This protocol is particularly relevant because it allows the detection and selection of single cells from a pool of mixed cells for subsequent downstream analysis at a single cell resolution. The main advantage of this technique is the potential to separate individual circulating tumor cells, or CTCs, from blood cells for a range of analysis that require high purity. Begin by starting up the micromanipulator software and switching on the micromanipulator.
Connect the micromanipulator to the computer and click Connect and Initialize Device to initialize the robotic arm in the microscope stage. Clean the external and internal surfaces of the protective cabinet with ethanol and close the protective cabinet after every manipulation of the machine to be able to maneuver the device through the computer. Install a new 20 to 30 micrometer glass capillary on the robotic arm and flush system oil through the system to remove any bubbles in the tubing.
Fill sterilization tank one with 70%ethanol, sterilization tank two with sterile nuclease-free water, and the buffer tank with sterile Dulbecco's PBS. Sterilize the capillary two times with 70%ethanol and replace sterilization tank one with sterilization tank two to use the sterilization function to wash the capillary in water at least three times. In the micromanipulator software, start a new experiment and select the type of picking experiment from the automatic and the manual selection modes.
Configure the deck tray to specify the positions of the sterilization tank, buffer tank, and depositing tray, and set the temperature of the liquid tanks in the selected depositing tray to four degrees Celsius. Position an ultra-low attachment plate containing the released CTC solution under the microscope inside the micromanipulator cabinet. Remove the lid from the plate and close the cabinet.
Centrifuge a 384-well plate containing 20 microliters of CTC culture medium per well to ensure that the medium is sequestered at the bottom of each well and place the plate into the target one position of the four degrees Celsius selected depositing tray. Manually select the microscope objective for picking and the exposure time of all of the necessary channels. Select Show Well Navigator to visualize and select the type of pickup plate.
Then calibrate a pickup position in the middle of the well containing the CTC solution without cells in the center of the field of view. Use the sensor to gently touch the bottom of the plate with a capillary and set the pickup position to 0.05 millimeters above the bottom of the plate. Carefully lower the robotic arm by 10 micrometer at a time to avoid damaging the capillary.
Select the cell type and picking parameters. For single cell picking, select the manual mode. Use the joystick to navigate the glass capillary within the well and place the capillary on top of a single cell of interest at least one millimeter from the well border.
Then manually add particles and select Pick Activated Particles to start the picking. When all of the cells have been picked, centrifuge the plate to sediment the cells at the bottom of each well. Bio-amino staining with anti-epithelial cell adhesion molecule antibody allows the visualization of cancer cells in the suspension with an accurate distinction from CD45 positive events.
Precise CTC isolation is characterized by the aspiration of only the desired target without the surrounding contaminant cells. As suspected, CTC clusters demonstrate an increase survival compared to single CTCs, giving rise to cell colonies within 56 days of in-vitro culture. Notably, CTC clusters also exhibit a higher proliferation rate and thus reach higher final cell numbers, indicating that the direct contact with other tumor cells has an impact on both tumor cell viability and proliferation rate.
Single-cell RNA sequencing of CTCs directly isolated from breast cancer patients reveals a T-distributed stochastic neighbor embedding of single-cells derived from single CTCs, CTC clusters, or CTC white blood cell clusters. Remember to install the glass capillary to remove air bubbles within the fluidic system of the micromanipulator and to calibrate the pickup position to ensure an efficient single-cell picking. The single-cells can be seeded or further processed for next generation sequencing to enable ex-vivo analysis and CTC characterization at a single-cell resolution to investigate the metastatic process.
