Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

Under suitable culture conditions, gram-negative bacteria release cargo-carrying outer membrane vesicles, or OMVs, into the supernatant. These vesicles are heterogeneously sized, small, spherical structures, which mediate cellular communication.

To fractionate differently-sized sub-populations of OMVs, take an empty size-exclusion chromatography column with an appropriate filter at its base to support the matrix.

Pack the column with gel filtration matrix consisting of chemically inert, spherical beads of defined pore size, ensuring the size is optimal to exclude large-sized OMVs from entering. Equilibrate the column with an appropriate buffer to prevent the beads from drying for optimum purification.

Load the bacterial culture supernatant containing differently-sized subpopulations of OMVs, protein aggregates, and nucleic acids on the top of the column. Allow the samples to run by the force of gravity.  

As the samples pass through the column, larger OMVs which cannot enter pores, move through the interparticle spaces between the gel beads to get eluted first. Meanwhile, the smaller OMVs penetrate the matrix pores, take a longer path, and elute later.

Due to their smaller size, the protein aggregates, and nucleic acids enter various pores in the matrix, causing them to have the longest retention time in the column and elute as the last fractions. Collect separate OMV eluates and store them for further analysis.

To begin, use a glass rod to mix a stock bottle of gel filtration medium, and pour the volume required to fill the column plus approximately 50% excess, into a glass bottle. Allow the beads to settle, and decant the excess liquid. Resuspend the beads in elution buffer to a final solution of approximately 70% gel and 30% buffer, and degas the solution under vacuum.

Mount the column on a ring stand in the vertical position, and fill the column with elution buffer to wet the walls, before draining the buffer until only about 1 centimeter remains. Then, carefully pipette the gel beads into the column, while simultaneously draining the excess buffer to prevent the beads from settling, until the column is packed to a height of approximately 2 centimeters below the bottom of the column reservoir.

Before loading the sample, degas the elution buffer under vacuum. Wash the column with approximately two times the column volume of elution buffer. Once the buffer reaches the top of gel layer, carefully add 2 milliliters of a 100 to 200-nanomoles per liter outer membrane vesicle sample on top of the gel layer without disturbing the surface, and let the sample enter the gel.

Slowly, add the elution buffer to the column without disturbing the gel layer, and place a 50-milliliter tube under the column. To prevent the column from drying, simultaneously add elution buffer to the top of the column.

After collecting 20 milliliters of eluate, place a rack of 1.5-milliliter tubes under the column. Collect a total of 96 1-milliliter fractions in each tube. While collecting the samples, continuously add elution buffer to the column. To clean the column, run 1 column volume of 0.1 molar sodium hydroxide through the column, followed by 2 column volumes of elution buffer.