Name: synthesis of biocompatible liquid crystal elastomer foams as cell scaffolds for 3d spatial cell cultures
Uploaded: 2017-04-11T15:00:00
Description: Watch this Scientific Journal Video about Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures at JoVE.com

Forfatterne vil gerne takke Kent State University (forskningssamarbejde tilskud og støtte til regenerativ medicin initiativet på Kent State - ReMedIKS) for økonomisk støtte til dette projekt.

BEMÆRK: Følgende trin til 3D LCE skumlignende præparat under anvendelse af 3-arm stjerne-blokcopolymer er vist i figur 1. For kernemagnetisk resonans (NMR) karakterisering, er det spektre registreret i deutereret chloroform (CDCI3) ved stuetemperatur på et Bruker DMX 400-MHz-instrument og internt refereres resterende toppe ved 7,26. Fouriertransformation infrarød (FT-IR) spektre er registreret ved anvendelse af et Bruker Vector 33 FT-IR spektrometer ved anvendelse svækket ...

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Flydende krystallinske elastomerer er for nylig blevet undersøgt som biokompatible celle scaffolds grund af deres stimuli reaktionsevne. De har vist sig at være ideelle platforme som celle stilladser. Men en vigtig faktor at huske på, når de forbereder og designe et nyt LCE stillads er porøsitet. Inkorporering af udvaskelige faste stoffer 23 eller gasser resulterer ikke altid i homogen porøsitet eller fuldstændigt indbyrdes forbundne porer. Brugen af ​​en metal skabelon, der kan ætset...

Der er adskillige eksempler på biologiske og biokompatible syntetiske materialer beregnet til påføring i celleundersøgelser og til vævsregeneration sigte på cellebinding og proliferation 1, 2, 3, 4, 5. Der har været nogle få eksempler på biokompatible materialer, der er kendt som flydende krystal elastomerer (afgørende for store komplekse), der kunne reagere på ydre stimuli med anisotropisk molekylær bestilling 6, 7. Afgørende for store komplekse er reagerer på stimulering, materialer, der kombinerer de mekaniske og elastiske egenskaber af elastomerer med den optiske funktionalitet og molekylær bestilling af flydende krystaller 8, 9. Afgørende for store komplekse kan opleve ændringer i form, mekanisk deformation, elastisk opførsel og optiske egenskaber som svar på ydre stimuli (dvs.., varme, stress, lys, etc.) 10, 11, 12, 13, 14, 15, 16. Tidligere undersøgelser har vist, at flydende krystaller (LCS) kan fornemme vækst og orienteringen af celler 4, 17. Det er muligt derefter at antage, at afgørende for store komplekse kan være egnet til biologisk og medicinsk relevante anvendelser, herunder celle stilladser og tilpasning. Vi har tidligere rapporteret fremstillingen af smektiske biokompatible, bionedbrydelige, støbt-støbte og tynde afgørende for store komplekse film med et &quot;Swiss-ostetype&quot; porøs morfologi 6, 18. Vi også forberedt nematiske biokompatible afgørende for store komplekse med kugleformet morfologi som skeletter til cellevækst 19 &lt;sup&gt;, 20. Vores arbejde var rettet mod tuning de mekaniske egenskaber af de materialer til at matche dem i vævet af interesse 21. Også disse undersøgelser fokuserer på forståelse elastomer-celle interaktioner samt cellulære respons, når de elastomerer er underlagt eksterne stimuli. De største udfordringer var delvist at skræddersy porøsitet afgørende for store komplekse at muliggøre cellevedhæftning og permeation gennem den elastomere matrix og for bedre massetransport. Porøsiteten af disse tynde film 6 tilladt for celle permeation gennem hovedparten af matrixen, men ikke alle porer var fuldt sammenkoblet eller havde en mere regelmæssig (homogen) porestørrelse. Vi rapporterede derefter på biokompatible nematic LCE elastomerer med kugleformede morfologier. Disse nematiske elastomerer tilladt for vedhæftning og proliferation af celler, men porestørrelsen varierede kun 10-30 um, som forebygges eller begrænset anvendelsen af ​​disseelastomerer med en bredere vifte af cellelinier 19, 20. Tidligere arbejde af Kung et al. om dannelse af graphene skum under anvendelse af en &quot;offer&quot; metal skabelon viste, at det opnåede graphene skum havde en meget regelmæssig porøs morfologi dikteret af det valgte metal skabelon 22. Denne metode giver fuld kontrol over porøsitet og porestørrelse. På samme tid, formbarhed og fleksibilitet i metal skabelon tillade dannelsen af ​​anden skabelon former forud for forberedelse skum. Andre teknikker, såsom materiale udvaskning 23, gas templating 24, eller elektro-spundne fibre 25, 26 tilbyder også potentialet til fremstilling af porøse materialer, men de er mere tidskrævende og i nogle tilfælde, er porestørrelsen begrænset til kun nogle få mikrometer. Skum-lignende 3D afgørende for store komplekse fremstillet ved anvendelse af metal skabeloner muliggør en højere celle belastning; en forbedret proliferationshastighed; co-dyrkning; og, at sidst men ikke mindst, bedre massetransport forvaltning (dvs. næringsstoffer, gasser, og affald) sikre fuld væv udvikling 27. Skum-lignende 3D afgørende for store komplekse synes også at forbedre cellejustering; dette er mest sandsynligt i forhold til LC vedhæng sensing cellevækst og celle orientering. Tilstedeværelsen af ​​LC-dele i LCE synes at forbedre cellejustering med hensyn til celle placering inden LCE stilladset. Celler justeret indenfor stiverne i LCE, mens der ikke observeres nogen klar orientering hvor stiverne slutte sig sammen (kryds) 27. Samlet set vores LCE celle stillads platform som en celle støtte medie giver muligheder for at tune den elastomere morfologi og elastiske egenskaber og til specifikt at lede tilpasningen af ​​(individuelle) celletyper til at skabe et bestilt, rumlige arrangementer of celler ligner levende systemer. Ud over det stillads stand til at opretholde og lede langsigtet cellevækst og proliferation, afgørende for store komplekse tillader også dynamiske forsøg, hvor celle orientering og interaktioner kan ændres i farten.

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			<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

Her præsenteres en trin-for-trin udarbejdelse af en 3D, biologisk nedbrydeligt, skum-lignende celle stillads. Disse skeletter blev fremstillet ved tværbinding stjerne blokcopolymerer featuring kolesterol enheder som sidekæde-sidegrupper, hvilket resulterer i smektisk-A (SMA) flydende krystaller elastomerer (afgørende for store komplekse). Skum-lignende stilladser, fremstillet ved anvendelse af metal-skabeloner, tilbyder indbyrdes forbundne mikrokanaler, hvilket gør dem egnede som 3D cellekultur stilladser. De kombinerede egenskaber af den regelmæssige struktur af metallet skum og af elastomeren resultat i en 3D celle stillads, der fremmer ikke kun højere celleproliferation sammenlignet med konventionelle porøse template film, men også bedre styring af massetransport (dvs. næringsstoffer, gasser, affald etc.). Karakteren af metal skabelon giver mulighed for let manipulation af skum former (dvs. ruller eller film), og for udarbejdelsen af stilladser i forskellige porestørrelser for forskellige celle undersøgelser samtidig bevare bindelsesudstyrted porøse natur af skabelonen. Ætsningsprocessen påvirker ikke kemien i elastomererne, bevare deres biokompatible og bionedbrydelige natur. Vi viser, at disse smektiske afgørende for store komplekse, når der dyrkes for omfattende tidsperioder, muliggøre studiet af klinisk relevante og komplekse væv konstruktioner og samtidig fremme vækst og spredning af celler.

Denne rapport viser fremstillingen fremgangsmåde en porøs 3D LCE som et skelet til celledyrkning under anvendelse af et nikkelmetal skabelon. Den opnåede 3D LCE demonstrerer et komplekst sammenhængende kanal netværk, der giver mulighed for nem celle infiltration, samt mere egnet massetransport 27. Det blev konstateret, at cellerne er i stand til fuldt ud at trænge det sammenkoblede kanal netværk og er også i stand til at tilpasse inden LCE. Her, et metal n...

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

Denne undersøgelse viser en metode til fremstilling af 3D, biologisk nedbrydelige, skumlignende celle scaffolds baseret på biokompatible sidekædeegenskaber flydende krystal elastomerer (afgørende for store komplekse). Konfokal mikroskopi eksperimenter viser, at skumlignende afgørende for store komplekse tillade cellevedhæftning, proliferation og den spontane tilpasning af C2C12s myoblaster.

Syntese af Biokompatible Liquid Crystal Elastomer Skum som Cell Stilladser til 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 ...

The goal of this procedure is to prepare biodegradable, three dimensional foam-like cell scaffolds based on biocompatible side chain liquid crystal elastomers. This method can help answer key questions in the liquid crystal and biomedical fields, such as the effects of liquid crystal elastomer properties on cell proliferation and alignment. The main advantage of this method of two dimensional cell scaffolds is it permits the study of spatial cell-cell interactions which is rarely possible in 2D macro environments.Generally, those new to this method will struggle with the tactile compression tests. We first had the idea for this method after having to change the media in hundreds of Petri dishes. We wondered if liquid crystal elastomers could support muscle cells on a 3D network, to eliminate the use of so many Petri dishes.Visual demonstration of this method is critical, as some steps require careful manipulation of chemicals. Further, shaping the metal foam template may be challenging. First, fill a 20 milliliter ampoule with a 2%by volume solution of PFOTES in toluene.Stir the solution in the ampoule for 24 hours to silanize the ampoule interior. Rinse the silanized ampoule with isopropyl alcohol and dry it at 140 degrees celsius for 30 minutes. Place 3.64 grams of distilled epsilon-caprolactone, 0.5 grams of alpha-chloro-epsilon-caprolactone, and 0.25 milliliters of glycerol in the dry ampoule.Vortex the mixture for one minute. Then, add 4.90 grams of DL-lactide to the mixture, and purge the ampoule atmosphere with nitrogen gas for one minute. Cover the ampoule opening with aluminum foil, and heat the mixture at 120 degrees Celsius for about two hours to melt the DL-lactide.Vortex the mixture to disrupt any unmelted solids, and then, add 66 microliters of 10-2-ethylhexanoate to the ampoule, and vortex again. Close the ampoule with aluminum foil and heat the mixture at 120 degrees Celsius for 10 minutes to remelt the DL-lactide. Once the DL-lactide has melted again, vigorously vortex the mixture, and purge the ampoule atmosphere with nitrogen gas.Seal the ampoule with a rubber septum. Insert a needle connected to a vacuum line through the septum, and start the vacuum. Flame-seal the neck of the ampoule, being careful not to melt the rubber stopper.Once the neck is sealed, heat the reaction mixture at 140 degrees Celsius for 48 hours. Then, allow the mixture to cool to room temperature. Break open the ampoule and dissolve the viscous reaction mixture in 10 milliliters of dichloromethane.Transfer the mixture to a separatory funnel. Place a flask containing 100 milliliters of methanol in a dry ice and acetone bath to cool down the methanol to approximately minus 78 degrees Celsius. Once the methanol has cooled down, secure the separatory funnel over the flask.Add the reaction mixture to the cold methanol at a rate of two drops every second. Collect the resulting white precipitate on filter paper. Dry the precipitate in a vacuum oven between 50 and 60 degrees Celsius to obtain the three-arm alpha-chloro SBC product.To prepare the alpha cholesteryl three-arm SBC, the pendant chlorine atom is replaced by an azide. A click reaction with cholesteryl 5-hexynoate results in a pendant cholesteryl as the liquid crystal moiety. To begin preparing the liquid crystal elastomer scaffold, combine 0.75 grams of alpha-cholesteryl three-arm SBC with 0.25 milliliters of HDI and 0.24 milliliters of distilled epsilon-caprolactone.Add to this 60 microliters of 10-2-ethylhexanoate and vortex the mixture. Then, cut out a one centimeter by four centimeter piece of nickel metal foam. Roll the nickel foam into a cylinder one centimeter in diameter and one centimeter tall to form a template for the liquid crystal elastomer foam scaffold.Place the template in a glass vial or aluminum foil enclosure, and pour the liquid crystal elastomer mixture over the template until it is completely covered. Allow the template to sit in the liquid crystal elastomer mixture for two minutes, and then, remove the excess with a pipette. Heat the mixture and template at 80 degrees Celsius overnight.Then, peel away the aluminum foil, or break the glass. Use a razor blade to remove excess liquid crystal elastomer to expose the nickel metal template. Place the foam in a flask and add 70 milliliters of a saturated aqueous solution of iron three chloride.Stir the foam in the solution for three days at room temperature to dissolve the nickel template. Every 24 hours, stir the foam in deionized water for 30 minutes, and then, resume stirring in a fresh iron three chloride solution. After the second day of stirring, perform a tactile compression test on the liquid crystal elastomer foam.Resistance to tactile compression indicates that the nickel template is still present in the foam. After the third day, all nickel template has been eliminated, and the resulting foam appears very soft and easy to fully compress. The nickel template must be completely eliminated.It is important to thoroughly rinse the liquid crystal elastomer foam in iron chloride until it's soft to the touch when a tactile compression test is performed. The liquid crystal elastomer foam scaffold is now ready to be characterized and used. Once the liquid crystal elastomer foam is soft, rinse it with 70%ethanol to sterilize it.To begin the seeding procedure, wash again the liquid crystal elastomer foam scaffolds twice in one milliliter of 70%ethanol to sterilize the elastomer surfaces. Then, irradiate the scaffolds with UV light for 10 minutes. Wash the scaffolds with another one milliliter portion of 70%ethanol.Rinse the scaffolds with one milliliter each of sterile water and phosphate-buffered saline. Load the sterilized liquid crystal elastomer scaffolds into 24-well culture plates. Prepare and count a suspension of the cells of interest in the appropriate cell growth medium with penicillin and streptomycin.Dilute the cell suspension to 1.5 times 10 to the fifth cells per 100 microliters using growth medium. Deposit a drop of diluted cell suspension on top of each liquid crystal elastomer scaffold. Incubate the seeded liquid crystal elastomer scaffolds at 37 degrees Celsius in a 5%CO2 atmosphere for two hours.Add another 0.5 milliliters of growth medium to each scaffold, and continue incubation. Every 48 hours, wash the seeded scaffolds with one milliliter of PBS, and add fresh growth medium. Continue incubating the scaffolds until the cells are ready for microscopy.These smetic LCs enable the study of complex tissue constructs while promoting cell growth and proliferation. In order to maintain cell viability, foams must be sterilized and cleanliness of cultures maintained at all times. To prepare for microscopy, fix the cells on the scaffolds with a solution of 4%paraformaldehyde in PBS for 15 minutes.Soak the samples three times in three milliliters of PBS for five minutes. Place one scaffold sample in an Eppendorf tube. Stain the sample with a solution of 0.1%DAPI in 500 microliters of PBS for 10 minutes.Soak the sample twice in one milliliter of PBS for five minutes. And then, immediately image the sample with confocal microscopy. Acquire image stacks spanning the sample and analyze the data in an image processing program.A three-arm SBC with liquid crystal moieties was cast on a nickel foam template with HDI as the cross-linker. The nickel template was removed by etching to obtain the liquid crystal elastomer foam. Compression deformation testing was periodically performed to monitor the etching process.Once the nickel had been completely dissolved, the compression deformation testing showed a 70%reduction in size when compressed. Upon releasing compression, the liquid crystal elastomer foam consistently recovered its original size and shape. This behavior is attributed to the liquid crystal moieties, as similar elastomer foams lacking the cholesterol epsilon-caprolactone moiety did not recover from compression.SCM of the internal foam morphology showed an interconnected network of hollow foam struts. The regular morphology was attributed to the structure of the nickel foam, indicating that pore size and overall shape can be controlled by selection of an appropriate metal template. The foam was seeded with neuroblastoma cells, which attached to the walls of the network within two days.30 days after seeding, confocal microscopy revealed that the cells extended over the liquid crystal elastomer network, and had formed multiple layers dispersed throughout the elastomer foam. Elongation of cell nuclei was observed, and was correlated to cell alignment. Once mastered, this technique can be done within three to four weeks if performed properly.After watching this video, you should have a good understanding of how to design and prepare liquid crystal elastomer cell scaffolds with specific pore sizes and morphologies. While attempting this procedure, it's important to fully characterize each product and to monitor cell viability and expansion. Further, proper staining technique is important to easily acquire confocal microscopy images.Following this procedure, liquid crystal elastomers can also be exposed to external stimuli, such as stress, or magnetic and electric fields, to answer additional questions about how the anisotropic molecular ordering of the liquid crystal elastomers affects cell response. The implications of this technique apply to the study of disease processes, as it provides a platform to dynamically study the effects of external agents on simulated disease activity, and subsequent repair processes. Though this smectic liquid crystal elastomer foam can provide insight into cell-cell interactions, they can also be used with other materials, such as semiconductor or metal nanostructures.After its development, this technique paved the way for researchers in biomedical fields to design long-term experimental platforms mirroring living systems.

Research

JoVE Journal

Bioengineering

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

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

Alginat mikrokapselsammensætning som en 3D-platform for Formering og differentiering af humane embryonale stamceller (embryonale) til forskellige linier

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

Udarbejdelse af 3D Fibrin stilladser til Stem celledyrkningsapplikationer

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

Selvrapportering Stilladser 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 ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Fabrikation af mekanisk Tunable og bioaktive Metal Stilladser til biomedicinske anvendelser

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

Vekslende magnetfelt-Responsive Hybrid Gelatine Mikrogeler for Controlled Drug frigivelse

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

Udarbejdelse af Monodomain Liquid Crystal elastomerer og Liquid Crystal Elastomer nanokompositter

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

Micropatterning og Samling af 3D mikrokar

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

Fabrikation af ekstracellulære matrix-afledte Skum og Mikrobærere som Tissue-specifikke cellekultur og Delivery Platforms

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

En Microcontroller drives enhed for Generation af flydende ekstrakter fra konventionelle cigaret røg og elektronisk cigaret Aerosol

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

3D analyse af multi-cellulære svar til Chemoattraktant gradienter

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