The isolation of suspension and adherent cells require different mechanical approaches. In the latter case, 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.
In case of suspension cells (e.g., Jurkat), adhesion to the surface does not have to be disrupted since the cells are only held to the substrate by weak non-specific forces – or not at all. When picking up such cells, the force-controlled approach, which works well with adherent samples, often results in the cell slipping away from below the cantilever. To enable the sorting of cells in suspension, we have developed a dedicated workflow that makes fast, easy, and efficient to pick and place cells in suspension.
The process begins with a force-controlled approach from content height with the goal of placing the cantilever at an ideal height for picking. This height should be roughly 2 um more than the average cell size in the sample. The default value of 17 um applies for cells with a diameter of 15 um. Once the cantilever is positioned at the ideal height, a gentle negative pressure is automatically switched on. At this point, the operator needs to position the cantilever above the target cell, which will be aspirated onto the aperture. If the cell is not aspired right away, the picking pressure can be increase or approach closer to the cell by using specialized buttons in the workflow. In case the cantilever touches the cell instead of hovering above it, the opposite button can be used to slightly move the cantilever upwards. Note that these non-force-controlled movements do not allow movements below a level of 1 um above the surface, therefore the cantilever is safe from breaking.
Once the cell is aspirated to the aperture, the cell can be transferred to a new position and the deposition process can start. Upon clicking “continue”, the probe will move to the target well while keeping the “hold-pressure” as defined in the first step. Note that to confidently achieve monoclonality, an extra washing might be necessary here.
When the probe arrives to the center of the target well the operator has the possibility to verify that the cell is still attached to the cantilever. Then, by clicking “OK”, the parameters of deposition can be set. The deposition itself is a force-controlled approach during which a 5 mbar pressure is applied. When the setpoint is reached, the cell is kept on the surface for a pre-defined contact time to allow for adhesion to the substrate, while applying the release pressure to break the tight seal with the aperture. Once the contact time is elapsed, the cantilever is retracted first slowly (0.5 um/s) to 20 um, then faster (20 um/s) to 50 um. The cell at this point should have been removed from the cantilever and remained on the bottom of the well. In case the cell is still adhered to the cantilever, the deposition process should be repeated. The application of a gradually increasing pressure pulse can remove the cell as a final solution. However, viability is seriously compromised upon application of > 200 mbar.
deposition, the cantilever is flushed with a given pressure to get rid of the occasional cell debris and the cantilever is ready to isolate the next cell.
List of parameters
Surface approach speed: The speed of initial force-controlled approach (50 um/s).
Surface approach setpoint: The setpoint of initial force-controlled approach (100 mV).
Cell Height: The distance to which the cantilever is retracted after touching the surface. This height should be roughly 2 um higher than the target cell. (17 um)
Holding pressure: The pressure value that will be applied when the cantilever is moving from the pick-up well to the deposition well.
Contact time: The amount of time for which the cell is brought into contact with the surface to allow for adhesion (10 s).
Release Pressure: The pressure that is applied in the cantilever during the entire deposition, from the beginning of the approach until the end of retraction (5 mbar).
Flushing pressure: The pressure value that is used to get rid of possible cell debris in the microchannel and around the aperture. (1000 mbar)
Flushing time: Duration of the above mentioned pulse. (10 s)