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1. Bacterial strains and culturing conditions
NOTE: Bacterial strains used in this study were Escherichia coli MP1, Akkermansia mucinophila, Bacteroides thetaiotaomicron, Bifidobacterium breve, and Bifidobacterium dentium.
1. For E. coli, use a sterile loop to inoculate single colonies into 250 to 1,000 mL of Luria-Bertani (LB) broth and incubate aerobically in a shaking incubator at 300 rpm and 37 °C for 48 h before processing the culture. For the recombinant E. coli MP1 strain harboring p114-mCherryClyluc, add chloramphenicol to the LB agar and broth at a final concentration of 17 µg/mL.
2. For A. mucinophila, B. thetaiotaomicron, B. breve, and B. dentium, streak on Brain Heart Infusion (BHI) agar plates and incubate anaerobically inside a vinyl anaerobic chamber. Inoculate single colonies into 100 mL of pre-reduced BHI broth and incubate for 48 h anaerobically.
2. EV isolation
1. Clarifying bacterial culture medium by centrifugation and filtration
1. Transfer the bacterial cell cultures inoculated in step 1 to clean 250 mL or 500 mL polypropylene centrifuge bottles by pouring. Centrifuge the bottles in a large-capacity, fixed-angle rotor at 4 °C and 5,000 × g for 15 min. Transfer the supernatant to clean centrifuge bottles by careful pouring, and centrifuge again at 10,000 × g for 15 min.
NOTE: Reuse the bottles after biosafety-appropriate cleaning and decontamination.
1. If large pellets of bacterial cells are present after the second centrifugation, repeat the centrifugation in a clean bottle to further remove cells.
2. Transfer the supernatant to a 0.22 µm polyethersulfone vacuum-driven filter device of appropriate size by pouring. Filter by connecting the filtration device to a vacuum wall supply. If the filtration rate drops significantly, simply move any unfiltered material to a new device. Store the filtered medium at 4 °C overnight and continue the protocol the following day if desired.
NOTE: The centrifugations above typically allow processing of ~2x the indicated volume of cell culture through each device. For example, a single 500 mL filter device could filter ~1,000 mL of precentrifuged culture. These devices are not typically reused. Using syringe filters at this step is not recommended without optimization, as significant losses were noted with the tested models. This is a potential stopping point.
3. Check for the complete removal of the viable cells at this point by spreading 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. If bacteria are detected, further optimize the procedure above by performing additional centrifugations and/ or filtrations.
2. Concentration of the filtered medium
1. If working with volumes significantly >100 mL, proceed to step 2.2.2. If working with volumes of ~100 mL, load 90 mL of filtered culture medium onto the reservoir of a respective capacity 100 kDa molecular weight cutoff (MWCO) centrifugal ultrafiltration device using serological pipettes. Always balance with a matching ultrafiltration device, and centrifuge in a swinging bucket rotor at 4 °C and 2,000 × g for 15-30 min intervals, until the volume of the medium in the top reservoir has been concentrated to <0.5 mL.
1. Top up the reservoir with any remaining filtered culture medium. If "topping up," remove the flow-through in the bottom of the device and rebalance any devices.
NOTE: It was observed that the maximum volume of filtered culture medium that can be concentrated using these devices is <2-fold the recommended volume.
2. If the viscosity of the concentrated medium in the reservoir is visibly increased (dark, viscous material), dilute with phosphate-buffered saline (PBS) and re-concentrate by centrifugation to dilute any non-EV proteins smaller than the MWCO of 100 kDa.
NOTE: This is a potential stopping point.
3. Transfer the concentrated medium to a low protein-binding tube, store at 4 °C overnight, and continue the protocol the following day if desired.
2. If working with volumes significantly >100 mL, select an appropriately sized tangential flow filtration (TFF) device (100 kDa MWCO) to accommodate the volume to be processed.
NOTE: Filtration devices for processing 100 mL to >1,000 mL are commercially available. Local availability, cost, and compatibility with the pump and tubing/connections will dictate which particular models will be most useful. Up to 2 L of culture medium were processed with the device indicated in the Table of Materials before needing to clean the filter (see step 2.3 below for the cleaning protocol).
1. Assemble a filtration circuit with #16 low-binding/low-leaching tubing, 1/8 inch hose-barb to Luer adapters, the TFF device, and a peristaltic pump.
NOTE: Perform TFF within a biosafety cabinet to minimize the risk of contaminating EV preparation with environmental bacteria.
2. At room temperature, begin circulating the filtered, conditioned medium at approximately 200 mL/min (minimum 100 mL/min). Determine the appropriate RPM corresponding to the desired flow rate by pumping 200 mL of PBS into a graduated vessel. When circulating filtered, conditioned medium, collect the molecules <100 kDa crossing the ultrafiltration membrane as waste in a separate vessel.
NOTE: The example below will be assuming a starting volume of 2 L of culture.
3. Continue to circulate the conditioned medium until its volume has been reduced to ~ 100-200 mL. Move to smaller vessels as needed. Dilute 2-fold with PBS, and continue to circulate with the pump, concentrating down to 75-100 mL. Dilute 2-fold with PBS and continue to circulate to a final volume of 25 mL. Dilute 2-fold with PBS and continue to circulate until <10 mL.
4. Lift the feed tubing out of the sample reservoir and continue to pump to purge the filter and recover the maximum amount of sample.
NOTE: This is a potential stopping point.
5. Transfer the concentrated sample to a conical tube and store overnight at 4 °C if desired. Alternatively, continue with the protocol.
6. Move the concentrated sample to a 15 mL capacity 100 kDa MWCO centrifugal ultrafiltration device. Centrifuge in a swinging bucket rotor at 4 °C and 2,000 × g for 15-30 min intervals until the volume of the medium in the top reservoir has been concentrated to <2 mL.
NOTE: This is a potential stopping point.
7. Transfer the concentrated medium to a low protein-binding tube, and store at 4 °C overnight, continuing the protocol the following day if desired.