Name: construction of modular hydrogel sheets for micropatterned macro-scaled 3d cellular architecture
Uploaded: 2016-01-11T10:30:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture at JoVE.com

This research was supported by a National Leading Research Laboratory Program (Grant NRF-2013R1A2A1A05006378) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The authors also acknowledge a KAIST Systems Healthcare Program.

1. Preparazione del micropatterned Stampi e idrogel <ol><li> Produrre i modelli micro scala desiderati utilizzando SU-8 photoresist sulla superficie di un wafer di silicio mediante due fasi tecnica fotolitografia 15,17 standard per colata stampi PDMS. L&#39;esempio illustrato utilizza un modello di rete fegato lobulo-like (Figura 1). </li><li> Pesare PDMS e una soluzione di catalizzatore con un rapporto di 1: 5 (cioè, 12.5 g di PDMS e 2,5 g di induritore). </li><li> Mescolare il 15 g della soluzione di fondo, degassare le bolle in una camera a vuoto, e poi diffondere la soluzione mista su una superficie micropatterned della fetta di silicio in modo uniforme in un piatto foil colata. </li><li> Posizionare il wafer di silicio su una piastra riscaldata 65 ° C per 90 min su una superficie piana per curare le PDMS. </li><li> Rimuovere le PDMS curate dal piatto di colata e il wafer di silicio. </li><li> Tagliare i bordi delle PDMS e posizionarlo su una capsula di Petri da 100 mm di diametro conil lato micropatterned up. </li><li> Lavare le PDMS curate micropatterned sulla capsula di Petri con il 70% di etanolo e acqua distillata per la sterilizzazione primario. Poi, asciugarle completamente per 10 minuti in forno a 65 ° C. </li><li> Sciogliere e miscelare O / N 3 g di un tensioattivo non ionico in polvere in 100 ml di acqua distillata, creando un 3% (w / v) di rivestimento. </li><li> Posizionare i PDMS micropatterned stampi in un pulitore plasma e pulirli per 1 min (85 W, 0,73 mbar) per creare una superficie idrofila, per facilitare l&#39;aggiunta di liquidi acquosi. Poi, rivestire la superficie delle PDMS con la soluzione di 100 ml di tensioattivo per almeno 3 ore (ORO / N) utilizzando un bilanciere di laboratorio. </li><li> Lavare la soluzione di tensioattivo dagli stampi PDMS e asciugare completamente in forno a 65 ° C per 10 min. Poi, sterilizzare ogni stampo micropatterned da esposizione a raggi ultravioletti (UV) oltre 30 min. </li></ol> 2. Preparare la sospensione cellulare in un idrogel Precursore <ol><li> Apreparare idrogel precursore, sciogliere 0,1 g di alginato di sodio in polvere a 10 ml di tampone fosfato salino (PBS), creando un 1% (w / v) precursore alginato. Per sciogliere completamente la polvere, incubare e mescolarle O / N. </li><li> Filtrare la soluzione attraverso un filtro da 0,22 micron collegato ad una siringa da 1 ml. </li><li> Cultura le cellule HepG2 in modificata mezzo di Eagle Dulbecco (DMEM) supplementato con 10% di siero fetale bovino e 1% di penicillina-streptomicina su un piatto di coltura di tessuto convenzionale fino 70% - 80% di confluenza in 5% CO 2 incubatore umidificato a 37 ° C . </li><li> Lavare le cellule una volta con PBS, e quindi trypsinize aggiungendo 1 ml di 0,05% tripsina-EDTA per 4 minuti in un incubatore umidificato CO 2 5% a 37 ° C. Centrifugare le cellule raccolte dalla piastra di coltura a 250 xg per 3 min e risospendere con 1 ml di PBS dopo la rimozione del supernatante. </li><li> Contare il numero di cellule singole distribuite in PBS usando una cella automatizzatacontatore. </li><li> Dopo centrifugazione a 250 xg per 3 min e la rimozione PBS, aggiungere 1 ml di 1% (w / v) di una soluzione di alginato di sodio al restante pellet e mescolare delicatamente con pipettamento. La densità di semina finale delle cellule dovrebbe essere di 5 x 10 6 - 10 7 cellule / ml. Incubare la sospensione cellulare / idrogel in 5% CO 2 incubatore umidificato a 37 ° C. </li></ol> 3. Caricamento e Cross-linking di cellule / Hydrogel Suspension <ol><li> Sciogliere 1,47 g di cloruro di calcio disidratare in 100 ml di DDH 2 O per produrre un reagente di reticolazione (ad esempio, 100 mM CaCl 2 · 2H 2 O in DDH 2 O). </li><li> Risciacquare l&#39;interno di un umidificatore con trasduttore ultrasonico utilizzando 70% di etanolo, e riempirlo con 200 ml di reagente di reticolazione. L&#39;umidificatore è largo 110 mm, 300 mm di lunghezza e 170 mm di profondità (ovvero 110 mm x 300 mm x 170 mm (L x A x P)). </li><li> Posizionare il PDMS micropatternedstampi in un pulitore plasma e pulirli per 1 min a 85 W per creare una superficie idrofila. </li><li> Caricare progressivamente 7,2 ml di sospensione cellulare / idrogel ben miscelato sul bordo del micropattern nello stampo. L&#39;esempio illustrato utilizza un modello di rete fegato lobulo-like (Figura 1). Il volume della sospensione dipende dal modello topografico. </li><li> Per raggiungere gelificazione della sospensione cellulare / idrogel, accendere l&#39;umidificatore e verificare che l&#39;umidificatore produce una nebbia del reagente di reticolazione. Spruzzare il reagente di reticolazione sul precursore idrogel per 5 min, coprendo la superficie topografica degli stampi PDMS in un intervallo di 5 cm. Nota: Le distanze più lunghe e più breve di 5 cm possono causare la gelificazione incompleta e irregolare, rispettivamente. Assicurarsi che l&#39;umidificatore ha un tasso di spruzzatura di 250 ml / h, producendo 20 ml di nebbia del reagente di reticolazione in 5 min. </li><li> A seguito del processo di cross-linking, spegnere l&#39;umidificatore e riempire il mo PDMSlds con PBS. </li></ol> 4. Trattamento delle fogli singoli modulare idrogel <ol><li> Staccare ogni foglio idrogel indurito dagli stampi micropatterned via pipettando PBS delicatamente intorno al foglio di idrogel con un puntale 200 l. </li><li> Pick up ogni foglio idrogel mobile usando un 1.000 ml fine punta della pipetta end-cut. Ogni maglia foglio idrogel lobulo-come il fegato ha dimensioni di 8 millimetri x 8,7 millimetri, ed essere 100 - 200 micron di spessore. </li><li> Trasferire un singolo strato di foglio idrogel in 1 ml di DMEM in un 12-pozzetti, e coltivare le cellule in vitro in modo flottante usando l&#39;idrogel costrutto come componente unità della 12-pozzetti più di una settimana in un 5 % di CO 2 umidificata incubatore a 37 ° C. Sostituire il terreno di coltura ogni altro giorno. </li></ol> 5. Fogli idrogel Assemblea di Sovrapposizione <ol><li> Ripetere i passaggi 1,1-1,5 per produrre un telaio PDMS con dimensioni di 18 millimetri x 18 mm x 4 mm (L xH x D), e che contiene strutture 170 micron-alto pilastro sulla superficie inferiore. Utilizzare 42 g della miscela di PDMS e agente reticolante, con lo stesso rapporto come al punto 1.2. Mettere uno stampo di policarbonato specializzata sul wafer di silicio per il telaio PDMS per creare un telaio interno con dimensioni di 8 millimetri x 9 mm x 2 mm (L x A x P). </li><li> Sterilizzare telaio PDMS e 180 micron-pori carte da filtro nylon sommerso in acqua distillata e pinzette in autoclave per 15 minuti a 121 ° C. </li><li> Posizionare il telaio PDMS sterilizzato su un quarto di un pezzo di carta da filtro in nylon (con un diametro di 5 cm) in una capsula di Petri del diametro di 60 mm. </li><li> Trasferire un foglio idrogel modulare nell&#39;interno del telaio PDMS utilizzando un 1.000 microlitri punta della pipetta fine taglio. I fogli idrogel utilizzati per l&#39;assemblaggio devono essere coltivati ​​per almeno un giorno dopo che sono state fabbricate. </li><li> Allineare il bordo di ciascun foglio idrogel modulare con il telaio PDMS utilizzando un puntale 200 microlitri vuoto. </li><li> Ripetere i punti 5.4 e 5.5 utilizzando fogli di idrogel modulari in modo da formare una pila di 4 - 6 strati. </li><li> Rimuovere il mezzo di coltura da fluisce fuori attraverso le strutture pilastro nella parte inferiore del telaio PDMS. Poi aggiungere 2 ml di soluzione di alginato (2% w / v) ad un angolo del costrutto multistrato. </li><li> Spray una nebbia del reagente reticolazione per 30 sec sul costrutto multistrato per fissare i bordi di ogni strato con quelli di un altro. Utilizzare 2 ml di nebbia del reagente di reticolazione (ad una velocità di 250 ml di spruzzatura / hr). </li><li> Sciacquare il costrutto multistrato delicatamente con 400 microlitri DMEM e rimuovere il telaio PDMS con una pinzetta. </li><li> Rimuovere il costrutto multistrato dalla carta da filtro strofinando delicatamente con una cella sollevatore dopo l&#39;aggiunta di 4 ml di DMEM. </li><li> Trasferire il multistrato costruire ad una piastra da 6 pozzetti contenente 3 ml di DMEM con carta da filtro, e coltivare le cellule in vitro in modo flottante, con l&#39;idrogel come costruttoun&#39;impalcatura cellulare multi-scala nella piastra da 6 pozzetti più di una settimana in un 5% CO 2 incubatore umidificato a 37 ° C. Sostituire il terreno di coltura ogni altro giorno. </li></ol>

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The authors have nothing to disclose.

Questo protocollo fornisce un semplice metodo per fabbricare fogli idrogel modulari, e loro assemblaggio per formare scaffold cellulari 3D. Per costruire strutture alginato modellata chiare in breve tempo, dobbiamo identificare un processo di reticolazione che può creare strutture sufficientemente rigido per mantenere le microdisegni complessi dallo stampo, così come mantenere la vitalità delle cellule e il metabolismo. Abbiamo sviluppato un processo di cross-linking, compresa una transiz...

Gli idrogel sono particolarmente promettenti biomateriali, e dovrebbero essere importante nella biologia di base, test farmacologici e la medicina. 1 Biofabrication di costrutti cellulari a base di idrogel-è stato suggerito di ridurre il ricorso alla sperimentazione animale, 2,3 sostituire tessuti trapiantabili, 4 e migliorare saggi cellulari. 5,6-contenenti acqua (idrocarburi) materiali viscoelastici (gel) permettono un gran numero di cellule da incapsulare e mantenuto in una struttura scaffold per controllare il microambiente cellulare 3D. In combinazione con la guida di tecnologie microfluidica o micropatterning, la geometria dei costrutti idrogel può essere controllata con precisione in scala cellulare. Fino ad oggi, una varietà di forme di idrogel, tra particelle, 7 - 9 fibre, 10 - 12 e fogli 13 - 15 sono stati utilizzati come unità edilizie in bottom-up approdolori alla fabbricazione di macro scala architetture multi-cellulari. Entrambe le particelle e fibre a base di idrogel-sono stati prontamente e rapidamente realizzati per applicazioni come ambienti cellulari micro-scala, con controlli fluidici che utilizzano dispositivi microfluidici. Tuttavia, come le unità di base dei tessuti ingegnerizzati, sarebbe complicato di riordinarli e per allargare il loro volume come costrutti macro-scala. 16 E &#39;più difficile da raggiungere costrutti macro-scala per la produzione di moduli base micron dimensioni. Unità lastriforme di costrutti idrogel a base-possono essere utilizzati per aumentare il volume di scaffold tramite un semplice processo di assemblaggio. Consequentially, strati sovrapposti di fogli idrogel forniscono non solo un aumento volumetrico ma anche un&#39;estensione geometrico in uno spazio 3D. Abbiamo precedentemente riportato un metodo per fabbricare fogli idrogel micropatterned, 13 - 15 insieme al loro assemblaggio in multi-layarchitetture cellulari strati sovrapposti. La tecnica consente micropatterning complessa e modularità di costrutti cellulari tramite un processo di sovrapposizione di strutture multistrato. Attraverso la realizzazione di fogli impilati idrogel modulari, che sono micropatterned, un sistema di coltura cellulare 3D con un microambiente cellulare macroscala controllato può essere realizzato. Questo protocollo video descrive un metodo di fabbricazione semplice ma potente che può essere utilizzato per costruire fogli idrogel modulari, in base alla linea cellulare di carcinoma epatico umano (HepG2). Dimostriamo qui semplice manipolazione di questi fogli di idrogel modulari fantasia, ed il loro assemblaggio in una struttura a più livelli.

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning Corporation</td><td>000000000001064291</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td>Powdered nonionic surfactant&amp;nbsp;</td></tr><tr><td>Alginic acid sodium salt, low viscosity</td><td>Alfa Aesar</td><td>B25266</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>Ultrasonic humidifier</td><td>MediHeim</td><td>MH-2800</td><td>Modified equipment, Maximum sprayed rate: 250 ml/hr</td></tr><tr><td>Nylon net filter hydrofilic, 180 &amp;mu;m</td><td>EMD Millipore</td><td>NY8H04700</td><td></td></tr><tr><td>Polycarbonate mold</td><td></td><td></td><td>Customized mold for fabrication of a PDMS frame pattern</td></tr></tbody></table>

construction of modular hydrogel sheets for micropatterned macro-scaled 3d cellular architecture

Idrogel possono essere modellati in micro-scala utilizzando tecnologie microfluidica o micropatterning di fornire un vivo like geometria tridimensionale dei tessuti (3D) in. I risultanti costrutti cellulari in 3D basato idrogel-sono stati introdotti come alternativa agli esperimenti sugli animali per studi biologici avanzati, saggi farmacologici e applicazioni trapianto di organi. Sebbene particelle, fibre a base di idrogel-possono essere facilmente realizzati, è difficile da manipolare loro per la ricostruzione del tessuto. In questo video, si descrive un metodo di fabbricazione dei fogli di idrogel alginato micropatterned, insieme al loro assemblaggio per formare un sistema di coltura cellulare 3D macro scala con un microambiente cellulare controllata. Utilizzando una forma di nebbia dell&#39;agente gelificante calcio, fogli sottili idrogel si generino facilmente con uno spessore dell&#39;ordine di 100 - 200 micron, e con microdisegni precise. Le cellule possono essere coltivate con la guida geometrica dei fogli in idrogelcondizioni freestanding. Inoltre, i fogli idrogel possono essere facilmente manipolati utilizzando una micropipetta con una punta fine-cut, e possono essere assemblati in strutture multistrato mediante sovrapposizione utilizzando un polidimetilsilossano modellata (PDMS) frame. Questi fogli idrogel modulari, che possono essere fabbricati con un processo facile, hanno potenziali applicazioni di test in vitro droga e di studi biologici, compresi studi funzionali di micro e macrostruttura e ricostruzione dei tessuti.

Abbiamo descritto la fabbricazione e la manipolazione di fogli di idrogel freestanding cellulari. Come mostrato in figura 1, abbiamo inventato PDMS micropatterned stampi, e idrogel contenenti cellule è stato caricato sulla superficie idrofila di questi stampi e reticolato utilizzando un umidificatore per generare una nebbia aerosol di agente gelificante. Dopo il rilascio dagli stampi, cellule HepG2 sono state coltivate in autoportante fogli idrogel con vari modelli <str...

Watch this Scientific Journal Video about Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture at JoVE.com

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Costruzione di fogli modulare idrogel per micropatterned macro-scala 3D Cellular Architettura

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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JoVE Journal

Bioengineering

10.2K Views.  Korea Advanced Institute of Science and Technology (KAIST). We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment. 

Video: Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Ricerca

Bioingegneria

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochimica

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 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 bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

La stampa termosensibile Stampi inversa per la Creazione di Patterned idrogeli a due componenti per coltura cellulare 3D

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

Immunologia e infezioni

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Gradiente di deformazione Chip per comportamenti stimolanti cellulari in cella-carichi idrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Un metodo versatile di patterning proteine ​​e cellule

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Un additivo tecnica di fabbricazione per la Facile e rapida realizzazione di Micromachines base di idrogel con componenti magneticamente reattivi

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

Controllo del Cluster iniziale delle cellule 3D in un ibrido Gel cubo dispositivo per reticolo ripetibile formazioni

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Protocolli di biostampa 3D di Gelatin Abasecryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

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.

Scaffold 3D macroporosi interconnessi da barre di microgel

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

Costruzione di fogli modulare idrogel per micropatterned macro-scala 3D Cellular Architettura

Riepilogo

Esplora Altri Video

Capitoli in questo video

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

La stampa termosensibile Stampi inversa per la Creazione di Patterned idrogeli a due componenti per coltura cellulare 3D

Gradiente di deformazione Chip per comportamenti stimolanti cellulari in cella-carichi idrogel

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Un metodo versatile di patterning proteine e cellule

Un additivo tecnica di fabbricazione per la Facile e rapida realizzazione di Micromachines base di idrogel con componenti magneticamente reattivi

Controllo del Cluster iniziale delle cellule 3D in un ibrido Gel cubo dispositivo per reticolo ripetibile formazioni

Protocolli di biostampa 3D di Gelatin Abasecryloyl Hydrogel Based Bioinks

Scaffold 3D macroporosi interconnessi da barre di microgel

Costruzione di fogli modulare idrogel per micropatterned macro-scala 3D Cellular Architettura

Riepilogo

Esplora Altri Video

Capitoli in questo video

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

La stampa termosensibile Stampi inversa per la Creazione di Patterned idrogeli a due componenti per coltura cellulare 3D

Gradiente di deformazione Chip per comportamenti stimolanti cellulari in cella-carichi idrogel

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Un metodo versatile di patterning proteine ​​e cellule

Un additivo tecnica di fabbricazione per la Facile e rapida realizzazione di Micromachines base di idrogel con componenti magneticamente reattivi

Controllo del Cluster iniziale delle cellule 3D in un ibrido Gel cubo dispositivo per reticolo ripetibile formazioni

Protocolli di biostampa 3D di Gelatin Abasecryloyl Hydrogel Based Bioinks

Scaffold 3D macroporosi interconnessi da barre di microgel

Un metodo versatile di patterning proteine e cellule