Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

Epithelial cell adhesion molecules, or EpCAMs, are surface transmembrane glycoproteins overexpressed in rapidly proliferating epithelial cells in prostate cancers. To isolate EpCAM-expressing cells, begin with a medical-grade wire bearing a functionalized gold-coated tip. Functionalization layers a polymeric gel containing covalently bound EpCAM-specific antibodies.

Immerse the wire into a peripheral blood suspension containing fluorescently labeled cancer cells. Ensure that the functionalized part remains dipped in the blood suspension. The EpCAMs on the cancer cells recognize and bind to their specific antibodies functionalized on the wire.

Wash with a buffer to remove any unbound cells from the wire surface. Bend the wire and mount it on a buffer-containing glass slide, with the functionalized part immersed in the buffer. Use a fluorescence microscope to check bright fluorescence from cell cytoplasm and nuclei and confirm the presence of captured cells on the wire.

Treat the wire with a release buffer. The proteolytic enzymes in the buffer cleave the polymeric gel, releasing the antibody-cancer cell complexes into the buffer. Centrifuge the wire assembly to collect the released antibody-cell complexes in the pellet. Remove the wire and the enzyme-containing supernatant. Resuspend the target cell-containing pellet in a suitable medium.

In 5 milliliters of peripheral blood, add about 500 to 500,000 cells and then mix by inverting the tube. After removing the wire from the storage compartment, remove the rubber cap that holds the wire and wash the wire in 1x phosphate-buffered saline and mount it in the cap of the tube. Then, incubate the tube on a tilted roller mixer at 5 rotations per minute for 30 minutes at room temperature. After incubation, rinse the wire in a 1x phosphate-buffered saline thrice. Then, store the wire in a 15-milliliter tube containing 1x phosphate-buffered saline in the dark.

Draw a rectangle area on a glass slide using a grease pen and add 500 microliters of 1x phosphate-buffered saline onto it. To immerse the functional part of the wire in 1x phosphate-buffered saline, bend the non-functional part of the wire and place it on the glass slide. To count the number of captured cells, visually inspect both sides of the wire. After inspecting, transfer the wire back in the 15-milliliter tube with 1x phosphate-buffered saline and keep in the dark.

In 1 milliliter of 1x phosphate-buffered saline, dissolve 4 milligrams of the release buffer component. Then, filter the solution through 0.2-micrometer sterile filter to obtain the ready-to-use buffer. Next, warm the release buffer at 37 degrees Celsius for 5 minutes. Once warmed, transfer 1.6 milliliters of release buffer to completely fill a 1.5-milliliter reaction tube. Next, incubate the functional part of the wire in the release buffer at 37 degrees Celsius in a water bath for 20 minutes.

After the incubation, place the wire on a shaker at 500 rotations per minute for 15 minutes. Then, centrifuge the wire at 300 x g for 10 minutes. Once the centrifugation is over, displace the wire from the tube, close the cap, and centrifuge the tube again at 300 x g for 10 minutes.