Preparation of Exosomes for siRNA Delivery to Cancer Cells

Harvesting exosome-enriched conditioned medium from cells cultured in bioreactor flasks and isolation by sucrose cushion ultracentrifugation result in exosome preparation of high yield with minimal contaminating proteins or non-exosomal vesicles. Using a single sucrose cushion prepared in deuterium oxide circumvents the laborious preparation of a discontinuous sucrose gradient and reduces the amount of sucrose needed to achieve the required density. Effective in vitro delivery of siRNA encapsulate in exosomes prepared in this protocol highlights the potential of this system as a new generation RNA interference-based therapy for pancreatic cancer.

To begin, culture HEK-293 cells in normal medium, as described in the manuscript. Expand the cells into four T75 flasks, and grow until they reach 90%confluency. To prepare a bioreactor flask, add 50 to 100 milliliters of normal medium into the medium reservoir of the bioreactor flask to wet the membrane.

Collect all HEK-293 cells from the four T75 flasks, and resuspend them in 15 milliliters of exosome-depleted medium in a centrifuge tube. Then, use a 20-milliliter syringe connected to a blunt fill needle to carefully add this cell suspension to the cell compartment of the bioreactor flask. Then, fill the medium reservoir of the bioreactor flask with the normal medium, up to 500 milliliters, and incubate it at 37 degrees Celsius, 5%CO2 for a week.

After one week of incubation, remove all the medium from the medium reservoir of the bioreactor flask by pouring it out. Using a 20-milliliter syringe connected to a blunt fill needle, remove all the medium from the cell compartment. Then, add 50 to 100 milliliters of normal medium to the medium reservoir and 15 milliliters of fresh exosome-depleted medium to the cell compartment using a 20-milliliter syringe connected to a blunt fill needle.

Then, fill the medium reservoir of the bioreactor flask with normal medium up to 500 milliliters. Incubate at 37 degrees Celsius, 5%CO2 for another week. To pre-clear the collected conditioned medium, centrifuge it at 500 times g for five minutes at four degrees Celsius.

Transfer the supernatant into a new tube, and discard the pellet. After repeating this centrifugation step with the recovered supernatant, recover the supernatant again and discard the pellet. Then, centrifuge the recovered supernatant at 2, 000 times g for 15 minutes and four degrees Celsius.

Keep the supernatant, and discard the pellet. Filter the supernatant once through a 22-micrometer filter attached to a 20-milliliter syringe into a fresh centrifuge tube. Meanwhile, to prepare a 25%sucrose solution in deuterium oxide, accurately weigh out 1.9 grams of sucrose in a universal tube.

Then, add deuterium oxide until the weight reaches 7.6 grams. Then, fill up an ultracentrifuge tube with 22.5 milliliters of pre-cleared conditioned medium. Place a glass pipette in the tube.

Add three milliliters of sucrose solution through the pipette so that the solution forms a separate layer beneath the conditioned medium. Carefully place the tube containing layered conditioned medium/sucrose solution into the bucket of a swing-out rotor. Secure the bucket into the rotor.

Place the rotor into the ultracentrifuge, and spin at 100, 000 times g at four degrees Celsius for 1.5 hours. Collect two milliliters of the sucrose layer from the tube, one milliliter at a time using a P1000 pipette, with its tip attached to a 10-microliter tip, and add it to an ultracentrifuge bottle containing 20 milliliters of filtered PBS for washing. Place the tube into a fixed-angle rotor, and centrifuge it at 100, 000 times g at four degrees Celsius for 1.5 hours.

Use a 10-milliliter serological pipette to carefully remove the supernatant. Resuspend the pellet with 400 microliters of filtered PBS. Before starting the electroporation, pre-chill the electroporation cuvette on ice for 30 minutes.

Mix seven micrograms of exosomes with 33 micrograms of siRNA in the microcentrifuge tube. Add citric acid buffer to achieve the volume of 150 microliters. Add the exosome-siRNA mixture to the electroporation cuvette using a plastic pipette, and cap the cuvette.

Place the cuvette in the right orientation in the cuvette holder of the electroporator, and rotate the turning wheel of the electroporator 180 degrees clockwise. Select the desired program, and press the Start button to start electroporation. The display will indicate a successful pulse.

Then, turn back the wheel 180 degrees counterclockwise, and remove the cuvette. Use a plastic pipette to remove the sample from the cuvette into a new microcentrifuge tube. Keep the tube on ice or in a fridge before further processing, if not used immediately.

To start free siRNA removal, pass 3.5 milliliters of filtered PBS through the size exclusion chromatography column twice to equilibrate it. Then, dissolve 150 microliters of electroporated sample in 350 microliters of filtered PBS, and transfer it to the SEC column. Collect the first 500-microliter fraction eluted from the column into a microfuge tube, and mark it as F0.Then, add 500 microliters of filtered PBS to the column.

Collect the next fraction, and mark it as F1.Repeat adding the PBS and collecting elution fractions until a total of 10 500-microliter fractions, or up to F9, is collected. Finally, to remove any sample residues, wash the column with filtered PBS at least twice, and then continue with experiments, as described in the manuscript. Morphological analysis using transmission electron microscopy showed that HEK-293 exosomes were spherical structures slightly larger than 100 nanometers.

This result agrees with that from nanoparticle tracking analysis measurement, which also showed the size distribution of the exosomes. Furthermore, they were positive for exosomal markers CD81, CD9, and CD63. The percentage recovery of exosomes purified using size exclusion chromatography and the percentage recovery of siRNA were calculated as described in the manuscript.

The post-purification recovery of exosome was 75%Using the siRNA standard curve, the encapsulation efficiency of siRNA in exosomes was calculated to be about 10 to 20%Flow cytometry qualitative analysis of in vitro uptake of exosomes loaded with fluorescent Atto655-siRNA showed that PANC-1 cells treated with siRNA-encapsulated exosomes had the largest shift in fluorescence signal. That was confirmed by the finding that PANC-1 cells treated with siRNA-encapsulated exosomes recorded a higher percentage of population positive for the Atto655 signal compared to that treated with unloaded exosomes and siRNA mixture. Cellular uptake of siRNA was also significantly higher in PANC-1 cells treated with siRNA-encapsulated exosomes compared to that treated with the exosome-siRNA mixture, confirming that the siRNA-encapsulated exosomes were internalized by the PANC-1 cells and that they effectively deliver the siRNA intracellularly.

It is important to weigh the sucrose and deuterium oxide very accurately when preparing the sucrose solution, and always use freshly prepared sucrose solution to avoid density alteration during storage. Confocal microscopy can be carried out to further validate the internalization of siRNA encapsulated in the exosomes into cells and study its intracellular trafficking following cellular uptake. This protocol allows us to assess the gene-specific knockdown of siRNA delivered by exosome and serves as a tool for new target validation in RNA interference-based cancer therapy.