The overall goal of this procedure is to isolate viable, circulating tumor cells from blood samples. Begin with preparation of the isolation micro tubes then isolate the buffy coat from the blood samples. Next process the Buffy coat through the functionalized micro tube device.
Harvest the captured cells from the micro tube and maintaining culture by using a device that physiologically mimics a venue to biomimetically capture flowing CTCs without inflicting cellular injury. This technique offers functionality in a clinical setting to develop personalized cancer therapies. The main advantages are that this is a straightforward technique that uses commonly available materials and it recreates the natural solid tissue process that occurs in blood vessels.
I'm Mike King, associate professor of biomedical engineering at Cornell University, demonstrating the procedure will be Jeff Madison, a senior technician from my laboratory. The following protocol is for the production of a single micro tube device, sonicate 250 microliters of 6.6%Halo site nano tube solution. For 30 seconds, call the solution with cold water.
After a second sonication draw the solution into a syringe. Filter the solution through a 0.45 micron syringe filter into a clean micro refuse tube vortex. The halo site nano tube solution periodically.
To maintain homogeneity, obtain a 50 centimeter long section of micro rehan micro tubing. Cut the micro tube inlet at a diagonal and insert one end of the micro tube into a small IDEXX adapter piece, and insert a syringe needle into the other end of the micro tube. To clean the micro tube, place the open end of the micro tube into 70%ethanol and draw around 50 microliters of ethanol into the syringe to fill the micro tube to rinse the ethanol outta the micro tube.
Draw a generous volume of distilled water into the syringe. Now detach the syringe from the micro tube, empty the syringe and then reattach to the micro tube. Draw 50 microliters of 0.02 weight per volume.
Polyol lysine into the micro tube and incubate for five minutes of room temperature. Then draw 100 microliter filtered nano tube solution into the micro tube and incubate for three minutes at room temperature. To rinse out the nano tube solution, draw 100 microliters of water through the micro tube and incubate overnight at room temperature, draw 50 microliters of PBS through the micro tube, followed by 50 microliters of 10 microgram per milliliter.
Protein G solution equilibrate for 90 minutes at room temperature. Next, draw 50 microliters of five microgram per milliliter, E selectin IgG, and 50 micrograms per milliliter antibody solution into the micro tube. Incubate for two hours at room temperature, block non-specific cellular adhesion with 5%milk protein.
Then incubating PBS at room temperature until the blood or buffy coat samples are ready for processing 10 minutes prior to using the micro tube for cell isolation. Draw 50 microliters of calcium saturated PBS to activate the selectin molecules in a heparinized tube. Collect a 10 milliliter blood sample from a patient for lymphocyte isolation.
Place 10 milliliter fol pack solution into a 50 milliliter centrifuge tube. Gently overlay the 10 milliliter whole blood sample centrifuge at 2000 times G for 15 minutes, four degrees Celsius with minimal deceleration. Next, transfer the buffy coat layer to a new tube.
Wash the buffy coat with PBS and centrifuge to lies the erythrocytes. Gently re suspend the cell pellets in one milli liter RBC lysis, buffer, and incubate for 10 minutes at room temperature. Then add 10 milliliters.
PBS mix gently and palate cells by centrification. Gently resuspend the cells in PBS plus using IDEXX adapters. Attach one end of the functionalized micro tube to a five milliliter syringe.
Insert the syringe onto a syringe pump and submerge the open end of the functionalized micro tube into the cell prevention. Process the cell suspension through the micro tube to remove the unbound and loosely bound cells. Transfer the open end of the micro tube to a tube containing PBS plus.
Draw 300 microliters PBS plus into the syringe. Next place the open end of the micro tube into a clean tube. Disconnect the syringe from the micro tube and attach a syringe containing acuate gently perfuse acuate to fill the micro tube.
After a 10 minute incubation at room temperature, attach a syringe containing one milliliter of growth media, perfuse the micro tube and collect the effluent into appropriate tissue. Culture plates proceed to culture. The isolated cells circulating tumor cells were isolated from blood samples of a breast cancer patient and a lung cancer patient after five days.
In culture. These preparations were characterized for CTC capture based on staining for epithelial cellular molecule epican, as well as the nucleus capture purity was calculated as the percentage of cells that were identified as CT C compared to the total number of captured cells. This image shows representative micrographs of CTCs isolated from donors A and B.After five days in culture, cells were fluorescently stained for EPAM with Alexa Fluor 4 88.
The nuclei were visualized by DPI staining. Following this procedure, viable CTCs and culture can be used to obtain patient specific information such as drug susceptibility or cell marker expression. Thanks for watching and good luck.
