Buoyant Density Fractionation of Small Extracellular Vesicle Sub-populations Derived from Mammalian Cells.

Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography-SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight differences in their buoyant density. Moreover, this technique also allows sEVs purification from vesicle-free RNA-protein complexes co-isolating in the high-speed pellet or by the use of crowding agents. This protocol describes cultivation of mammalian cells for sEV collection, sEV sedimentation, buoyant density fractionation of sEV sub-populations and immunoblots for sEV markers. This protocol can be used to fractionate distinct sEV sub-populations produced by a variety of mammalian cells.

have been proposed to play a variety of roles ranging from immunity to cancer metastasis (Pegtel et  . Therefore the sedimentable fraction or specific vesicle species that promotes a particular physiological response remains unknown (Shurtleff et al., 2018). A methodology that allows purification of sEVs away from contaminants and fractionation of distinct sEV sub-populations is of broad utility. The procedure explained below uses differential ultracentrifugation followed by buoyant density flotation to allow: 1) Purification of particles associated with a lipidic membrane (sEVs) from non-vesicular particulate material and 2) Fractionation of distinct sEV sub-populations based on slight differences in their buoyant densities. The purified sEV fractions are of suitable purity for use in physiological and/or characterization experiments. 2 www.bio-protocol.org/e3706   Note: The maximum volume of medium that can be processed in the steps below is 420 ml.

Note: Do not allow cells to become overconfluent, we have observed a dramatic decrease in sEVs from overconfluent cultures.
B. Conditioned medium collection and extracellular vesicle sedimentation 1. Collect conditioned medium from fourteen 15 cm plates by pouring it off in adequate containers.
The total amount of conditioned medium collected is ~420 ml. 9. Centrifuge at ~100,000 x g (29,500 RPM) for 1.5 h using a SW32 Ti rotor at 4 °C at maximum acceleration and brake. 10. Immediately after, aspirate the supernatant from the top to just above the sucrose cushion using an aspirating pipet fixed to a vacuum system. Be careful not to aspirate the interface at the cushion and the conditioned medium (this is where the sEVs have sedimented). Aspirate until leaving ~4-5 ml of conditioned medium resting on top of the sucrose cushion. Repeat this step 5 times, for a total of six 38.5 ml ultra-clear tubes.
11. Very carefully add the remaining conditioned medium (from Step B6) to each 38.5 ml ultra-clear tube. Do so by using transfer pipettes (see materials) and adding the conditioned medium very slowly and smoothly along the tube wall. Disturbance of the sucrose cushion must be avoided.
Any fast pouring can disturb the sucrose cushion.
13. Using a 1 ml micropipette carefully collect and discard ~2-3 ml of the ~4-5 ml remaining on top of the sucrose cushion after aspiration. Do so until there is ~2 ml of conditioned medium remaining on top of the sucrose cushion.
Note: For this step, the use of a 1ml micropipette is recommended over vacuum aspiration.
Using a 1 ml micropipette at this step allows a more controlled/accurate medium collection.
14. Collect the ~2 ml of remaining conditioned medium, plus the top-most 1 ml of sucrose cushion and save the 3 ml into a 50 ml conical tube. Repeat this step 5 times with the 5 remaining tubes and pool them together in a single 50 ml conical tube (final volume ~18 ml).

Measure the sucrose concentration of the solution from
Step B14 by using a refractometer.
Make sure the concentration does not exceed 21% sucrose (w/v). If the concentration exceeds 21% sucrose dilute with EV buffer. Generally an adequate sucrose concentration ranges from 15 to 20%.
Note: If the sucrose concentration from B15 exceeds 21% sucrose (w/v), sEVs will not sediment in Step B18 and instead they will equilibrate throughout the 13.2 ml ultra-clear tubes. 23. Use the refractometer to measure sucrose concentration. The sucrose concentration after this step is generally higher than 42% sucrose (w/v). Values equal or higher than 42% are adequate.

Note: If the sucrose concentration does not exceed 42% add more concentrated sucrose cushion until it is equal or exceeds this value. If the concentration is lower, the OptiPrep layer from
Step C3 will have higher density and sink, impeding the proper linear gradient formation.
24. Save 10% from B23 (500 μl) to use as "100,000 x g pellet" in Procedures D and E.
5. Centrifuge at ~160,000 x g (36,000 RPM) for 15 h using a SW41 Ti rotor at 4 °C at minimum acceleration and NO brake.

Note: It is important to have no brake during deceleration. Having the brake on can cause
disruption of the gradient and mixture of sEV sub-populations. 6. Once stopped, collect 400 μl fractions from top to bottom using of a 1 ml micropipette. A schematic summarizing Steps B1 to C6 is shown in Figure 1.

Note: Two different sEV sub-populations can be observed against a black background and with the appropriate lighting (application of an artificial or natural source of light can aid visualization).
The 2 distinct sEV sub-populations will appear as 2 separate white bands contrasting with the black background.  6. Incubate membrane(s) with the desired primary antibodies overnight in the cold room (4 °C).