Name: printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture
Uploaded: 2013-07-10T14:00:00
Description: Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Vi takker Deborah Studer for hjælp med bioprinter. Arbejdet blev finansieret af Den Europæiske Union syvende rammeprogram (FP7/2007-2013) under tilskudsaftale nr. NMP4-SL-2009 til 229.292.

1.. Fremstilling af Poloxamer 407 Løsning Hvis det er tilgængeligt, skal du udføre forberedelsen af ​​poloxameren opløsning i et koldt rum (4 ° C). Hvis det ikke er tilgængelig, skal du placere en glasflaske i et bæger fyldt med iskoldt vand. Ved højere temperaturer poloxameren vil ligge over gelpunktet og ikke vil blive opløst. <ol><li> 60 ml iskold PBS-opløsning i en glasflaske og omrøres kraftigt ved hjælp af en magnetisk omrører. </li><li> Afvejes 24,5 g poloxamer og t...

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Her præsenterer vi for første gang, at anvendelsen af ​​en termoresponsiv polymer til en opofrende støbeform, der hurtigt kan elueres i koldt vand på grund af gel-sol-overgang poloxamer på ~ 20 ° C. Hastigheden af ​​hele processen gør poloxamer interessant for en hurtig oprettelse af biopolymer strukturer, der ikke kan udskrives med tilstrækkelig opløsning. Teknikken beskrevet her kan anvendes til mønsterdannelse en hydrogel i en anden hydrogel eller til oprettelse af mikrofluidkanaler som det tidliger...

Tissue engineering tilgange har gjort store fremskridt i de seneste år med hensyn til regenerering af humane væv og organer 19,20. Men indtil nu har fokus for tissue engineering ofte blevet begrænset til væv, som har en simpel struktur eller små dimensioner såsom blæren 21,22 eller huden 23-25. Det menneskelige legeme, indeholder imidlertid mange komplekse tredimensionale væv, hvor celler og ekstracellulære matrix er arrangeret i et rumligt defineret måde. At fremstille disse væv, er en teknik, kræves der kan placere celler og ekstracellulær matrix stilladser i en tredimensional konstruktion ved specificerede positioner. Bioprinting har potentialet til at være sådan en teknik, hvor visionen om fremstilling komplekse tre-dimensionelle væv kan realiseres 10,11,26-28. Bioprinting defineres som &quot;brug af materiale overførsel processer for mønstret og montage biologisk relvante materialer - molekyler, celler, væv og biologisk nedbrydelige biomaterialer -. med en fastsat organisation til at udrette et eller flere biologiske funktioner &quot;4 Det omfatter flere forskellige teknikker, der arbejder på forskellige opløsninger og længde skalaer, der spænder fra sub-micron opløsning på to -foton polymerisering 29 til en opløsning på 150 um til 420 um for ekstrudering udskrivning 1,12,30. Ikke en eneste, materiale kombination vil opfylde kravene i hver metode 31.. til ekstrudering udskrivning de vigtigste parametre er viskositet og gelering tid 32, hvor høj viskositet og hurtig gelering er ønskelige. 3D-print er en teknik, der gør det muligt nemt at oprette offer forme til at skabe komplekse geometrier 30,33,34. Denne proces er baseret på konstruktionen af ​​en støbeform med en rapid prototyping teknik, såsom en ekstrudering bioprinter. Den oprettede opofrende støbeform anvendesat danne komplekse strukturer af materialer, som er vanskelige at udskrive pga. deres lave viskositet og langsom gelering tid. Fremgangsmåden præsenteres her indebærer etablering af en opofrende støbeform bestående af et materiale, der opløses hurtigt ved lav temperatur og kan ekstruderes præcist. Blokcopolymeren poly (ethylenglycol) 99-poly (propylenglycol) 67-poly (ethylenglycol) 99 (også kendt som Pluronic F127 eller poloxamer 407) opfylder disse krav. Det er allerede blevet anvendt i en modificeret udgave i ekstrudering udskrivning 1, men til vores viden, er aldrig blevet brugt til udskrivning i sin umodificerede udgave på grund af sin ustabilitet i flydende miljøer. Poloxamer 407 viser også en invers termisk reagerende opførsel 18 dvs. det skifter fra en gel til en sol ved afkøling. Vigtigst er det, kan den trykkes i komplekse vilkårligt buede strukturer med meget stor nøjagtighed. Dette giver mulighed for etablering af et struktureret hydrogel fra enlav viskositet materiale, i dette tilfælde langsomme gelering agarose. ved pipettering af opløsningen i den trykte offer skimmel Kombinationen af ​​at udskrive opofrende formen med high fidelity og dens praktiske eluering fra støbt strukturerede hydrogel gør det en hurtig og fleksibel metode til at skabe forme med forskellige geometrier uden brug af en maske eller et stempel som det ofte kræves i litografiske metoder. Den støbt strukturerede hydrogel kan yderligere fyldes med et andet materiale, der ikke er egnet til ekstrudering trykning på grund af sin lave viskositet. Dette er i vores tilfælde en lav viskositet alginat methacrylate løsning. Her præsenterer vi metoden til termoresponsiv reverse offer forme til hydrogel mønstret ved hjælp af eksempel på en søjle array.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Poloxamer (Pluronic F127)</td>
 <td>Sigma</td>
 <td>P2443</td>
 <td ></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=10010015" target="_blank">10010-015</a></td>
 <td ></td>
 </tr>
 <tr>
 <td>CAD software</td>
 <td>regenHU</td>
 <td>BioCAD</td>
 <td ></td>
 </tr>
 <tr>
 <td>Alginate methacrylate</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
 <td>Invitrogen</td>
 <td>F13191</td>
 <td ></td>
 </tr>
 <tr>
 <td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Lonza</td>
 <td>50004</td>
 <td ></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Bioprinter</td>
 <td>regenHU</td>
 <td>Biofactory</td>
 <td ></td>
 </tr>
 <tr>
 <td>Valve</td>
 <td>regenHU</td>
 <td>300 &mu;m Nozzel Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>regenHU</td>
 <td>150 &mu;m Inner Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Zeiss Axioobserver with ApoTome</td>
 <td>Zeiss</td>
 <td></td>
 <td ></td>
 </tr>
 <tr>
 <td>UV Light Source</td>
 <td>UVP</td>
 <td>Blak-Ray B-100AP High Intensity UV Lamp</td>
 <td>100 W</td>
 </tr>
 </tbody>
</table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

Bioprinting er en ny teknologi, der har sin oprindelse i rapid prototyping industrien. De forskellige grafiske processer kan opdeles i kontakt bioprinting 1-4 (ekstrudering, dip pen og blød litografi), kontaktløs bioprinting 5-7 (laser fremad overførsel, ink-jet deposition) og laser-baserede teknikker såsom to foton fotopolymerisation 8.. Den kan bruges til mange applikationer såsom tissue engineering 9-13, biosensor microfabrication 14-16 og som et redskab til at besvare grundlæggende biologiske spørgsmål såsom påvirkninger af co-dyrkning af forskellige celletyper 17.. I modsætning fælles fotolitografiske eller blød litografiske metoder har ekstrudering bioprinting den fordel, at det ikke kræver en særskilt maske eller stempel. Ved hjælp af CAD-software, kan udformningen af ​​strukturen hurtigt ændres og justeres i henhold til kravene for den enkelte operatør. Dette gør bioprinting mere fleksibelt end litografi-baseredetilgange. Her viser vi trykning af en opofrende støbeform til at skabe en multi-materiel 3D struktur ved hjælp af en vifte af søjler inden for en hydrogel som et eksempel. Disse søjler kunne repræsentere hule strukturer til en vaskulær netværk eller rørene i en nerve guide ledning. Det valgte materiale til det hellige formen var poloxamer 407, en termoresponsiv polymer med fremragende trykegenskaber som er flydende ved 4 ° C og en fast over sin geleringstemperatur ~ 20 ° C i 24,5% w / v opløsninger 18. Denne egenskab giver poloxamer-baserede blote støbeform til elueres på efterspørgsel og har fordele i forhold til langsom opløsning af et fast materiale, især for smalle geometrier. Poloxamer var trykt på mikroskop objektglas til at skabe det hellige mug. Agarose blev pipetteret ind i formen og afkøles indtil gelering. Efter eluering af poloxameren i iskoldt vand, blev hulrummene i agarose formen fyldt med alginat methacrylat spiked med FITC mærket fibrinogen. De fyldte hulrum blev derefter tværbundet med UV og konstruktion blev afbildet med en epi-fluorescensmikroskop.

De repræsentative resultater viser, at den omvendte skimmel teknik (afbildet i figur 2) vil skabe en struktureret gel, der kan fyldes med et andet materiale. Ved begyndelsen af ​​hver trykprocessen trykning parametrene er først optimeres. Trinvis justeringer af disse parametre vil resultere i trykte flerlags konstruktioner afbildet i figur 3 og 4, når enkelte linjer udskrives. Om tykkelsen (nålen lift efter én udskrevet layer) er for lav, vil man bemærke, at n...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

En bioprinter blev brugt til at skabe mønstrede hydrogeler baseret på en opofrende støbeform. Poloxamer Formen blev tilbagefyldt med en anden hydrogel og derefter elueret, hvilket efterlader hulrum, som blev fyldt med en tredje hydrogel. Denne metode bruger hurtig eluering og god trykbarhed af poloxamer at generere komplekse arkitekturer fra biopolymerer.

Udskrivning termoresponsive Reverse forme til Oprettelse af Mønstret To komponent Hydrogeler til 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

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