The overall goal of this procedure is to stretch pattern cells on an elastic membrane of poly dimethyl suboxane or PDMS. This is accomplished through the ation of A-P-D-M-S sheet, followed by the application of a micro pattern on the PDMS. The micro patterned PDMS sheet is then mounted on the stretching device and a medium retaining pool is placed on top.
Next, the cells are plated on the surface of the micro patterned PDMS sheet and allowed to attach to the pattern before flushing to remove unattached cells. The final step is to apply an elongation to the cell containing PDMS to stretch the cells. Ultimately, video microscopy is used to show that application of forces on the retraction fibers of mitotic mammalian cells in cell stretching results in deformation of the cell.
This method can help answer key questions in the biomechanics field, such as what are the effects of external forces on cellular processes like division? We first had the idea for this method when we wanted to apply forces on a dividing cell to begin cut a piece of PDMS approximately 35 by 20 millimeters from a pre-made sheet. Here, a commercially available thin PDMS sheet is used because it is more reproducible and is less likely to break as compared to custom-made.PDMS.
Remove the top and the bottom protective layers of plastic and use tweezers to place the PDMS in a plastic petri dish. Then wash the PDMS with 70%ethanol for five minutes on a rotator at 30 oscillations per minute. Following the wash, dry the surface by flowing air over it.
Next illuminate the PDMS with deep ultraviolet light for five minutes at a distance from the ultraviolet bulbs of about five centimeters. Meanwhile, prepare an E-D-C-N-H-S solution as described in the text protocol. The solution must be prepared just before use because the reactivity of the solution decays in a matter of hours.
Transfer the PDMS sheets from the Petri dish to the lid of the Petri dish, which has not been illuminated. This will ensure that the surroundings of the PDMS are very hydrophobic and facilitate. The next step.
Pipette the E-D-C-N-H-S solution over the PDMS and incubate for 15 minutes at room temperature. Following the incubation. Rinse the excess E-D-C-N-H-S off with water.
Next, add the PLL grafted PEG solution and incubate from three hours to overnight at room temperature following incubation, rinse the excess PLL grafted peg off the PDMS with water. The PDMS is now passivated or functionalized with PLL grafted PEG and can be stored for several days at four degrees Celsius. To pattern the PDMS place, A-P-D-M-S sheet on a synthetic quartz photo mask bearing the micro features for patterning.
Place the PDMS side bearing the PLL grafted PEG facing the chrome side of the photo mask. Then illuminate for seven minutes through the photo mask at a distance from the ultraviolet bulbs of about five centimeters. Following illumination.
Add water onto the mask and PDMS then peel the PDMS slowly off the mask mount the previously passivated PDMS onto the stretching device. Attach one side of the PDMS to the fixed part of the stretcher. Fix the other side to the mobile part of the stretcher without clamping too much of the PDMS sheet.
If the PDMS is clamped too much, it becomes shorter and this can lead to braking as the tension will be greater for the same distance of stretch. Make sure the screws are well tightened, or the PDMS sheet will slip as soon as the stretch begins, or later on during the experiment. Next, incubate the PDMS with fibronectin solution for one hour.
At room temperature, it is possible to use other extracellular matrix proteins, but these have not been tested with this protocol. As a final step, rinse the PDMS with phosphate buffered saline. Next, cut a rectangle of PDMS in a thick PDMS slab.
Then cut another rectangle inside. In order to have a pool that will retain the cells and medium, add silicone grease under this rectangular cut PDMS and place it on top of the PDMS sheet to create a medium retaining pool. The grease will allow the gliding of the pool over the PDMS sheet during stretching to pattern the cells.
First, detach the cells from a culture flask at 50%co fluency. Once the cells are in suspension, pipette them several times with a 200 microliter tip to break up aggregates, which will ensure that individual cells bind to the pattern. Next, add one milliliter of the cell suspension into the pool.
Let the cells bind to the patterns for 10 to 30 minutes, depending on the cell. Type in a 37 degree Celsius incubator. Once the cells have attached to the patterns, gently flush the floating cells with equilibrated medium to flush the cells.
Remove the medium with an aspirating pipette while simultaneously adding medium. It is critical to maintain sufficient flux to wash away unattached cells while keeping the cells submerged in medium. Next, add a cover slip on top of the pool to avoid evaporation and medium leakage in case the PDMS breaks and allow the cells to spread for a few hours on the patterns.
Then put the device on an inverted microscope and start imaging. Avoid the use of oil immersion objectives as they will not work due to focusing problems. To stretch the micro pattern cells turn the micrometric screw while correcting the stage position in the x, y, and Z axes.
The stage position needs to be corrected to counteract the widening of the PDMS and the loss of focus. The technique presented in this video protocol allowed the application of forces on the retraction fibers of mitotic mammalian cells by stretching the substrate at the onset of metaphase. Some of these retraction fibers were pulled away from the cell body, resulting in a mechanical force applied on the mitotic cells cortex before stretching, the cell is plated on an oval pattern to achieve this.
The patterning is done with the PDMS being stretched during the impression of round patterns through the photo mask. After stretching, the substrate is stretched such that the pattern becomes a circle. After watching this video, you should have a good understanding of how you can plate cells on macro patterns and then apply stretching forces to the substrate.
