FluidFM Micropipettes can be used to sort single cells from a cell population. This allows for separation for cells with specific properties and avoids the need for serial/limit dilutions to obtain single cells. The method can be used in adherent cell cultures, since FluidFM offers the possibility to locally detach cells using the so called “trypsin shower” method, where the cell of interest is locally exposed to a concentrated trypsin solution. Due to this exposure, the cell detaches from the surface and can be picked by a force-controlled approach and negative pressure. This protocol was first released in the ARYA version 2.1.0, and is designed for the isolation of CHO cells growing in 12-well plates. This protocol is designed for adherent cells, in case of cells in suspension, please follow the protocol linked here.
- FluidFM Micropipette (8 or 4 µm aperture, 0.3 N/m stiffness).
Note: The size of the aperture depends on the size of the cell and needs optimisation.
- 1 ul of 10x Trypsin solution.
- Medium without phenol red (DMEM/F-12 ) , with 1% penicillin/streptomycin , filtered (for the already existing cell culture), serum free. In case of no CO2 control within the system use CO2 independent medium (e.g. Leibovitz's L15 medium).
- Complete growth medium (e.g. DMEM-F12), with 10% FBS and 1% penicillin/streptomycin , filtered (for the newly isolated cells).
- CHO cells, at 70% confluence.
- 2x 12-well plates (ThermoFisher-Nunc).
- Poly-L-lysine (PLL)
Initialization of the system
1. Turn on the microscope components. Turn on the system control unit and the pressure controller.
2. In the computer, open ARYA and create a new experiment.
3. Align the x/y/z axis if necessary.
4. Select the respective plate on the left port (e.g., 12 well plate). Set the content height at 1 mm if the plate used is not listed.
Preparation of the probe
1. Load the probe with 1ul of 10x Trypsin.
2. Use the Exchange Plates workflow to move to the right port, place the loading plate inside and place the probe in the loading plate (position A1, A3, C1 or C3)
3. Follow the Preparation or Preparation Advanced workflow until the Go to Sample step.
4. We highly recommend coating the cantilever before the experiment. Follow the protocol “COATING WITH PAcrAm inside FluidFM system” indicated in this link in order to avoid debris and cells to adhere on the surface of the probe.
Preparation of the cells
1. Plate cells at least the day before the experiment, so on the day of the experiment they grow to 70% confluency.
2. Shortly before the experiment, remove the medium from the cells and wash them once with PBS. Add then 1 ml of CO2 independent growth medium without FBS to the cells.
Note: FBS inhibits trypsin activity!
3. Use the Exchange Plates workflow, move to the left port and place this plate in the left port of the FluidFM OMNIUM system.
Preparation of the destination plate
1. Coat a 12 well plate with poly-L-lysine (PLL) according to the manufacturers protocol.
2. Fill the wells with 1ml of standard growth medium containing FBS.
3. Once the probe has been loaded into the system, use the Exchange Plates workflow to move to the right port and remove the loading plate.
4. Place the destination plate in the right port of the system.
Preparation of the system
1. Move the probe into the well from which the cells will be isolated
2. Use the Maximize Signal step of the Preparation workflow. Measure the sensitivity of the specific probe after the coating on the picking well.
3. To align the probe, use the Align Probe step in the Preparation workflow and drag the crosshair to the center of the aperture.
Trypsinization of the target cells
1. Use the Probe Control tool to approach the surface and retract 20 µm above the cells.
2. With the Pressure Control tool, apply 200 - 400 mbar pressure to release 10x Trypsin solution around the targeted cells. Move the probe around the desired cells. When trying to detach only 1 or 2 cells, apply lower pressure (50 mbar) and approach a bit closer to the cell.
3. Cells will slowly change their shape into a more round and bright morphology, which indicates cell detachment. Keep applying pressure until the cells are detached from the surface.
1. Open the Cell Isolation workflow and press on step 1. Pick-Up Cell.
2. Select Lift up.
3. Input desired parameters for the force-controlled approach, speed at which the probe is approaching the cell as well as the time that the probe will be over
the cell and the negative pressure with which the cell will attach to the probe.
Note: These parameters are given as an indication and can differ according to the experimental conditions (cell type, confluency, surface of the plate, etc.).
4. Move the crosshair over the center of the target cell that will be lifted. Press OK for the process to start.
5. The cell that has been picked up will appear round, will move with the cantilever, and will stay in the same focal plane as the cantilever. If that is the case, then verify that the cell has been picked up and press Continue. If the cell did not lift-up then press Retry to repeat the process. This is the good time to adjust the parameters if needed to improve the cell attachment and lifting.
6. Once the cell has been picked up, select the well in the “destination plate”. The plate is depicted on the right side as it is in the right port. The green label indicates that the well has already been used, but they can also be reused if needed. The blue label depicts a well that will be selected for cell deposition. White wells are empty and free to use. All wells where cells will be deposited need to contain media as mentioned before. Once the well is selected, press Continue.
Note: To confidently achieve monoclonality, an extra washing might be necessary here. You can find out how to wash the extra cells off here.
7. The system will move to deposit the cell.
8. Once the cell is transferred into the destination well check if the cell is still attached to the cantilever. It will appear round as after the picking, in the focal plane of the cantilever. If the cell is still attached the transfer was successful, therefore verify the transfer by pressing OK in the step 2. Verify.
1. In the step 3. Place Cell adjust the parameters with which the cell will be deposited on the bottom of the destination well. These include the force with which the cantilever will approach the surface, the time that it will keep once the surface has been reached and the pressure that will allow for release of the cell in the given time. Once the desired parameters are set press Place.
2. At the end of the release process, the XY stage will perform an automatic movement of 50 um to the left, which allows the release of the cell.
3. After the cell has been stably deposited move away from the cell to the content height using the Navigation Tool.
4. In the step 4. Flush Probe, there is a pressure pulse applied to release the membrane content from the microfluidic channel, that might have been aspirated during the transfer process.
5. After the probe has been flushed move to the step 5. Go to Source. Use the history menu from the Navigation Tool to go back to the initially trypsinized cells. To pick another cell simply follow again the same workflow from Cell picking and Cell deposition.
Once finished, incubate the cells inside the system at 37°C for 1 h. After this time, exchange the medium gently, place fresh cell culture growth media to the isolated cells and move them into the usual incubator.