and set the UV detector to 280 nanometers.
When all of the programs have been set, inject 300 microliters of the sample and record the UV and light scattering signals in the ASTRA software. After 12 and a half minutes, begin collecting one milliliter fractions until 27 minutes post injection. Then perform a glucuronidase assay and a nano particle tracking analysis as demonstrated.
Here, the particle recovery and the size of the extracellular vesicles isolated from human vascular endothelial cells after seven days of storage under different conditions are shown in comparison with a fresh sample. Vesicles were subsequently subjected to AF4 purification and the glucuronidase activity per particle was measured. Both size exclusion chromatography and AF4 are successful in separating extracellular vesicle from free glucuronidase.
Due to the higher degree of separation for AF4 between particles and free enzyme, contamination of the fractions containing vesicles is less probable. AF4 purification after storage removes free enzyme from the samples and thus lowers the amount of recovered enzyme activity per particle. This effect is most prominent after storage at four degrees Celsius, for which a 2/3 reduction is observed.
Omitting this additional purification step can lead to wrong assumption about the enzyme stability. Transmission electron microscopy does not reveal big differences in shape for extracellular vesicles lyophilized without a cryoprotectant. Scanning electron microscopy imaging, however, reveals the presence of aggregates within the lyophilized samples that are not found in the minus 80 degrees stored samples.
It is crucial that free enzyme is removed from the vesicles before the glucuronidase assay. Thus, the technique used for vesicle purification needs to be thoroughly evaluated. The purified particles could also be looked at in terms of their surface charge to find out whether the storage changed the natural composition of the membrane proteins or sugars.
With our technique, it is possible to get a first indication about extracellular vesicle storage stability, even when there are no known specific markers or assays for the activity available.
