The overall goal of this model is to create a clinically relevant neurotrauma phenotype in a 96-well format to facilitate the efficient and simultaneous comparison of injury sustained under multiple experimental conditions. Primary purpose of this model is to enable high-content phenotypic drug discovery screens in neurotrauma, with human-induced pluripotent stem cell-derived neuron. The plates used in this model retain the industry-standard 96-well plate geometry, maintaining their compatibility with existing high-content screening machinery, like robotic arms and automated microscopes.
To begin, place a bottomless 96-well plate bottom side up inside a 200 watt plasma cleaner for 60 seconds on high power. Next, place the plate in 200 milliliters of freshly prepared 1.5%APTES in deionized water, bottom side down, for 20 minutes in a fume hood. 10 minutes into the incubation, use forceps to position a plasma-treated silicone membrane on top of a 7.5 by 11 centimeter parchment paper rectangle, and align a 7.5 by 11.05 centimeter aluminum slab on the lower portion of the plate fabrication clamp.
Then use forceps to align the silicone membrane and parchment paper on the aluminum slab. At the end of the coating period, remove the bottomless plate from the APTES bath, and shake off the excess solution. Dip the bottomless plate into two consecutive 200 milliliter deionized water baths for five seconds each dip, and shake off the excess water.
After complete drying with compressed air, place the bottomless plate into the upper portion of the plate fabrication clamp, and gently close the clamp to press the bottomless plate and silicone together. To align the indenter block, use stage clamps to secure the plate onto the stage of the stretching device under a camera with a live feed, and place a dome light over the device. Launch the instrument control software on the computer attached to the injury device, and click on the Launch window to run the in-vitro neurotrauma project.
From the project window, double-click on the Motion Control and position tracker virtual instruments. Close the cage surrounding the injury device, and click on the arrow in the motion control virtual instrument to run the instrument. Click near bottom to lower the stage to within two millimeters of contact with the indenters, then click stop to stop the virtual instrument.
In the project window, right click on Axis One, and click on Interactive Test Panel. Enter 500 units in the Target Position field in the window that appears to set the step size to 50 micrometers. Then click Go repeatedly, until the plate first makes contact with any indenters, checking for contact on the live image displayed by the camera.
The vertical stage position will be reported in the top left of the interactive test panel window. Lower the plate further until every well has made contact with the indenters, moving the camera and stage as necessary to view the entire plate. Note the reported vertical stage position when all of the posts are in contact, and close the interactive test panel.
Next, run the motion control virtual instrument, and click Top to raise the stage, using the stop button to stop the virtual instrument when the stage is in position. Open the door to deactivate the device, then loosen the seth screw on the corner that made contact first to lower the corner, and tighten the opposite screw to raise the corner that made contact last. Repeat the block lowering, observing, and adjusting until the plate makes contact with all indenters simultaneously.
Careful alignment of the indenter block is important for achieving a uniform injury across all of the wells of the plate. Open the door to deactivate the device again, and insert the tie-down screws into their holes on the indenter block. Then tighten the screws into position, and note the stage position reported by the interactive test panel when the plate makes contact.
To stretch the plate, change the file name in the File Path field in the position checking virtual instrument to a unique file name, and click the Run arrow. Set the depth and duration of the indentation and 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 the 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 three-by-four grid. With the place 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 two to three 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. An optimal uninjured culture will have few if any clumps of more than five cells, and the neurites will be individual, long, slender, and curved, with little or no beading. Under ideal conditions, the viability of the cultures should closely approach the viability specified in the manufacturer's data sheet, and cultures on silicone should resemble those maintained on conventional rigid culture substrates.
Increasing the cell density increases the number and size of clumps that form in culture, while increasing the laminin concentration typically counteracts this effect. In cultures injured with a 57%peak strain, four hours after injury, the neurites are shortened or missing, and some have beads with clumping demonstrated by the remaining viable cells. Notably, higher dose laminin treatments reduce the severity of injury to the cell cultures in a concentration-dependent manner, as evidenced by increases in neurite length and cell viability in the higher-dose laminin treated cultures.
While attempting this procedure, it is important to consider lead times. For example, membrane detoxification takes three days, and the manufactured plates should cure overnight before use. Don't forget that the injury device contains a powerful voice coil to drive its motion, and one should always keep clear of the device when it is active, and only power down the device when the stage is at the top of its travel.
