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>

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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><tbody><tr><td>1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane</td><td>Alfa Aesar</td><td>L16606</td><td>Silanizing agent</td></tr><tr><td>2-bis(4-hydroxy-cyclohexyl)propane</td><td>TCI</td><td>&lt;a href=&quot;http://www.tcichemicals.com/eshop/en/us/commodity/B0928/&quot;&gt;B0928&lt;/a&gt;</td><td>Reagent</td></tr><tr><td>2-chlorohexanone&amp;nbsp;</td><td>Alfa Aesar</td><td>A18613</td><td>Reagent</td></tr><tr><td>2-heptanone&amp;nbsp;</td><td>Sigma Aldrich</td><td>W254401</td><td>Solvent</td></tr><tr><td>2-propanol&amp;nbsp;</td><td>Sigma Aldrich</td><td>278475</td><td>Solvent</td></tr><tr><td>3-chloroperbenzoic acid, m-CPBA</td><td>Sigma Aldrich</td><td>273031</td><td>Reagent</td></tr><tr><td>4-dimethylaminopyridine</td><td>Alfa Aesar</td><td>A13016</td><td>Reagent</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole, DAPI&amp;nbsp;</td><td>Invitrogen</td><td>D1306</td><td>Nuclear Stain</td></tr><tr><td>5-hexynoic acid&amp;nbsp;</td><td>Alfa Aesar</td><td>B25132-06</td><td>Reagent</td></tr><tr><td>Acetic acid</td><td>VWR</td><td>36289</td><td>Solvent</td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td>34850</td><td>Solvent</td></tr><tr><td>Alcohol 200 proof ACS Grade&amp;nbsp;</td><td>VWR</td><td>71001-866</td><td>Reagent</td></tr><tr><td>Benzene</td><td>Alfa Aesar</td><td>AA33290</td><td>Solvent</td></tr><tr><td>&amp;epsilon;-caprolactone&amp;nbsp;</td><td>Alfa Aesar</td><td>A10299-0E</td><td>Reagent</td></tr><tr><td>Chloroform</td><td>VWR</td><td>BDH1109</td><td>Solvent</td></tr><tr><td>Cholesterol</td><td>Sigma Aldrich</td><td>C8503</td><td>Reagent</td></tr><tr><td>Chromium(VI) oxide</td><td>Sigma Aldrich</td><td>232653</td><td>Reagent</td></tr><tr><td>Copper(I) iodide</td><td>Strem Chemicals</td><td>100211-060</td><td>Reagent</td></tr><tr><td>&lt;em&gt;D&lt;/em&gt;,&lt;em&gt;L&lt;/em&gt;-Lactide&amp;nbsp;</td><td>Alfa Aesar</td><td>L09026</td><td>Reagent</td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>320269</td><td>Solvent</td></tr><tr><td>Diethyl ether&amp;nbsp;</td><td>Emd Millipore</td><td>EX0190</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma Aldrich</td><td>270547</td><td>Solvent</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium, DEME&amp;nbsp;</td><td>CORNING Cellgo</td><td>10-013</td><td>Cell Media</td></tr><tr><td>Ethanol</td><td>Alfa Aesar</td><td>33361</td><td>Solvent</td></tr><tr><td>Formaldehyde&amp;nbsp;</td><td>SIGMA Life Science</td><td>F8775</td><td>Fixative</td></tr><tr><td>Fetal bovine serum, FBS&amp;nbsp;</td><td>HyClone</td><td>SH30071.01</td><td>Media Component</td></tr><tr><td>Filter paper, Grade 415, qualitative, crepe</td><td>VWR</td><td>28320</td><td>Filtration</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td>Central node (3-arm)</td></tr><tr><td>Hexamethylene diisocyanate, HDI</td><td>Sigma Aldrich</td><td>52649</td><td>Crosslinker</td></tr><tr><td>Iron(III) chloride&amp;nbsp;</td><td>Alfa Aesar</td><td>12357</td><td>Etching agent</td></tr><tr><td>Isopropyl alcohol</td><td>VWR</td><td>BDH1133</td><td>Solvent</td></tr><tr><td>Methanol</td><td>Alfa Aesar</td><td>L13255</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&#039;&lt;/em&gt;-dicyclohexylcarbodiimide</td><td>Aldrich</td><td>D80002</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma Aldrich</td><td>270547</td><td>Solvent</td></tr><tr><td>Nickel metal template</td><td>American Elements</td><td>Ni-860</td><td>Foam template</td></tr><tr><td>Neuroblastomas cells (SH-SY5Y)</td><td>ATCC</td><td>CRL-2266</td><td>Cell line</td></tr><tr><td>Penicillin streptomycin&amp;nbsp;</td><td>Thermo SCIENTIFIC</td><td>15140122</td><td>Antibiotics</td></tr><tr><td>Polyethylene glycol 2000, PEG</td><td>Alfa Aesar</td><td>B22181</td><td>Reagent</td></tr><tr><td>Sodium azide&amp;nbsp;</td><td>VWR</td><td>97064-646</td><td>Reagent</td></tr><tr><td>Sodium bicarbonate</td><td>AMRESCO</td><td>865</td><td>Drying salt</td></tr><tr><td>Sodium chloride</td><td>BDH</td><td>BDH9286</td><td>Drying salt</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Fisher Scientific</td><td>S-374</td><td>Drying salt</td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma Aldrich</td><td>S9638</td><td>Drying salt</td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>239313</td><td>Drying salt</td></tr><tr><td>Tetrahydrofuran</td><td>Alfa Aesar</td><td>41819</td><td>Solvent</td></tr><tr><td>Thiosulfate de sodium</td><td>AMRESCO</td><td>393</td><td>Drying salt</td></tr><tr><td>Tin(II) 2-ethylhexanoate</td><td>Aldrich</td><td>S3252</td><td>Reagent</td></tr><tr><td>Toluene</td><td>Alfa Aesar</td><td>22903</td><td>Solvent</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>471283</td><td>Reagent</td></tr><tr><td>Trypsin</td><td>HyClone</td><td>SH30042.01</td><td>Cell Detachment</td></tr><tr><td>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

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 ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Chemistry

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin adhesives. Two different poly-(dimethyl siloxanes) (PDMS and soft skin adhesive (SSA)), have been used for in depth investigation of biological effects and adhesive characteristics. The composite films consist of a flexible backing layer and an adhesive top coating. Both layers have been manufactured by doctor blade application technique. In the present investigation, the adhesive behavior of the composite films has been investigated as a function of the layer thickness or a variation of the Young&#39;s modulus of the top layer. The Young&#39;s modulus of PDMS has been changed by varying the base to crosslinker mixing ratio. In addition, the thickness of SSA films has been varied from approx. 16 &#181;m to approx. 320 &#181;m. Scanning electron microscopy (SEM) and optical microscopy have been used for thickness measurements. The adhesive properties of elastomer films depend strongly on the film thickness, the Young&#39;s modulus of the polymers and surface characteristics. Therefore, normal adhesion of these films on glass substrates exhibiting smooth and rough surfaces has been investigated. Pull-off stress and work of separation are dependent on the mixing ratio of silicone elastomers.
Additionally, the thickness of the soft skin adhesive placed on top of a supportive backing layer has been varied in order to produce patches for skin applications. Cytotoxicity, proliferation and cellular adhesion of L929 murine fibroblasts on PDMS films (mixing ratio 10:1) and SSA films (mixing ratio 50:50) have been conducted. We have shown here, for the first time, the side by side comparison of thin composite films manufactured of both polymers and present the investigation of their biological- and adhesive properties.

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young&#39;s moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

Developmental Biology

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...