3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

The goal of this procedure is to prepare biodegradable, three dimensional foam-like cell scaffolds based on biocompatible side chain liquid crystal elastomers. This method can help answer key questions in the liquid crystal and biomedical fields, such as the effects of liquid crystal elastomer properties on cell proliferation and alignment. The main advantage of this method of two dimensional cell scaffolds is it permits the study of spatial cell-cell interactions which is rarely possible in 2D macro environments.

Generally, those new to this method will struggle with the tactile compression tests. We first had the idea for this method after having to change the media in hundreds of Petri dishes. We wondered if liquid crystal elastomers could support muscle cells on a 3D network, to eliminate the use of so many Petri dishes.

Visual demonstration of this method is critical, as some steps require careful manipulation of chemicals. Further, shaping the metal foam template may be challenging. First, fill a 20 milliliter ampoule with a 2%by volume solution of PFOTES in toluene.

Stir the solution in the ampoule for 24 hours to silanize the ampoule interior. Rinse the silanized ampoule with isopropyl alcohol and dry it at 140 degrees celsius for 30 minutes. Place 3.64 grams of distilled epsilon-caprolactone, 0.5 grams of alpha-chloro-epsilon-caprolactone, and 0.25 milliliters of glycerol in the dry ampoule.

Vortex the mixture for one minute. Then, add 4.90 grams of DL-lactide to the mixture, and purge the ampoule atmosphere with nitrogen gas for one minute. Cover the ampoule opening with aluminum foil, and heat the mixture at 120 degrees Celsius for about two hours to melt the DL-lactide.

Vortex the mixture to disrupt any unmelted solids, and then, add 66 microliters of 10-2-ethylhexanoate to the ampoule, and vortex again. Close the ampoule with aluminum foil and heat the mixture at 120 degrees Celsius for 10 minutes to remelt the DL-lactide. Once the DL-lactide has melted again, vigorously vortex the mixture, and purge the ampoule atmosphere with nitrogen gas.

Seal the ampoule with a rubber septum. Insert a needle connected to a vacuum line through the septum, and start the vacuum. Flame-seal the neck of the ampoule, being careful not to melt the rubber stopper.

Once the neck is sealed, heat the reaction mixture at 140 degrees Celsius for 48 hours. Then, allow the mixture to cool to room temperature. Break open the ampoule and dissolve the viscous reaction mixture in 10 milliliters of dichloromethane.

Transfer the mixture to a separatory funnel. Place a flask containing 100 milliliters of methanol in a dry ice and acetone bath to cool down the methanol to approximately minus 78 degrees Celsius. Once the methanol has cooled down, secure the separatory funnel over the flask.

Add the reaction mixture to the cold methanol at a rate of two drops every second. Collect the resulting white precipitate on filter paper. Dry the precipitate in a vacuum oven between 50 and 60 degrees Celsius to obtain the three-arm alpha-chloro SBC product.

To prepare the alpha cholesteryl three-arm SBC, the pendant chlorine atom is replaced by an azide. A click reaction with cholesteryl 5-hexynoate results in a pendant cholesteryl as the liquid crystal moiety. To begin preparing the liquid crystal elastomer scaffold, combine 0.75 grams of alpha-cholesteryl three-arm SBC with 0.25 milliliters of HDI and 0.24 milliliters of distilled epsilon-caprolactone.

Add to this 60 microliters of 10-2-ethylhexanoate and vortex the mixture. Then, cut out a one centimeter by four centimeter piece of nickel metal foam. Roll the nickel foam into a cylinder one centimeter in diameter and one centimeter tall to form a template for the liquid crystal elastomer foam scaffold.

Place the template in a glass vial or aluminum foil enclosure, and pour the liquid crystal elastomer mixture over the template until it is completely covered. Allow the template to sit in the liquid crystal elastomer mixture for two minutes, and then, remove the excess with a pipette. Heat the mixture and template at 80 degrees Celsius overnight.

Then, peel away the aluminum foil, or break the glass. Use a razor blade to remove excess liquid crystal elastomer to expose the nickel metal template. Place the foam in a flask and add 70 milliliters of a saturated aqueous solution of iron three chloride.

Stir the foam in the solution for three days at room temperature to dissolve the nickel template. Every 24 hours, stir the foam in deionized water for 30 minutes, and then, resume stirring in a fresh iron three chloride solution. After the second day of stirring, perform a tactile compression test on the liquid crystal elastomer foam.

Resistance to tactile compression indicates that the nickel template is still present in the foam. After the third day, all nickel template has been eliminated, and the resulting foam appears very soft and easy to fully compress. The nickel template must be completely eliminated.

It is important to thoroughly rinse the liquid crystal elastomer foam in iron chloride until it's soft to the touch when a tactile compression test is performed. The liquid crystal elastomer foam scaffold is now ready to be characterized and used. Once the liquid crystal elastomer foam is soft, rinse it with 70%ethanol to sterilize it.

To begin the seeding procedure, wash again the liquid crystal elastomer foam scaffolds twice in one milliliter of 70%ethanol to sterilize the elastomer surfaces. Then, irradiate the scaffolds with UV light for 10 minutes. Wash the scaffolds with another one milliliter portion of 70%ethanol.

Rinse the scaffolds with one milliliter each of sterile water and phosphate-buffered saline. Load the sterilized liquid crystal elastomer scaffolds into 24-well culture plates. Prepare and count a suspension of the cells of interest in the appropriate cell growth medium with penicillin and streptomycin.

Dilute the cell suspension to 1.5 times 10 to the fifth cells per 100 microliters using growth medium. Deposit a drop of diluted cell suspension on top of each liquid crystal elastomer scaffold. Incubate the seeded liquid crystal elastomer scaffolds at 37 degrees Celsius in a 5%CO2 atmosphere for two hours.

Add another 0.5 milliliters of growth medium to each scaffold, and continue incubation. Every 48 hours, wash the seeded scaffolds with one milliliter of PBS, and add fresh growth medium. Continue incubating the scaffolds until the cells are ready for microscopy.

These smetic LCs enable the study of complex tissue constructs while promoting cell growth and proliferation. In order to maintain cell viability, foams must be sterilized and cleanliness of cultures maintained at all times. To prepare for microscopy, fix the cells on the scaffolds with a solution of 4%paraformaldehyde in PBS for 15 minutes.

Soak the samples three times in three milliliters of PBS for five minutes. Place one scaffold sample in an Eppendorf tube. Stain the sample with a solution of 0.1%DAPI in 500 microliters of PBS for 10 minutes.

Soak the sample twice in one milliliter of PBS for five minutes. And then, immediately image the sample with confocal microscopy. Acquire image stacks spanning the sample and analyze the data in an image processing program.

A three-arm SBC with liquid crystal moieties was cast on a nickel foam template with HDI as the cross-linker. The nickel template was removed by etching to obtain the liquid crystal elastomer foam. Compression deformation testing was periodically performed to monitor the etching process.

Once the nickel had been completely dissolved, the compression deformation testing showed a 70%reduction in size when compressed. Upon releasing compression, the liquid crystal elastomer foam consistently recovered its original size and shape. This behavior is attributed to the liquid crystal moieties, as similar elastomer foams lacking the cholesterol epsilon-caprolactone moiety did not recover from compression.

SCM of the internal foam morphology showed an interconnected network of hollow foam struts. The regular morphology was attributed to the structure of the nickel foam, indicating that pore size and overall shape can be controlled by selection of an appropriate metal template. The foam was seeded with neuroblastoma cells, which attached to the walls of the network within two days.

30 days after seeding, confocal microscopy revealed that the cells extended over the liquid crystal elastomer network, and had formed multiple layers dispersed throughout the elastomer foam. Elongation of cell nuclei was observed, and was correlated to cell alignment. Once mastered, this technique can be done within three to four weeks if performed properly.

After watching this video, you should have a good understanding of how to design and prepare liquid crystal elastomer cell scaffolds with specific pore sizes and morphologies. While attempting this procedure, it's important to fully characterize each product and to monitor cell viability and expansion. Further, proper staining technique is important to easily acquire confocal microscopy images.

Following this procedure, liquid crystal elastomers can also be exposed to external stimuli, such as stress, or magnetic and electric fields, to answer additional questions about how the anisotropic molecular ordering of the liquid crystal elastomers affects cell response. The implications of this technique apply to the study of disease processes, as it provides a platform to dynamically study the effects of external agents on simulated disease activity, and subsequent repair processes. Though this smectic liquid crystal elastomer foam can provide insight into cell-cell interactions, they can also be used with other materials, such as semiconductor or metal nanostructures.

After its development, this technique paved the way for researchers in biomedical fields to design long-term experimental platforms mirroring living systems.