Our research focuses on small vesicles produced by a type of immune cells that are naturally effective at killing cancer cells. These vesicles hold promising therapeutic potential for treating solid tumors, both at the primary and secondary sites such as the brain. In this study, we use a bimodal resin to isolate vesicles via size exclusion chromatography, maximizing product purity by physically separating the vesicles from contaminants while further trapping contaminants within the resin through electrostatic forces.
We then sterilize, concentrate, and buffer exchange the vesicles before use The biomanufacturing workflow details in this video uses clinically relevant conditions and is scalable for cost, effectively producing large quantities of high purity extracellular vesicles. This makes the technique accessible for academic labs to confidently invest resources in studying such therapeutics with a reliable production and isolation process. We're currently investigating the therapeutic potential of these vesicles and preclinical models and further strengthening our understanding of how they work.
To begin, perform fast protein liquid chromatography system initiation according to the manufacturer's instructions. Make connections using the drip-to-drip method to ensure no air is introduced inside the column. Set up the fraction collector with appropriate collection tubes to collect the samples.
Change the fractionation settings to the desired collection volume. Remove 40 to 80 milliliters of previously prepared extracellular vesicle or EV-rich conditioned media from the 80 degrees Celsius freezer and thaw quickly at 37 degrees Celsius. Spin the sample at 10, 000 G for 30 minutes at four degrees Celsius and transfer the supernatant to a new tube.
To reduce double stranded DNA levels, treat the EV-rich conditioned media with five millimoles of magnesium chloride and 50 units per milliliter of endonuclease. Incubate the sample for two to four hours at 37 degrees Celsius with moderate mixing. Once the chromatography system is ready, load the EV-rich conditioned media into a 60-milliliter syringe and connect it to the sample line.
Click Manual Run to start the system and set the flow rate to zero. Follow the software prompts to save the run preemptively. Then click Start.
Select Line Beat Double-Filtered PBS and run at a flow velocity of two milliliters per minute, ensuring the solution runs through the column. Once the absorbent stabilizes, press Auto Zero UV.Change the flow path to direct the sample to the waste bottle before the column. After five to 20 seconds, direct the sample to the column.
Once the UV readings reach approximately 230 milliabsorbance units, click Fractionation. Upon complete injection of the sample, switch the buffer system over to Double-Filtered PBS to continue purification. When the UV value reaches approximately 1, 600 milliabsorbance units, again, click Fractionation.
Once the run has stopped, save the chromatogram as a PDF document. Rinse every component of the ultra filtration apparatus with 20 to 30 milliliters of 90%ethanol. Spin at 4, 000 G for five to 10 minutes and discard the flow through.
Then perform the rinse with sterile PBS to equilibrate the device and repeat the centrifugation steps as before. Combine all diluted fractions of NK-EVs. To maximize sterility, filter the diluted NK-EV solution using a 0.22-micrometer syringe filter, pre-wet with double-filtered PBS.
Collect the filtrate into the sterilized concentration apparatus. Spin the filtrate at 4, 000 G for 15 to 40 minutes. After spinning, mix the solution within the top filter compartment using a serological pipette.
Again, spin at 4, 000 G for 10 minutes. Invert the filtration device and attach it to the collecting device to collect the NK-EV. Spin the NK-EV at 2, 000 G for two minutes and transfer the purified product to a sterile tube.
Initiate the nanoparticle tracking analysis system according to the manufacturer's instructions. Observe the flow cell for air bubbles. If an air bubble is present, remove it by rinsing the flow cell with 20%ethanol and double-filtered water.
Once clear, carefully reinsert the laser module into the nanoparticle tracking analysis instrument. Close the door and click Start Camera. Change capture settings to a screen gain of two and a camera level of 14.
Turn on the heater to temperature stabilize the flow cell. Click Standard Measurement to create a script under the SOP tab for collecting one capture over one minute at a flow rate of 30 at 23 degrees Celsius. Add the folder and file name to the pathway name to save the data.
Vortex the purified NK-EV product and prepare their dilution using double-filtered PBS. After vortexing the diluted sample, load it into the 1-milliliter acquisition syringe without introducing air bubbles. Carefully connect the syringe to the instrument loading line.
Slowly push half of the sample, leaving approximately 0.5 milliliters in the syringe. Once particles are visible on the screen, focus the camera to have a maximum of one halo around each particle, Under the Hardware tab, click Infuse and set a rate of 1, 000 for five seconds. Then bring the rate down to 30.
Press Run Script. After ensuring the settings are correct, click Yes and follow the software prompts. After completing the capture, click Cancel when the software asks to process or export files.
Upon recording five captures per dilution, import all five captures for analysis. Highlight the files and click Process Selected Files. Under the Process tab, adjust the analysis settings to a screen gain of two and a detection threshold of 15.
Check and click OK for analysis. After processing, click Yes without clicking additional boxes or click Export to export the files. Purified NK-EV particle size ranged from 76.30 to 174.30 nanometers with an average concentration of 1.39 times 10 to the power of 12 EVs per milliliter.
Additionally, fluorometer quantification showed a protein concentration of approximately 300 milligrams per milliliter and a double-stranded DNA concentration of 225 milligrams per milliliter. Human K562 leukemia cell treatment with NK-EVs showed a dose dependent decrease in cell viability with an EC 50 of 9.33 times 10 to the power of nine EVs per milliliter.
