The authors would like to thank Kent State University (collaborative research grant and support for the Regenerative Medicine Initiative at Kent State &#8722; ReMedIKS) for the financial support of this project.

NOTE: The following steps for the 3D LCE foam-like preparation using the 3-arm star block copolymer are shown in Figure 1. For nuclear magnetic resonance (NMR) characterization, the spectra are recorded in deuterated chloroform (CDCl3) at room temperature on a Bruker DMX 400-MHz instrument and internally referenced residual peaks at 7.26. Fourier transform infrared (FT-IR) spectra are recorded using a Bruker Vector 33 FT-IR spectrometer using attenuated total reflectance mode. For each step of the following protocol, it is important to wear appropriate personal protective clothing (PPE).
1. Synthesis of &#945;-Chloro-&#949;-caprolactone (Monomer) (According to the Procedure in J&#233;r&#244;me et al.28)
<ol>
	<li>Before beginning the synthesis, purify 27 g of 3-chloroperbenzoic acid as follows:
	<ol>
		<li>In 800 mL of distilled water, add 1.28 g of sodium phosphate monobasic monohydrate and 8.24 g of sodium phosphate dibasic heptahydrate. Adjust the pH to 7.4 (using sodium hydroxide or hydrochloric acid) and reserve 30 mL of this solution; this is the buffer solution.</li>
		<li>Using a separatory funnel, dissolve 3-chloroperbenzoic acid in 35 mL of diethyl ether. Wash the organic solution with 10 mL of buffer solution (prepared in step 1.1.1.). Repeat the wash three times.</li>
		<li>Add 3 g of sodium sulfate directly to the organic solution; this drying agent absorbs water from the organic solution.</li>
		<li>Filter the solution to remove the drying agent. Concentrate the filtrate under reduced pressure by using a rotary evaporator at 850 mbar and 40 &#176;C.</li>
	</ol>
	</li>
	<li>Solubilize 18.5 g of purified 3-chloroperbenzoic acid in 150 mL of dry dichloromethane by stirring in an ultrasonic bath; this process typically takes 20 min. Place the solution inside a separatory funnel.</li>
	<li>Inside a two-neck, round-bottom flask, dissolve 13.1 g of 2-chlorocyclohexanone in 15 mL of dry dichloromethane using a magnetic stirrer under nitrogen gas. Keep stirring.</li>
	<li>Fit the separatory funnel containing 3-chloroperbenzoic acid solution (from step 1.2) to the two-neck flask in step 1.3. Flush the system with nitrogen gas. Adjust the opening of the separatory funnel so that the chloroperbenzoic acid solution falls dropwise into the 2-chlorocyclohexanone solution (1 drop every other second) and continue stirring the mixture under nitrogen gas for 96 h.</li>
	<li>Cool the reaction mixture to -20 &#176;C for 1 h to precipitate the m-chlorobenzoic acid (m-CBA) byproduct.</li>
	<li>Filter the m-chlorobenzoic acid (m-CBA) and wash the remaining solution with saturated solutions of sodium thiosulfate, sodium bicarbonate, and sodium chloride.</li>
	<li>Remove the solvent under reduced pressure using a rotary evaporator at 850 mbar and 40 &#176;C. Purify the pale yellow, viscous liquid by distillation under reduced pressure at 2.3 Torr and 96 &#176;C.</li>
	<li>Monitor the success of the synthesis using the following 1H NMR peaks. 1H NMR (400 MHz, CDCl3, &#948;[ppm]): 4.75-4.68 (m, 1H, CHClCO), 4.37-4.26 (m, 1H, CH2O), 4.18-4.05 (m, 1H, CH2O), and 2.06-1.58 (m, 6H, &#8722;CH2&#8722;)6,27.</li>
</ol>
2. Synthesis of &#945;-Three-arm Star Block Copolymer (SBC-&#945;Cl) by Ring Opening Copolymerization (Sharma et al.6 and Amsden et al.29)
<ol>
	<li>Before synthesis, silanize a 20-mL ampoule by filling it with a 2% (v/v) solution of 1H,1H, 2H,2H-perfluorooctyltriethoxysilane in toluene and stirring for about 24 h. Rinse with isopropyl alcohol and dry it by placing it in an oven at 140 &#176;C for 30 min.</li>
	<li>Add 3.64 g of distilled &#949;-caprolactone, 0.5 g of &#945;-chloro-&#949;-caprolactone, and 0.25 mL of glycerol to the ampoule. Mix using a vortex for 1 min.</li>
	<li>Add 4.90 g of D,L-lactide to the ampoule and purge it with nitrogen. Place the ampoule in an oven at 120 &#176;C to melt the D,L-lactide; this process typically takes about 2 h. Mix again using a vortex to make sure that all contents are well mixed and add 66 &#181;L of tin(II)2-ethylhexanoate (Sn(Oct)2) to the ampoule. 
	NOTE: D,L-lactide will cool down during this process and will need to be re-heated in the oven to melt.</li>
	<li>Vigorously mix one last time using the vortex and flush with nitrogen.</li>
	<li>Close the ampoule with a rubber stopper. Place a needle connected to a vacuum tube (the house vacuum is usually enough) through the rubber stopper. Turn on the vacuum and, using a flame, melt the long neck of the glass, twisting slowly until the glass collapses on itself. Be careful to not melt the rubber stopper. Once the ampoule is flame-sealed, place it in a sand bath, an oven, or appropriate heating element at 140 &#176;C for 48 h.</li>
	<li>Take out the ampoule and let it cool at room temperature.</li>
	<li>Break the ampoule at the sealed mark and dissolve the highly viscous liquid by adding 10 mL of dichloromethane. Transfer the solution to a separatory funnel.</li>
	<li>Prepare a flask containing 100 mL of cold methanol (chilled using a dry ice/acetone bath at a temperature around -78 &#176;C). Fix the separatory funnel (step 2.7) on top of the flask. Adjust the opening of the separatory funnel so that about two drops fall every other second (dropwise).</li>
	<li>Collect the white precipitate by filtration (using a paper filter) and dry it in a vacuum oven between 50 and 60 &#176;C.</li>
	<li>Monitor the success of the synthesis using the following 1H NMR and FT-IR peaks. 1H NMR (400 MHz, CDCl3, &#948; [ppm]): 5.29-5.03 (m, COCHCH3), 4.43-4.25 (m, CHCl), 4.24-4.12 (m, CH2O), 4.11-4.03 (t, J = 4.6 Hz, CH2O), 3.80-3.68 (m, CHCH2), 3.09-2.64 (broad, s, OH), 2.39 (t, J = 4.5 Hz, &#945;-H), 2.33 (t, J = 5.1 Hz, &#945;-H), and 1.77-1.25 (m, CH2, CH3); FT-IR (1/&#955; [cm-1]): 2,932 (s), 2,869 (m), 1,741 (s), 961 (s), 866, and 735 (m)6,27. 
	Note: To prepare a more hydrophilic 3D LCE, prepare a linear block copolymer (LBC) instead of an SBC, following the ring opening copolymerization (ROP) procedure described above.
	<ol>
		<li>Add 0.3 g of polyethylene glycol (PEG), 3.15 g &#949;-caprolactone, 1.0 g of &#945;-chloro-&#949;-caprolactone, and 5.0 g of D,L-lactide to the silanized vial mix.</li>
	</ol>
	</li>
</ol>
3. Synthetic Modification of &#945;-Cl-Three Arm SBC to &#945;-N3-Three Arm SBC (SBC-&#945;N3) (According to Sharma et al.6)
<ol>
	<li>In a round-bottom flask and under nitrogen, dissolve 5 g of SBC-&#945;Cl in 30 mL of dry N,N&#39;-dimethylformamide.</li>
	<li>Add 0.22 g of sodium azide and allow to react overnight at room temperature. 
	Caution: Sodium azide is toxic; wear appropriate personal protective clothing (PPE).</li>
	<li>Remove the N,N&#39;-dimethylformamide under reduced pressure using a rotary evaporator at 11 mbar and 40 &#176;C. Dissolve the mixture in 30 mL of toluene. Centrifuge the solution thrice at 2,800 x g for 15 min to remove the salt formed. Evaporate the toluene under reduced pressure using a rotary evaporator at 77 mbar and 40 &#176;C.</li>
	<li>Monitor the success of the azide substitution using 1H NMR and FT-IR peaks. 
	NOTE: 1H NMR spectrum was similar to the parent SBC-&#945;Cl, with the exception of the appearance of a peak at 3.90 ppm related to the azide group; FT-IR (1/&#955; [cm-1]): 2,928, 2,108 (s, azide), 1,754 (s), 1,450 (s), 960 (m), 865 (s), 733 (s), and 696 (m).</li>
</ol>
4. Synthesis of Cholesteryl 5-Hexynoate (LC Moiety) (According to Sharma et al.6 and Donaldson et al.30)
<ol>
	<li>In a round-bottom flask, mix 3 g of 5-hexynoic acid and 130 mL of dichloromethane. Cool to 0 &#176;C using an ice bath.</li>
	<li>In another round-bottom flask, mix 8.28 g of N,N&#39;-dicyclohexylcarbodiimide, 10.3 g of cholesterol, and 0.2 g of 4-dimethylaminopyridine.</li>
	<li>Transfer the 5-hexynoic acid solution dropwise to the flask containing the cholesterol mixture and maintain the final mixture at 0 &#176;C for 1 h.</li>
	<li>Allow the mixture to warm up to room temperature overnight.</li>
	<li>Remove the resulting dicyclohexylurea precipitate by filtration using a grade 415 paper filter and discard it.</li>
	<li>Concentrate the filtrate under reduced pressure using a rotary evaporator at 850 mbar and 40 &#176;C. Dissolve the collected residue in 150 mL of hexane.</li>
	<li>Evaporate the solvent under reduced pressure using a rotary evaporator at 335 mbar and 40 &#176;C. Add 350 mL of ethanol to the oily residue to collect the final product. Wash the off-white solid formed with ethanol and dry the solid product under vacuum at 50 &#176;C.</li>
	<li>Monitor the synthesis using the following 1H NMR and FT-IR peaks. 1H NMR (400 MHz, CDCl3, &#948; [ppm]): 5.39 (d, J = 4.7 Hz, 1H, CH=C), 4.70-4.58 (m, 1H, CHOCO), 2.44 (t, J = 2.5 Hz, 2H, CH2CO), 2.34 (m, 2H, CH2-CH=), 2.31 (m, 2H, CH2CH2-COO), 2.28 (s, 1H, HC&#8801;C), 2.27 (d, J = 2.3 Hz, 2H, &#8801;C-CH2), 2.07-1.06 (m, 2H, CH2, CH), 0.94 (s, 3H, CH3), 0.89 (d, J = 1.8 Hz, 3H, CH3CH), and 0.88 (d, J = 1.8 Hz, 6H, CH3-CH), 0.67 (s, 3H, CH3). FT-IR (1/&#955; [cm-1]): 2,830-2,990 (broad and strong peak), 2,104 (m), 1,695 (s), 1,428 (m), 1,135 (m), 999 (s), 798 (s), and 667 (s).</li>
</ol>
5. Synthetic Modification of &#945;-N3-Three Arm SBC to &#945;-Cholesteryl-Three Arm SBC (SBC-&#945;CLC) via an Azide-Alkyne Huisgen Cyclo-addition Reaction (&#34;Click&#34; Reaction) to Obtain SBC-Chol (According to Sharma et al.6)
<ol>
	<li>In a round-bottom flask, dissolve 1 molar equivalent of SBC-&#945;N3 (1.5 g) in 100 mL of freshly distilled tetrahydrofuran (THF). Add 1.2 molar equivalent of cholesteryl 5-hexynoate (1.94 g), 0.1 molar equivalent of copper(I)iodide (0.06 g), and 0.1 molar equivalent of triethylamine (0.03 g). Stir the mixture overnight at 35 &#176;C under nitrogen.</li>
	<li>Evaporate the solvent under reduced pressure using a rotary evaporator at 357 mbar and 40 &#176;C.</li>
	<li>Dissolve the residual mixture in 80 mL of dichloromethane and centrifuge for 5 min at 2,800 x g at room temperature to remove unreacted materials and side products.</li>
	<li>Monitor the synthesis using the following 1H NMR and FT-IR peaks. 1H NMR (400 MHz, CDCl3, &#948; [ppm]): 7.54 (s, CH=C-triazole), 5.43-5.34 (m, C=CH cholesterol), 5.10-5.06 (m, COCHCH3), 4.68-4.55 (m, O-CH cholesterol), 4.24-4.19 (m, CH2O), 4.18-4.13 (t, J=5.0 Hz, CH2O), 4.11-4.05 (t, J=4.4 Hz, CH2O), 2.47-2.41 (t, J=4.9 Hz, COCH2), 2.31-2.25 (m, COCH2), 2.07-1.02 (m, CH2, CH3), 1.05-1.03 (s, CH2, CH3), 0.96-0.92 (d, J=3.3 Hz, CH2, CH3), 0.91-0.87 (dd, J=1.9 Hz, J=1.8 Hz, CH3), and 0.71-0.68 (s, CH3). FT-IR (1/&#955; [cm-1]): 3,260 (s), 2,920 (s), 1,710 (s), 1,460 (s), 1,370 (s), 1,240 (m), 1,190 (s), 733 (s), and 668 (s).</li>
</ol>
6. Synthesis of 2,2-Bis(1-caprolactone-4-yl)propane (Crosslinker, BCP) (According to Gao et al.27 and Albertsson et al.31)
<ol>
	<li>In a round-bottom flask, prepare a solution containing 10.8 g of 2-bis(4-hydroxy-cyclohexyl)propane and 52 mL of acetic acid.</li>
	<li>Prepare a solution containing 11 g of chromium trioxide in 50 mL of acetic acid and 8 mL of distilled water. Add this solution dropwise to the solution prepared in step 6.1 while maintaining the mixture temperature between 17 and 20 &#176;C (e.g., in a water bath); this dropwise process takes 2 h. Once the process is complete, allow the solution to stir for about 30 min.</li>
	<li>Add 50 mL of 2-propanol. Stir the solution overnight at room temperature.</li>
	<li>Concentrate the dark purple solution under reduced pressure using a rotary evaporator at 137 mbar and 40 &#176;C. Add 300 mL of distilled water to precipitate; the precipitate should be light purple.</li>
	<li>Filtrate the crude product using a grade 415 paper filter. Wash the solid material with ~ 250 mL of distilled water or until the solid becomes white.</li>
	<li>Dissolve the solid material in 15 mL of benzene at 40 &#176;C and let it recrystallize at 25 &#176;C.</li>
	<li>Add 8.34 g of dry diketone dissolved in dry dichloromethane and a solution containing 6.0 g of 3-chloroperbenzoic acid in 75 mL of dichloromethane to the flask.</li>
	<li>Reflux the solution at 40 &#176;C for 24 h.</li>
	<li>Cool the reaction mixture to -20 &#176;C for 10 min to precipitate the m-chlorobenzoic acid byproduct.</li>
	<li>Remove m-chlorobenzoic acid by filtration (using a paper filter) and concentrate the solution under reduced pressure.</li>
	<li>Wash the viscose crude product with 200 mL of 2-heptanone and dry the precipitate under vacuum at 50 &#176;C overnight.</li>
	<li>Monitor the synthesis using the following 1H NMR and FT-IR peaks. 1H NMR (400 MHz, CDCl3, &#948; [ppm]): 4.42-4.37 (dd, J = 14.2, 7.4 Hz, 2H, CH2OC=O), 4.21-4.15 (t, 2H, J = 11.3 Hz, CH2OC=O), 2.80-2.75 (ddt, J = 14.3, 6.5, 1.6 Hz, 2H, CH2COO), 2.63-2.57 (tt, J = 13.3, 2.1 Hz, 2H, CH2COO), 2.04-1.93 (M, 4H, -CH2CH2OC=O), 1.70-1.56 (m, 4H, -CH2 CH2COO), 1.48-1.38 (m, 2H, -CHC(CH3)2), and 0.84 (s, 6H, CH3C-).</li>
</ol>
7. Creation of Porous 3D Elastomer Scaffold Using either Hexamethylene Diisocyanate (HDI) or 2,2-Bis(1-caprolactone-4-yl)propane (BCP)27 as Crosslinkers (According to Gao et al.27)
<ol>
	<li>Prepare three-arm elastomer mixture using 0.75 g of SBC-&#945;CLC by adding 0.25 mL of HDI (or 0.45 mL of BCP) and 0.24 mL of distilled &#949;-caprolactone monomer. Add 60 &#181;L of Sn(Oct)2. If using BCP instead of HDI, mix SBC-&#945;CLC and BCP using a vortex and place them in an oven at 140 &#176;C until the BCP fully melts and dissolves (this step can take up to 2 h). Once the BCP has been dissolved, take it out of the oven and, the Sn(Oct)2, and vortex.</li>
	<li>Prepare a &#34;sacrificial&#34; nickel foam template by cutting a 1 x 4 cm metal piece. Roll it from one of the short ends so that the final roll is approximately a 1 x 1 cm metal piece (see Figure 4).</li>
	<li>Put the nickel foam in a glass vial or aluminum foil pack and pour the mixture prepared in step 7.1 to fully cover the foam for 2 min. Remove the excess mixture with a Pasteur pipet. Leave it in an oven overnight at 80 &#176;C.</li>
	<li>Peel off the aluminum foil or break the glass. Using a razor blade, shave the elastomer around the metal foam to expose the nickel metal.</li>
	<li>Prepare 1 M iron(III)chloride (FeCl3) solution in 100 mL of water. Place the foam in a flask and add 70 mL of FeCl3 solution. Stir for three days at room temperature and change the FeCl3 solution every day. Before each change, stir the foam with ionized water for 0.5 h. 
	NOTE: The etching process is typically completed after three days. To ensure that the etching process is completed, perform tactile compression tests until the foams feel soft. Foam resistance to tactile compression tests indicates the presence of a residual metal template.</li>
	<li>Rinse the elastomer foam with ethanol and place in a vacuum oven overnight at 40 &#176;C.</li>
	<li>Characterize the materials using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), mechanical compression tests, and thermal gravimetric analysis (TGA)27. 
	NOTE: For preparing a more hydrophilic 3D LCE, replace the SBC with LBC (making sure that the LBC also contains an LC moiety) following the steps described in step 7.5.</li>
</ol>
8. Seeding of Elastomer Scaffold with SH-SY5Y Neuroblastoma Cells and Culture Using Sterile Techniques
<ol>
	<li>Sterilize the elastomer by washing it twice with 1 mL of 70% ethanol. Perform UV irradiation for 10 min and wash with 1 mL of 70% ethanol. Rinse it twice with 1 mL of sterile water and 1 mL of phosphate-buffered saline (PBS). Place the elastomers in 24-well culture plates.</li>
	<li>Prepare cell growth medium for SH-SY5Y containing 90% Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Pen-Strep).</li>
	<li>After counting the cells using a hematocytometer, prepare 1.5 x 105 SH-SY5Y cells suspended in 100 &#956;L of growth medium (seed solution). Add the solution on top of the elastomers, making sure to form a drop.</li>
	<li>Incubate the seeded elastomers at 37 &#176;C and 5% CO2 for 2 h to promote cell adhesion. Add 0.5 mL of growth medium. Incubate again at 37 &#176;C with 5% CO2.</li>
	<li>Change the medium every other day after washing with 1 mL of PBS.</li>
</ol>
9. Microscopic Imaging of Elastomer Construct
<ol>
	<li>Fix the cells grown on elastomers with 4% paraformaldehyde (PFA) in PBS for 15 min. Rinse with 3 mL of PBS three times for 5 min each and place the elastomer with the fixed cells in a culture dish with an attached coverslip. 
	Caution: PFA is toxic. Wear appropriate personal protective clothing (PPE).</li>
	<li>Stain the fixed samples with 0.1% of 4&#39;,6-diamidino-2-phenylindole (DAPI) in 500 &#181;L of PBS for 10 min and rinse twice with 1 mL of PBS for 5 min.</li>
	<li>Immediately image the elastomers using confocal microscopy with DAPI fluorescence, acquiring image stacks that span the sample. 
	NOTE: Here, image stacks were acquired using a 20X objective and a 60X objective.</li>
	<li>Analyze the optical confocal image stacks using ImageJ32.</li>
</ol>

<ol>
	<li>Khor, E., Lim, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantable+applications+of+chitin+and+chitosan.">Implantable applications of chitin and chitosan.</a> Biomaterials. 24 (13), 2339-2349 (2003).</li><li>Chung, H. J., Park, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+engineered+and+drug+releasing+pre-fabricated+scaffolds+for+tissue+engineering.">Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering.</a> Adv. Drug Deliv. Rev. 59 (4-5), 249-262 (2007).</li><li>Yakacki, C. M., Gall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-Memory+Polymers+for+Biomedical+Applications.">Shape-Memory Polymers for Biomedical Applications.</a> Shape-Memory Polymers. 226, 147-175 (2010).</li><li>Agrawal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+liquid+crystal+elastomers+for+dynamic+cell+culture.">Stimuli-responsive liquid crystal elastomers for dynamic cell culture.</a> J. of Mat. Res. 30 (4), 453-462 (2015).</li><li>Agrawal, A., Yun, T. H., Pesek, S. L., Chapman, W. G., Verduzco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-responsive+liquid+crystal+elastomer+bilayers.">Shape-responsive liquid crystal elastomer bilayers.</a> Soft Matter. 10 (9), 1411-1415 (2014).</li><li>Sharma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+and+Porous+Liquid+Crystal+Elastomer+Scaffolds+for+Spatial+Cell+Cultures.">Biodegradable and Porous Liquid Crystal Elastomer Scaffolds for Spatial Cell Cultures.</a> Macromol. Biosci. 15 (2), 200-214 (2015).</li><li>Yakacki, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailorable+and+programmable+liquid-crystalline+elastomers+using+a+two-stage+thiol-acrylate+reaction.">Tailorable and programmable liquid-crystalline elastomers using a two-stage thiol-acrylate reaction.</a> RSC Adv. 5 (25), 18997-19001 (2015).</li><li>deGennes, P. G., Hebert, M., Kant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+muscles+based+on+nematic+gels.">Artificial muscles based on nematic gels.</a> Macromolecular Symposia. 113, 39-49 (1997).</li><li>Fleischmann, E. -. K., Zentel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-Crystalline+Ordering+as+a+Concept+in+Materials+Science:+From+Semiconductors+to+Stimuli-Responsive+Devices.">Liquid-Crystalline Ordering as a Concept in Materials Science: From Semiconductors to Stimuli-Responsive Devices.</a> Angew. Chem. Int. Ed. 52 (34), 8810-8827 (2013).</li><li>Finkelmann, H., Kim, S. T., Munoz, A., Palffy-Muhoray, P., Taheri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+mirrorless+lasing+in+cholesteric+liquid+crystalline+elastomers.">Tunable mirrorless lasing in cholesteric liquid crystalline elastomers.</a> Adv. Mater. 13 (14), 1069-1072 (2001).</li><li>Artal, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHG+characterization+of+different+polar+materials+obtained+by+in+situ+photopolymerization.">SHG characterization of different polar materials obtained by in situ photopolymerization.</a> Macromolecules. 34 (12), 4244-4255 (2001).</li><li>Camacho-Lopez, M., Finkelmann, H., Palffy-Muhoray, P., Shelley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+liquid-crystal+elastomer+swims+into+the+dark.">Fast liquid-crystal elastomer swims into the dark.</a> Nat. Mater. 3 (5), 307-310 (2004).</li><li>Yamada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photomobile+polymer+materials:+Towards+light-driven+plastic+motors.">Photomobile polymer materials: Towards light-driven plastic motors.</a> Angew. Chem. Int. Ed. 47 (27), 4986-4988 (2008).</li><li>Ohm, C., Brehmer, M., Zentel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Crystalline+Elastomers+as+Actuators+and+Sensors.">Liquid Crystalline Elastomers as Actuators and Sensors.</a> Adv. Mater. 22 (31), 3366-3387 (2010).</li><li>Fleischmann, E. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-piece+micropumps+from+liquid+crystalline+core-shell+particles.">One-piece micropumps from liquid crystalline core-shell particles.</a> Nat. Commun. 3, (2012).</li><li>Herzer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printable+Optical+Sensors+Based+on+H-Bonded+Supramolecular+Cholesteric+Liquid+Crystal+Networks.">Printable Optical Sensors Based on H-Bonded Supramolecular Cholesteric Liquid Crystal Networks.</a> J. Am. Chem. Soc. 134 (18), 7608-7611 (2012).</li><li>Lockwood, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermotropic+liquid+crystals+as+substrates+for+imaging+the+reorganization+of+matrigel+by+human+embryonic+stem+cells.">Thermotropic liquid crystals as substrates for imaging the reorganization of matrigel by human embryonic stem cells.</a> Adv. Funct. Mater. 16 (5), 618-624 (2006).</li><li>Sharma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+structural+variations+on+the+cellular+response+and+mechanical+properties+of+biocompatible,+biodegradable,+and+porous+smectic+liquid+crystal+elastomers.">Effects of structural variations on the cellular response and mechanical properties of biocompatible, biodegradable, and porous smectic liquid crystal elastomers.</a> Macromol. Biosci. , (2016).</li><li>Bera, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Crystal+Elastomer+Microspheres+as+Three-Dimensional+Cell+Scaffolds+Supporting+the+Attachment+and+Proliferation+of+Myoblasts.">Liquid Crystal Elastomer Microspheres as Three-Dimensional Cell Scaffolds Supporting the Attachment and Proliferation of Myoblasts.</a> ACS Appl. Mater. Interfaces. 7 (26), 14528-14535 (2015).</li><li>Bera, T., Malcuit, C., Clements, R. J., Hegmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Surfactant+during+Microemulsion+Photopolymerization+for+the+Creation+of+Three-Dimensional+Liquid+Crystal+Elastomer+Microsphere+Spatial+Cell+Scaffolds.">Role of Surfactant during Microemulsion Photopolymerization for the Creation of Three-Dimensional Liquid Crystal Elastomer Microsphere Spatial Cell Scaffolds.</a> Front. Mater. 3 (31), (2016).</li><li>McKee, C. T., Last, J. A., Russell, P., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indentation+Versus+Tensile+Measurements+of+Young's+Modulus+for+Soft+Biological+Tissues.">Indentation Versus Tensile Measurements of Young's Modulus for Soft Biological Tissues.</a> Tissue Eng. Part B Rev. 17 (3), 155-164 (2011).</li><li>Kung, C. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+three+dimensional+graphene+foam+supported+platinum-ruthenium+bimetallic+nanocatalysts+for+hydrogen+peroxide+based+electrochemical+biosensors.">Preparation and characterization of three dimensional graphene foam supported platinum-ruthenium bimetallic nanocatalysts for hydrogen peroxide based electrochemical biosensors.</a> Biosens. Bioelectron. 52, 1-7 (2014).</li><li>Amsden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curable,+biodegradable+elastomers:+emerging+biomaterials+for+drug+delivery+and+tissue+engineering.">Curable, biodegradable elastomers: emerging biomaterials for drug delivery and tissue engineering.</a> Soft Matter. 3 (11), 1335-1348 (2007).</li><li>Sinturel, C., Vayer, M., Morris, M., Hillmyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Vapor+Annealing+of+Block+Polymer+Thin+Films.">Solvent Vapor Annealing of Block Polymer Thin Films.</a> Macromolecules. 46 (14), 5399-5415 (2013).</li><li>Riboldi, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+myogenesis+on+highly+orientated+microfibrous+polyesterurethane+scaffolds.">Skeletal myogenesis on highly orientated microfibrous polyesterurethane scaffolds.</a> J. Biomed. Mater. Res. A. 84 (4), 1094-1101 (2008).</li><li>Chung, S., Moghe, A. K., Montero, G. A., Kim, S. H., King, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrous+scaffolds+electrospun+from+elastomeric+biodegradable+poly(L-lactide-co-epsilon-caprolactone)+copolymer.">Nanofibrous scaffolds electrospun from elastomeric biodegradable poly(L-lactide-co-epsilon-caprolactone) copolymer.</a> Biomed. Mater. 4 (1), 9 (2009).</li><li>Gao, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+3D+Liquid+Crystal+Elastomer+Cell+Scaffolds+and+Foams+with+Primary+and+Secondary+Porous+Architecture.">Biocompatible 3D Liquid Crystal Elastomer Cell Scaffolds and Foams with Primary and Secondary Porous Architecture.</a> ACS Macro Lett. 5 (1), 14-19 (2016).</li><li>Lenoir, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ring-opening+polymerization+of+alpha-chloro-is+an+element+of-caprolactone+and+chemical+modification+of+poly(alpha-chloro-is+an+element+of-caprolactone)+by+atom+transfer+radical+processes.">Ring-opening polymerization of alpha-chloro-is an element of-caprolactone and chemical modification of poly(alpha-chloro-is an element of-caprolactone) by atom transfer radical processes.</a> Macromolecules. 37 (11), 4055-4061 (2004).</li><li>Younes, H. M., Bravo-Grimaldo, E., Amsden, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization+and+in+vitro+degradation+of+a+biodegradable+elastomer.">Synthesis, characterization and in vitro degradation of a biodegradable elastomer.</a> Biomaterials. 25 (22), 5261-5269 (2004).</li><li>Donaldson, T., Henderson, P. A., Achard, M. F., Imrie, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+liquid+crystal+tetramers.">Chiral liquid crystal tetramers.</a> J. Mater. Chem. 21 (29), 10935-10941 (2011).</li><li>Palmgren, R., Karlsson, S., Albertsson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+degradable+crosslinked+polymers+based+on+1,5-dioxepan-2-one+and+crosslinker+of+bis-epsilon-caprolactone+type.">Synthesis of degradable crosslinked polymers based on 1,5-dioxepan-2-one and crosslinker of bis-epsilon-caprolactone type.</a> J. Pol. Sci. A Polym. Chem. 35 (9), 1635-1649 (1997).</li></ol>

The authors have nothing to disclose.

Liquid crystalline elastomers have recently been studied as biocompatible cell scaffolds due to their stimuli responsiveness. They have been proven to be ideal platforms as cell scaffolds. However, an important factor to keep in mind when preparing and designing a new LCE scaffold is porosity. The incorporation of leachable solids23 or gases does not always result in homogeneous porosity or fully interconnected pores. The use of a metal template that can be etched out not only offers the opportuni...

There are several examples of biological and biocompatible synthetic materials designed for application in cell studies and for tissue regeneration aiming at cell attachment and proliferation1,2,3,4,5. There have been a few examples of biocompatible materials, known as liquid crystal elastomers (LCEs), that could respond to external stimuli with anisotropic molecular ordering6,7. LCEs are stimuli-responsive materials that combine the mechanical and elastic properties of elastomers with the optical functionality and molecular ordering of liquid crystals8,9. LCEs can experience changes in shape, mechanical deformation, elastic behavior, and optical properties in response to external stimuli (i.e., heat, stress, light, etc.)10,11,12,13,14,15,16. Earlier studies have shown that liquid crystals (LCs) can sense the growth and orientation of cells4,17. It is possible then to assume that LCEs may be suitable for biologically and medically relevant applications, including cell scaffolding and alignment. We have previously reported the preparation of smectic biocompatible, biodegradable, cast-molded, and thin LCEs films featuring a &#34;Swiss-cheese type&#34; porous morphology6,18. We also prepared nematic biocompatible LCEs with globular morphology as scaffolds for cell growth19,20. Our work was aimed at tuning the mechanical properties of the materials to match those of the tissue of interest21. Also, these studies focus on understanding elastomer-cell interactions, as well as cellular response when the elastomers are subject to external stimuli.
The main challenges were in part to tailor the porosity of the LCEs to allow for cell attachment and permeation through the elastomer matrix and for better mass transport. The porosity of these thin films6 allowed for cell permeation through the bulk of the matrix, but not all pores were fully interconnected or had a more regular (homogeneous) pore size. We then reported on biocompatible nematic LCE elastomers with globular morphologies. These nematic elastomers allowed for the attachment and proliferation of cells, but the pore size ranged only from 10-30 &#181;m, which prevented or limited the use of these elastomers with a wider variety of cell lines19,20.
Previous work by Kung et al. relating to the formation of graphene foams using a &#34;sacrificial&#34; metal template showed that the obtained graphene foam had a very regular porous morphology dictated by the chosen metal template22. This methodology offers full control of porosity and pore size. At the same time, the malleability and flexibility of the metal template allow for the formation of different template shapes prior to foam preparation. Other techniques, such as material leaching23, gas templating24, or electro-spun fibers25,26 also offer the potential for the preparation of porous materials, but they are more time consuming and, in some cases, the pore size is limited to only a few micrometers. Foam-like 3D LCEs prepared using metal templates allow for a higher cell load; an improved proliferation rate; co-culturing; and, last but not least, better mass transport management (i.e., nutrients, gases, and waste) to ensure full tissue development27. Foam-like 3D LCEs also appear to improve cell alignment; this is most likely in relation to the LC pendants sensing cell growth and cell orientation. The presence of LC moieties within the LCE appears to enhance cell alignment with respect to cell location within the LCE scaffold. Cells align within the struts of the LCE, while no clear orientation is observed where the struts join together (junctions)27.
Overall, our LCE cell scaffold platform as a cell support medium offers opportunities to tune the elastomer morphology and elastic properties and to specifically direct the alignment of (individual) cell types to create an ordered, spatial arrangements of cells similar to living systems. Apart from providing a scaffold capable of sustaining and directing long-term cellular growth and proliferation, LCEs also allow for dynamic experiments, where cell orientation and interactions may be modified on the fly.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:132px;">L16606</td>
			<td style="width:153px;">Silanizing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-bis(4-hydroxy-cyclohexyl)propane</td>
			<td>TCI</td>
			<td><a href="http://www.tcichemicals.com/eshop/en/us/commodity/B0928/">B0928</a></td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-chlorohexanone&#160;</td>
			<td>Alfa Aesar</td>
			<td>A18613</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-heptanone&#160;</td>
			<td>Sigma Aldrich</td>
			<td>W254401</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-propanol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>278475</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-chloroperbenzoic acid, m-CPBA</td>
			<td>Sigma Aldrich</td>
			<td>273031</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-dimethylaminopyridine</td>
			<td>Alfa Aesar</td>
			<td>A13016</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole, DAPI&#160;</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Nuclear Stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-hexynoic acid&#160;</td>
			<td>Alfa Aesar</td>
			<td>B25132-06</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid</td>
			<td>VWR</td>
			<td>36289</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td>Sigma Aldrich</td>
			<td>34850</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol 200 proof ACS Grade&#160;</td>
			<td>VWR</td>
			<td>71001-866</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzene</td>
			<td>Alfa Aesar</td>
			<td>AA33290</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#949;-caprolactone&#160;</td>
			<td>Alfa Aesar</td>
			<td>A10299-0E</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>VWR</td>
			<td>BDH1109</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Sigma Aldrich</td>
			<td>C8503</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromium(VI) oxide</td>
			<td>Sigma Aldrich</td>
			<td>232653</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Copper (I) iodide</td>
			<td>Strem Chemicals</td>
			<td>100211-060</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D,L-Lactide&#160;</td>
			<td>Alfa Aesar</td>
			<td>L09026</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>320269</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethyl ether&#160;</td>
			<td>Emd Millipore</td>
			<td>EX0190</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>270547</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle medium, DEME&#160;</td>
			<td>CORNING Cellgo</td>
			<td>10-013</td>
			<td>Cell Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Alfa Aesar</td>
			<td>33361</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde&#160;</td>
			<td>SIGMA Life Science</td>
			<td>F8775</td>
			<td>Fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum, FBS&#160;</td>
			<td>HyClone</td>
			<td>SH30071.01</td>
			<td>Media Component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper, Grade 415, qualitative, crepe</td>
			<td>VWR</td>
			<td>28320</td>
			<td>Filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>Central node (3-arm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexamethylene diisocyanate, HDI</td>
			<td>Sigma Aldrich</td>
			<td>52649</td>
			<td>Crosslinker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron(III) chloride&#160;</td>
			<td>Alfa Aesar</td>
			<td>12357</td>
			<td>Etching agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl alcohol</td>
			<td>VWR</td>
			<td>BDH1133</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Alfa Aesar</td>
			<td>L13255</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N&#39;-dicyclohexylcarbodiimide</td>
			<td>Aldrich</td>
			<td>D80002</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>270547</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nickel metal template</td>
			<td>American Elements</td>
			<td>Ni-860</td>
			<td>Foam template</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neuroblastomas cells (SH-SY5Y)</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin&#160;</td>
			<td>Thermo SCIENTIFIC</td>
			<td>15140122</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol 2000, PEG</td>
			<td>Alfa Aesar</td>
			<td>B22181</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide&#160;</td>
			<td>VWR</td>
			<td>97064-646</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>AMRESCO</td>
			<td>865</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>BDH</td>
			<td>BDH9286</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>S-374</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>239313</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran</td>
			<td>Alfa Aesar</td>
			<td>41819</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiosulfate de sodium</td>
			<td>AMRESCO</td>
			<td>393</td>
			<td>Drying salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tin(II) 2-ethylhexanoate</td>
			<td>Aldrich</td>
			<td>S3252</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toluene&#160;</td>
			<td>Alfa Aesar</td>
			<td>22903</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>471283</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin&#160;</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>Cell Detachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV1000</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of biocompatible liquid crystal elastomer foams as cell scaffolds for 3d spatial cell cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This report shows the preparation method of a porous 3D LCE as a scaffold for cell culture using a nickel metal template. The obtained 3D LCE demonstrates a complex interconnected channel network that allows for easy cell infiltration, as well as more suitable mass transport27. It was found that cells are able to fully penetrate the interconnected channel network and are also able to align within the LCE. Here, a metal nickel foam (99% Ni, density of 860 g/cm2...

Watch this Scientific Journal Video about Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures at JoVE.com

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

synthesis-biocompatible-liquid-crystal-elastomer-foams-as-cell

Research

JoVE Journal

Bioengineering

9.4K Views.  Kent State University. This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Video: Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...