Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis

In video, we present a protocol for freeze-fracturing of extracellular vesicles originating from bladder cancer cells in vitro. Extracellular vesicles are membrane-limited vesicles that derived from cells are released into extracellular space and have a role in the intercellular communication. Within extracellular vesicles, we discriminate three populations, exosomes, microvesicles, and apoptotic bodies, which differ by their origin, size and molecular composition.

Molecular composition of extracellular vesicles reflects molecular profile of the donor cell and interfere with biological processes in the recipient cell. However, the composition of limiting membrane of cellular vesicles is crucial for the interaction with the recipient cell membrane. To analyze organization of limiting membrane, extracellular vesicles have to be first isolated, and then approached to freeze-fracture technique.

Freeze-fracture is an electron microscopic technique dedicated to studied the two-dimensional organization of biological membranes, including vesicles. The steps of freeze-fracturing are rapid freezing of the specimen, fracturing the specimen, making the replica of the frozen-fractured surface, and cleaning the replica to remove biological materials. Replicas are finally visualized with transmission electron microscope.

Our aim was to use freeze-fracture technique to characterize microvesicles in terms of shape, size, and the composition of membrane. Start with growing cells in the cell incubator. Inspect cells under the microscope to confirm their variability and confluence.

Collect culture media with microvesicles into tubes, and centrifuge at 300 g-forces for 10 minutes. Collect supernatant. Subsequently, centrifuge supernatant at 2000 g-forces and 10, 000 g-forces.

After that, observe the tip of the tube for a whitish pellet indicating microvesicles. Carefully remove the supernatant. Add 1.5 milliliters of PBS, and re-suspend pellet containing microvesicles.

Then centrifuge at 10, 000 g-forces. Observe for the whitish pellet. Remove the supernatant, and gently add fixative.

Leave for 20 minutes at 4 degrees Celsius. Next remove the fixative, and add washing buffer to rinse the pellet without re-suspending it. Leave for 10 minutes at 4 degrees Celsius.

Remove washing buffer, and add 30%glycerol to pellet. Re-suspend pellet into homogeneous suspension, and incubate for 30 minutes at 4 degrees Celsius. Prepare clean copper carriers with a center pit and develop flask with frown and liquid nitrogen.

Under microscope, transfer the sample into the center pit of copper carrier. Mix solidified freon to become liquified. Grab outer ring of the carrier with tweezers and immerse it into cooled freon.

After eight seconds quickly transfer the carrier into develop flask with liquid nitrogen. Mark cryo vile and cool it. Under liquid nitrogen collect the carriers into the vile, close it, and store in the liquid nitrogen container until freeze fracturing.

Prepare freeze-fraction unit according to the manufacturers operating instructions. Start the vacuum system and cool down the unit chamber to minus 150 degrees Celsius. Transfer copper carriers with frozen sample into the freeze-fractioning unit.

Set the knife temperature to minus 100 degrees Celsius and wait for the vacuum. Begin sectioning the sample by turning on motorized movement of the knife until the surface of the sample is smooth. For fracturing turn off motorized movement of knife and proceed with slow manual control of the knife.

Proceed with the platinum shadowing at the 45 degrees angle. Turn on high tension and increase the current to the platinum gun. Sparks of platinum should begin to shadow the sample.

Supervise the position of platinum on the quartz crystal thickness monitor. Recommended thickness of platinum is 2.5 nanometers. Turn off current and high tension.

Proceed with carbon shadowing at a 90 degrees angle. Turn on high tension and increase current to carbon gun. Sparks of carbon should begin to shadow the sample.

When 2.5 nanometers of carbon is obtained, turn off current and high tension. Transfer those samples with the replicas from the freeze-fracture unit to a 12-valve plate filled with double-distilled water. Replicas will float on the water surface while the carriers sink.

Transfer replicas with wire loop to a 12-valve plate filled with sodium hypoclorite and incubate overnight. Wash replicas in double-distilled water. Collect them on a mesh copper TM grate and let them dry on air for two hours.

Use a transmission electron microscope to image replicas and obtain micrographs. For accurate interpretation of replicas and micrographs, follow guidelines for the proper orientation on nomenclature. Analyzes of replicas showed that isolated-conditioned medium contained enriched fraction of vesicles.

Isolated vesicles were either gathered in clusters of three or more or very individually distributed. Vesicles were spherically shaped. The diameter of vesicles corresponded to the diameter of microvesicles.

Freeze-fracturing also revealed two-dimensional organization of microvesicles membrane. Exoplasmic phase of microvesicles had smooth, uniform appearance while in the protoplasts phase we observed inter-membrane particles. Inter-membrane particles appeared sporadically, which implies that microvesicles isolated from cancer material cells contain only low amount of membrane proteins or protein assemblies.

To sum up, freeze-fracture technique used in the presented protocol prove to be optimal technique for the characterization of extracellular vesicles and can be used for the evaluation of other populations of extracellular vesicles. A crucial advantage of freeze-fracture technique is its power to resolve internal organization of vesicles-limiting membrane, which determines biological function of extracellular vesicles.