The overall goal of this procedure is to prepare aligned, shape responsive, liquid crystal elastomers and nanocomposites. So liquid crystal elastomers are stimuli responsive materials that change shape in response to stimuli in the environment. And they can be used in a variety of applications, such as tissue engineering, where we know cells respond to stresses and strains in the environment.
In this procedure that we're gonna demonstrate, we rely on materials that are low cost. The method we demonstrate is reliable and it can be implemented in any lab without the need for expensive or specialized equipment. These materials can be applied to the development of cardiac cell sheets, where we know that stresses and strains can affect the differentiation, the maturation and the function of cardiac muscle cells.
Visual demonstration of this procedure is important because the process can be difficult to learn and the samples especially delicate when we're hanging, loading and crosslinking it. So, demonstrating the procedure will be Hojin Kim, Bohan Zhu and Huiying Chen, students from our laboratory and the Jeff Jaycot Laboratory. Begin by combining 166.23 milligrams of a reactive mesogen, 40 milligrams of siloxane and 12.8 milligrams of crosslinker with 0.6 milliliters of anhydrous toluene in a small vial charged with a stirring bar.
Stir the solution at 35 degrees Celsius for 25 minutes for the reagents to completely dissolve. In a separate vial, prepare a one weight percent solution of a platinum catalyst in dichloromethane. Then, add 30 microliters of the catalyst solution to the previously prepared solution and stir the mixture with the stir bar.
Next, pour the solution into a custom made, rectangular polytetrafluoroethylene mold. Cover the mold loosely with a glass slide and place it into an oven at 60 degrees Celsius for 30 minutes. Shake the mold periodically to remove bubbles during the first 15 minutes.
Remove the mold from the oven and cool it quickly with liquid nitrogen by pouring liquid nitrogen into a small container and contacting the bottom of the PTFE mold with the liquid nitrogen for 2 seconds. Once the mixture has cooled, set down a PTFE sheet and use a metal spatula to carefully remove the elastomer from the mold and place it on top of the sheet. Next, use a razor blade to trim the edges of the liquid crystal elastomer and cut it along its length into three equal sized pieces.
Hang each piece to a horizontal rod and hold the elastomer in place using tape. Add one paper clip at a time to the end of the elastomers in 10 minute increments. Allow them to hang for seven days at room temperature and note any changes in length or uniformity.
Discard any sample that tears or breaks. To prepare an electrically responsive, liquid crystal elastomer nanocomposite, add 4.38 milligrams of carbon black nanoparticles to the reaction solution containing the reactive mesogen, the crosslinker and siloxane. During the hanging step of the fabrication, use a total of five paperclips instead of 10 to load the sample.
Add additional carbon black nanoparticles to the liquid crystal elastomer surface. Prepare a 1%weight per volume solution of carbon black nanoparticles in toluene. Sonicate the solution for 20 minutes to disperse carbon nanoparticles and then pour the dispersion into a petri dish.
Then immerse the liquid crystal elastomers into the nanoparticle dispersion for six hours. Once removed from the solution, use the pipette to withdraw excess solution from the petri dish and allow the elastomer to dry in air. Gently clean excess carbon particles on the surface using tape or a cotton swab.
To prepare reversible epoxy-based liquid crystal elastomers, mix 246.15 milligrams of four four prime diglycidyl diloxy biphenyl, 101 milligrams of sebacic acid, 71.6 milligrams of hexadecanedioic acid and 76 milligrams of carboxydecyl terminated PDMS in the rectangular PTFE mold. Heat the samples by placing them on a hotplate at 180 degrees Celsius. Then, add 11.48 milligrams of catalyst and use metal tweezers preheated to 180 degrees Celsius to stir the mixture.
Continue to stir the mixture periodically to remove bubbles generated by the reaction. Continue reacting the mixture for approximately 20 minutes. Once gelled, remove the PTFE boat from the hotplate and allow the mixture to cool to room temperature.
Then, use a razor blade to separate the elastomer from the PTFE mold. Next, place two PTFE sheets in a polymer press at 180 degrees Celsius. Place the elastomer between the PTFE sheets and compress the sample to a thickness of 0.3 to 0.5 millimeters.
Continue heating the elastomer at 180 degrees Celsius for four hours. After four hours, remove the sample and cool it to room temperature. Then, cut the sample into rectangular pieces, 2.5 centimeters in length and 0.5 centimeters in width.
Hang the sample at one end using polyimide tape inside a heating oven. Attach 12 paper clips with a total weight of 8.88 grams to the free end of the sample. Then set the temperature of the oven to 165 degrees Celsius and heat the samples overnight.
Remove the elastomer from the oven and note the change in length. Next, heat the sample to 80 degrees Celsius on a hotplate to remove residual stress, then cool back to room temperature. Measure reversible strain by placing the samples onto a hotplate and heating the plate back up to 120 degrees Celsius.
Image the sample at room temperature after heating to 120 degrees Celsius and after cooling back to room temperature. Use the images to measure the sample length at each step. For thermomechanical measurements, use a razor blade to manually cut samples to dimensions of two centimeters by 0.3 centimeters and carefully fasten them, one at a time, in between the tension clamps.
Apply a force of one millinewton to the sample in order to first remove any slack. Then, thermally equilibrate samples at 30 degrees Celsius, followed by heating and cooling cycles. Heat the sample at five degrees Celsius per minute from 30 degrees Celsius up to 120 degrees Celsius.
Record the changes in the length and width of the sample during the temperature change. Treat one surface of liquid crystal elastomer nanocomposites under oxygen plasma for 30 seconds. Then, spin cast 300 microliters of a 1%weight per volume solution of polystyrene in toluene at 3, 300 rpm for one minute on top of the plasma cleaned surface.
Dry the elastomer under vacuum for 12 hours to remove toluene. And then treat the polystyrene coated surface of the LCE nanocomposite using oxygen plasma for 30 seconds. Sterilize the plasma treated elastomer nanocomposites in 70%ethanol for 30 minutes.
And then wash them with phosphate buffered saline. Transfer the washed elastomer nanocomposites to a dry petri dish with a polystyrene coated side facing up. Next, add five milliliters of type one collagen solution to a dish and coat the entire surface of the samples by immersing them in the collagen solution.
Then, cover the samples and incubate them at 37 degrees Celsius and 5%carbon dioxide for at least 30 minutes. Plate isolated neonatal rat ventricular cardiomyocytes on top of the substrates at a density of 100, 000 to 600, 000 cells per square centimeter. Culture the cells as described in the accompanying text protocol.
Once the cells have firmly attached, transfer the seeded liquid crystal elastomer nanocomposites to a custom, 3D printed vessel. Next, insert a rectangular plastic piece through the notches in the 3D vessel to hold the liquid crystal elastomer in place at one or both ends. Place the sample across the carbon rods and fix it on one end to ensure a good electrical contact.
Ensure that the vessel is filled with cell culture maintenance media and is equipped with parallel conductive carbon rods connected to an electric source. Electrically stimulate liquid crystal elastomers with a 40 volt A/C electrical potential, which has a five second on off time for a total of 24 hours. Liquid crystal elastomers contract and elongate reversibly when heated from room temperature to above the nematic to isotropic transition temperature, roughly 80 degrees Celsius.
Dynamic mechanical analysis provides a quantitative measure of the liquid crystal elastomer shape change as a function of temperature. The sample described by the graph shown here was heated and cooled through four cycles. Once heated, the samples display about a 35%shortening in the direction it was hung during fabrication.
The sample also expands 25 to 30%in the perpendicular direction. Liquid crystal elastomer nanocomposites contract on the application of an electrical potential. When exposed to 40 volt A/C, the liquid crystal elastomer nanocomposites shorten about 22 1/2%in the direction they were hung during fabrication.
Here, the samples were exposed to an on off cycle every 15 seconds. So once the procedure is mastered, it can be completed in two hours and the entire synthesis will be complete after one week after hanging the sample. While attempting this procedure, it is important that you remember to hang the LC with correct amount of weight.
Otherwise, you may not align the sample properly or break it during the fabrication. Following this procedure, you can add other types of additives to LCE like carbon nanotubes to understand what stimuli response it may have. We hope that this research will pave the way for biologists and bioengineers to shape responsive substrates to investigate the effect of stress and strain on cell growth, cell alignment and cell behavior.
So after watching this video, you should have a good understanding of how to make aligned, shape responsive liquid crystal elastomers and liquid crystal elastomer nanocomposites.
