This protocol is important because it provides a way to isolate extracellular vesicles from a variety of bacteria in a highly reproducible manner. The great thing about this technique is it can be used to isolate EVs from quite large environments of bacterial cultures, which will enable in vivo studies. While the data we show reflects preclinical applications, it is foreseeable that this protocol could be adapted to manufacturing bacterial EVs for therapeutics.
This method can be applied to any scalable cell culture system with minor modification. Because of the protocol scalability, it's important to have appropriately-sized filters and SEC columns to handle the desired starting volumes of bacterial cell culture. Demonstrating the procedures are Sadie Johnson, an assistant technologist in Dr.Justin Lathia's lab, Dionysius Watson, an oncology fellow in Dr.Justin Lathia's lab, and Akeem Santos, a research technologist in my laboratory.
Begin by inoculating single colonies of Escherichia coli into 250 to 1000 milliliters of Luria-Bertani, or LB broth, using a sterile loop. Then, incubate the culture aerobically in a shaking incubator at 300 rotations per minute and 37 degrees Celsius. After 48 hours of incubation, clarify the bacterial culture medium by transferring the cell cultures to clean 500-milliliter polypropylene centrifuge bottles to centrifuge in a large-capacity, fixed-angle rotor at four degrees Celsius and 5, 000 times G for 15 minutes.
Transfer the supernatant to clean centrifuge bottles by carefully pouring to centrifuge at 10, 000 times G for 15 minutes. Next, transfer the supernatant to a 0.2-micron polyethersulphone vacuum-driven filter device of an appropriate size and connect the filtration device to a vacuum wall supply. If the filtration rate significantly drops, move the unfiltered material to a new device.
The filtered medium can be stored at four degrees Celsius overnight or can be further processed immediately. To check for the complete removal of the viable cells, spread an aliquot of the filtered supernatant on suitable agar plates and ensure the absence of any colonies after incubation at optimum conditions for the bacterial strain. For concentrating the filtered medium with a volume of 100 milliliters, load 90 milliliters of filtered culture medium onto the reservoir of centrifugal ultrafiltration device with a 100 kilodaltons molecular weight cutoff and centrifuge the medium in swinging bucket rotor at four degrees Celsius and 2, 000 times G for 15 to 30 minute intervals until the volume of the in the top reservoir has been concentrated to less than 0.5 milliliters.
To top up the reservoir with any remaining filtered culture medium, remove the flow-through in the bottom of the device to rebalance. If the viscosity of the concentrated medium in the reservoir is visibly increased, Dilute the medium with PBS and reconcentrate by centrifugation to reduce any non-extracellular vesicle, or EV proteins, smaller than the molecular weight cutoff of 100 kilodaltons. Then, transfer the concentrated medium to a low-protein binding tube to store at four degrees Celsius overnight For concentrating the filtered medium with a volume larger than 100 milliliters, select an appropriately-sized tangential flow filtration, or TFF device, with a molecular weight cutoff of 100 kilodaltons.
After assembling a filtration circuit as explained in the manuscript, pump 200 milliliters of PBS into a graduated vessel to determine the appropriate speed corresponding to the desired flow rate. When the speed is set, circulate the filtered conditioned medium at approximately 200 milliliters per minute at room temperature and collect the molecules smaller than 100 kilodaltons, crossing the ultrafiltration membrane as waste in a separate vessel until the volume of the conditioned medium has been reduced to 100 to 200 milliliters. Sequentially dilute the filtered volume twofold with PBS and continue circulating the medium with the pump in smaller vessels to concentrate the volume to 75 to 100 milliliters, and then to 25 milliliters and 10 milliliters.
Lift the feed tubing out of the sample reservoir and pump to purge the filter to recover the maximum amount of the sample. Move the concentrated sample to a 15-milliliter centrifugal ultrafiltration device with molecular weight cutoff of 100 kilodaltons and centrifuge in a swinging bucket rotor at four degrees Celsius and 2, 000 times G for 15 to 30 minute intervals until the volume of the medium in the top reservoir is less than two milliliters. Transfer the concentrated medium to a low-protein binding tube to store at four degrees Celsius overnight.
For the isolation of the EVs, perform size exclusion chromatography, or SEC, using a small column with a 10-milliliter bed volume for less than 100 milliliters of starting material and a larger column with a 47-milliliter bed volume for more than 100 milliliters of starting material. Over several hours before the experiment, bring the SEC column and PBS to room temperature, followed by stabilizing the column in a vertical position using a standard laboratory stand and holder. Before connecting to the SEC column, hydrate the sample reservoir by allowing five milliliters of PBS to flow through the frit into a waste container.
Then, unscrew the inlet cap of the column to add two milliliters of PBS to the sample reservoir and carefully connect the reservoir to the column as the PBS is dripping out through the frit. For equilibration of the column, add 47 milliliters of PBS to the sample reservoir, followed by uncapping the bottom of the SEC column. Then, allow all the loaded sample buffer to flow through the column and discard the flow-through.
After loading two milliliters of the sample onto the sample reservoir, allow the sample to enter the column completely and collect the flow-through. Immediately add PBS to the sample reservoir at a volume of 14.25 milliliters minus the sample volume to allow the solution to flow through the column, while discarding the amount equal to the column void volume. Next, position a two-milliliter low-binding microtube directly below the SEC column to collect the first flow-through or fraction one after allowing two milliliters of PBS to run through the column.
Continue to add two milliliters of PBS to the sample reservoir to collect each subsequent fraction. Store the collected fractions at four degrees Celsius for short term storage or minus 80 degrees Celsius for long term storage. For sterility testing of the collected fractions of EVs, inoculate three milliliters of the culture medium with 100 microliters of the fractions to be used in assays.
Then, culture the medium under optimal conditions while observing turbidity for at least three days. Alternatively, the fraction samples can be applied to agar plates containing the medium used to grow the producing bacteria, then check for colony formation. Next, store the EVs by transferring 25 to 50%of the individual or pooled fractions to low-protein binding tubes at minus 80 degrees Celsius to avoid freeze-thaw cycles.
The representative analysis shows the elution of Escherichia coli MP1 EVs in the early chromatography fractions. The fractions one to six contained the most EVs, with an average diameter lower than 100 nanometers. At the same time, the subsequent fractions contained EV-free proteins and very few EVs.
The EV enrichment and size were confirmed by transmission electron microscopy, particularly in fractions two to six. It was observed that EV-enriched fractions two to seven had high nanoluciferase activity, but only a very low fluorescent signal from non-EV-associated mCherry compared to that of the later fractions. The immunogold labeling confirmed the EV association of nanoluciferase in fractions two to five.
The applicability of the protocol to the other bacterial species was assessed by isolating EVs from the cultures of diverse anaerobic bacteria. It was observed that the early chromatography fractions one to four were enriched for EVs, with a diameter size lower than 100 nanometers. The complex brain-heart infusion, or BHI, contained the EV-sized particles at less than 25%of the total EV yield.
It is important to use TFF devices capable of handling the starting volume of the bacterial cell culture. This technique allows us to generate enough EVs to ask biological questions on their function in complex animal models.
