We have previously designed a device to apply mechanical strain to adherent cells. In the current work, we have redesigned the geometry of the substratum and customized the setup for high-resolution imaging of these strain cells. The main advantage of this cell-stretching system is that it provides a high-resolution subcellular imaging at 100x of the strain cells.
Demonstrating the substratum fabrication will be Vicki, a research assistant from my lab. Begin by designing a single-well polydimethylsiloxane mold by following along in the accompanying text protocol. Next, mix 20 parts of a polydimethylsiloxane base with one part curing agent in a disposable cup.
Once mixed, place the polydimethylsiloxane mix in a vacuum de-gasser at 0.8 bar for 30 minutes to remove the bubbles from the mix. Then, pour the polydimethylsiloxane mix into the mold and the dish. Remove any additional bubbles at this stage by de-gassing it again at 0.8 bar vacuum for 30 minutes.
When de-gassing is complete, bake the samples at 80 degrees Celsius on a leveling table for two hours. Next, carefully remove the samples from the oven and let them cool down to room temperature. Cut the edges with a blade and gently peel out the cured polydimethylsiloxane.
On the 150 millimeter diameter polydimethylsiloxane, draw a two centimeter by two centimeter grid with a marker. Within each square, draw a one centimeter by one centimeter square, leaving a margin of 0.5 centimeters on all sides. Use a blade to carefully cut along the lines and obtain square compartments.
Next, clean the samples by incubating them in 100%acetone for four hours, 100%ethanol for an additional four hours, and then in autoclaved water for four hours. Place the cleaned samples on paraffin film backing paper inside a 150 millimeter diameter plastic dish and dry the sample in an 80 degree Celsius oven for four hours. When finished, seal the dishes with paraffin film and store them in a cold room until further use.
Prepare progenitor cells and suspend them in proliferation medium. Seed these cells at a density of 35, 000 cells per square centimeter onto PDL-coated plastic surfaces. Adjust the volume of the proliferation medium to three milliliters per ten centimeters squared of surface area.
A lower volume of media may lead to cell clumping, arising from surface tension forces, while a higher volume of media in the dishes may cause the media to spill over. On the third day after seeding, harvest the cells and count them using a hemocytometer. Then, seed 35, 000 cells per polydimethylsiloxane plate with 700 microliters of proliferation medium.
Next, add a plasmic construct to labeling the cell's histone H2B complexes with green fluorescent protein by following manufacturer's instructions. After 24 hours, mount the polydimethylsiloxane samples with cells to be stretched on the uniaxial strain device. Then, change the medium in all samples to differentiation medium.
Measure the unstrained cell compartment length and turn the stage micrometer screw to increase the cell compartment length by the desired amount of strain. Leave the stretched and unstretched polydimethylsiloxane samples in the incubator at 37 degrees Celsius until imaging. Prepare the microscope incubator for live imaging by setting the incubator temperature to 37 degrees Celsius.
Next, bring a 100x oil immersion objective to the central position, as the objective turnit will not be accessible later. Once in position, unscrew the objective and screw it back together with an objective ring, so that the objective can be brought closer to the cells. Then, add a drop of oil to the objective.
Using a custom-printed holder, coat the holder with vacuum grease at the periphery of the window and place a number zero thickness glass coverslip onto the top surface of the holder. Tape the plastic window onto the microscope stage. Next, screw a Z translation stage onto the microscope stage and move it to the top-most Z position.
Remove 500 microliters of the medium from the stretched polydimethylsiloxane plate to be imaged, and add this medium onto the glass coverslip in the white plastic window. Then, use a pair of sterile tweezers to carefully detach the square compartment from the polydimethylsiloxane plate. Hold the strain device in an upright position so that the cells are facing up, above the white plastic window and carefully invert the strain device, so as to let any extra medium drop directly in the middle of the glass coverslip with the cells facing down.
Place the bottom part of the strain device onto the Z translation stage using double-sided tape to hold it in place. While looking through the eyepiece under bright-field, very slowly bring the strain device down and move the objective up to focus on the cells. Perform this focusing step slowly, alternating between lowering of the Z translation stage and raising of the objective.
If the Z translation stage lowers too much, the cells could compress and hence die. If we raise the objective too high, it could break the glass coverslip and cause the medium to leak. Scan the polydimethylsiloxane plate in X and Y directions to find a cell that has a fluorescent nucleus, using epiflorescence at 488 nanometers excitation.
Also make sure that the cell has an appropriate morphology when looking at the sample under bright-field. Record wide-field or open-pinhole images of the nucleus with 488 nanometers of wavelength excitation and of the cell with bright-field excitation at 30-second intervals per frame for a total duration of at least 30 minutes. The redesigned geometry of the polydimethylsiloxane substratum and the imaging setup shown in this video minimizes the distance between the cells and the objective.
This enables time-lapse imaging of fluorescently labeled cell nuclei using a 100x oil immersion objective. To measure the nuclear fluctuations, first plot the nuclear area versus time. Then, detrend the data using a third-order polynomial.
And finally, plot residual fluctuations and calculate standard deviation of the residual fluctuations. The nuclear fluctuations of the oligodendrocyte progenitor cells, following chemical induction of differentiation with and without a 10%tensile strain, show that with chemical induction alone, the amplitude of nuclear fluctuations showed a significant decrease at 48 hours, but not at 24 hours. On the other hand, chemical induction together with a 10%tensile strain showed a significant decrease at 24 hours, which remained constant without further reduction at 48 hours.
The most important thing to remember is that the working distance of the chosen objective should be equal to or greater than the depth of the cell compartment plus the thickness of the coverslip. The substratum can be redesigned to incorporate a grid, which would facilitate imaging the same cell before and after application of strain, since a before and after type of dataset reduces the noise coming from experimental error. Using this imaging setup, many subcellular phenomena within adherent live cells, such as protein localization or dynamics of cellular components, can be imaged within seconds of applying the mechanical strain.
