This article details a method for inducing mechanical stress on human induced pluripotent stem cell-derived neurons (hiPSCNs) to study cellular responses. The process involves using a multi-well plate with a silicone membrane to apply controlled indentation, simulating stretch injury.
Begin with a multi-well bottomless plate attached to a silicone membrane. The membrane is coated with a biopolymer to facilitate cell adhesion.
Introduce human induced pluripotent stem cell-derived neurons, or hiPSCNs, suspended in a medium into the wells. The medium is supplemented with extracellular matrix, or ECM proteins to enhance cell adhesion.
Incubate the plate, allowing the cells to adhere to the ECM proteins and biopolymer, promoting uniform interaction with the membrane surface.
Next, secure the plate on the stage of an injury device.
Using the device, apply indentation on the membrane to generate controlled mechanical stress on the neurons.
The stress disrupts the cytoskeleton, which causes the degeneration of cellular protrusions termed neurites and the formation of small swellings along the neurites.
The stress also damages the cell membrane integrity, resulting in cell death.
The hiPSCNs exhibiting a stretch injury phenotype are now ready for analysis.
To stretch the plate, change the "File name" and the "File path" field in the "Position Tracking" virtual instrument to a unique file name and click the Run arrow. Set the depth and duration of the indentation in the "Injury" and "Injury duration" fields in the movement control panel virtual instrument. With the plate at the zero position, run the "Movement Control Panel" virtual instrument, and click Injure to indent the plate.
Click Top to move the stage up, clicking Stop when the plate is in the top position, and open the door to deactivate the injury device. Then, inspect the displacement history of the stage presented in the "Position Tracker" virtual instrument to confirm that the specified maximum displacement was applied, and export the data to an appropriate spreadsheet program.
To assess the stretch of the silicone membrane after plasma treatment, place the plate on a soft surface and prime the small protrusion on a stamp with ink from a permanent marker pen. Insert the stamp into the first well to be tested, and tap to ensure a good transfer of ink. When all of the wells have been stamped, place a high-speed camera on a boom stand over the injury device, with the lens set to the smallest numbered F-stop so that the field of view contains 12 indenters in a 3-by-4 grid.
With the plate in the zero point position, one click the Record button on the camera software so that it reads "Trigger In" and turn on the bright, diffuse axial light, then indent the plate as just demonstrated, and turn off the axial light.
To injure a cell culture, add 100 microliters of laminin-treated human induced pluripotent stem cell-derived neurons to each well of the plasma-treated plate, and allow the cells to attach to the plate bottom for 15 minutes at room temperature, before placing the plate in the cell culture incubator. After 2 to 3 days of culture, clamp the plate on the injury device stage and adjust the position of the lid to secure the plate without exposing the cultures to the ambient air. Then, with the plate in the zero point position, indent the plate as just demonstrated.
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