The overall goal of this procedure is to culture cells in a substrate with a topography that changes over time. This is accomplished by first programming a shape memory polymer into a permanent shape that can be remembered. The shape memory polymer is then programmed into a temporary shape onto which cells are later plated.
The final step of the procedure is to trigger the shape memory polymer to return to its permanent shape by increasing the temperature, ultimately results can be obtained that show a change in cell behavior through fluorescence microscopy. Hi, my name's Pat Mather. I'm a professor of biomedical and chemical Engineering and director of Syracuse Biomaterials Institute here at Syracuse University.
The main advantage of this technique over commercial or custom apparatus for stretching cells is that in a single polymeric system, we're able to apply a wide variety of complex strains to cells. My name is Jay Henderson. I'm an assistant professor of Biomedical and Chemical Engineering and a member of the Syracuse Biomaterials Institute.
This method can help answer key questions in the field of Mechanobiology, such as how cells respond to dynamic changes in their physical environment. My name is Kevin Davis and I'm a PhD student at Syracuse University in the Department of Biomedical Engineering and Chemical Engineering. Though this method can provide insight into 2D mechanical biology, it can also be used for other systems such as 3D shape changing scaffolds for tissue engineering.
Prior to the start of this procedure, prepare a custom curing chamber using a glass slide, a one millimeter thick Teflon spacer and an aluminum plate. Secure the chamber using small binder clips to begin, inject NOA 63 into the chamber through a hole in the Teflon spacer using an 18 gauge needle. Then place the chamber in a hot plate set at 125 degrees Celsius.
Allow the chamber to heat to a uniform temperature for five minutes following chamber heating. Procure the NOA 63 for 20 minutes by positioning a UV lamp 6.5 centimeters from the surface of the glass. Remove the NOA 63 from the chamber while still warm using a razor blade.
Then post cure the shape memory polymer under the UV light for three hours and 40 minutes directly on the hot plate at 125 degrees Celsius. To characterize the shape memory of the NOA 63, prepare a dumbbell specimen by hot pressing the cured film with a customized punch. Finally, loathe the specimen into a dynamic mechanical analyzer with tensile fixture.
Set the instrument to force controlled mode and program the testing procedure as found in the written protocol accompanying this video. The result of the procedure is isothermally cured shaped memory polymer or SMP film place CSMP in a hot plate and set the temperature higher than the glass transition temperature in order to reduce the modulus and ease cutting. To prepare individual samples from the isothermally Cured s and p film.
Cut the s and p film with a razor to the desired sample size. The temporary shape can be fixed in a number of different ways.Here. A benchtop hydraulic press with plats that can be heated and cooled is used to emboss a temporary topography.
First, set the temperature of the PLAs at a temperature above the tg. For this demonstration, an embosser was made by curing epoxy on a vinyl record to produce a temporary shape of parallel grooves. The embosser can be made from other materials and with different topographies, but must be stiffer than the NOA 63.
At the embossing temperature place the s and p samples face down on the embosser. Then place the samples and embosser into the press. Apply approximately 100 kilo pascals preload to make contact between the heating PLAs and the samples hold for about five minutes to allow the samples to reach a uniform temperature.
Once a uniform temperature is reached, apply one to six megapascals to the samples and hold for one minute. The SMP will fracture. If a larger stress is applied.
Apply smaller stresses to introduce topographies with smaller amplitudes. After pressure is applied, reduce the temperature to below the TG by using the water cooling capability of the press plaats. When the temperature is below the tg, remove the applied force prior to starting the active cell culture experiment.
The UV light of the biological safety cabinet is used to sterilize the samples. Arrange the samples face down in sterile dishes without lids, and turn on the UV light for six hours. Then flip the samples to new sterile dishes in the face-up position.
Turn on the UV light for an additional six hours. The samples need to be equilibrated to a relatively stable state before plating with cells. Place the samples in a 96 well plate and add 150 microliters of complete growth.
Medium per well. Place the plate in a 30 degree Celsius incubator with 5%CO2 until reaching the desired partial recovery. For this demonstration, the samples are incubated for 30 hours to produce a large enough amplitude to align the cells.
Once partial recovery is achieved, the samples can be plated with cells. Place the samples in a new 96 well plate. Then add 150 microliters of cell solution to each of the samples here, C3 H 10 t and a half.
Most embryonic fibroblasts are used at 20, 000 cells per milliliter. To achieve isolated cells, allow the cells to attach and spread on the temporary topography by incubating at 30 degrees Celsius for 9.5 hours. Next assay cell morphology before transition by removing samples and performing staining and fluorescence imaging of the samples.
This material exhibits autofluorescence through most of the UV and visible range fluoro fours in the far red end of the spectrum, such as Alexa Fluor 6 47 are recommended to reduce background to trigger samples to recover, move the plate to a 37 degree Celsius incubator and continue culture for 19 hours. This allows the material to recover and the cells to adapt their morphology to the new topography. Following incubation, apply the appropriate stains such as fallin for filamentous actin imaging.
As the final step, the cells can be imaged on the new topography shown here is the bulk one way shape memory of a single NOA 63 cure. Repeated three times where the asterisk indicates experimental onset cured. NOA 63 is a transparent, glassy solid that has excellent shape memory properties.
In this case, the material was cured and shows a uniform TG of 51.1 degrees Celsius as determined from the onset of storage modulus drop. The drop in strain seen here occurs at the measured tg. A large percentage of the 4.7%applied strain was fixed after unloading a 20 degrees Celsius corresponding to a fixing ratio of 89.3%The fixed strain was then recovered at a ratio of 84.4%in a relatively small temperature range during heating.
The shape memory performance showed no deterioration up to three cycles as evidenced by all curves following almost exactly the same path. The amplitude of the temporary topography decreases over time at 30 degrees Celsius by 30 hours. At 30 degrees Celsius.
The amplitude has been reduced by approximately 50%indicated here at time zero. It reduces another 10%over the next 9.5 hours. When recovery is triggered by increasing the temperature to 37 degrees Celsius, the amplitude reduces to 0.5%of the initial amplitude within 9.5 hours for embosser used and an embossing stress of 4.9 megapascals.
This corresponds to a functional change of 13 micrometer grooves to a nearly flat surface. This polymer recovers in the temperature range of 30 to 37 degrees under cell culture conditions. This is due to water uptake from the culture medium, the water plastic sizes the polymer, which lowers the tg, allowing for recovery at temperatures below the dry TG of 51 degrees.
An example of a cell behavior controlled through the use of active cell culture substrates is a change in cytoskeleton organization on temporary grooved substrates. Before recovery is triggered, the actin micro filaments align along the direction of the grooves. After recovery by temperature increase, the micro filaments have reorganized and are randomly oriented.
Conversely, control samples that have static, flat or static grooves do not reorganize after increase in temperature. While attempting this procedure, it's important to remember that the geometry of the curing chamber, the transition temperature of the polymer, the method of strain fixing and the composition of the polymer itself can all be adjusted to achieve the transition desired in your own experiment Following this procedure. Other methods such as real time PCR, can be used to answer additional questions such as how gene expression changes in response to the mechanical stimulation from the substrate After its development.
This technique paved the way for researchers in the field of cell mechanobiology to investigate cell cytoskeletal, rearrangement, and soft matter physics in two dimensions. Thanks for your interest in our work and thanks for watching.
