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. Herstellung der mikrostrukturierten Formen und Hydrogele <ol><li> Die gewünschten Mikrostufenmuster mit Su-8 auf der Oberfläche eines Silizium-Wafers über eine Standard zweistufige photolithographischen Technik 15,17 zum Gießen PDMS Schimmelpilze. Das gezeigte Beispiel verwendet eine Leberläppchens artigen Maschenmuster (1). </li><li> Abwiegen PDMS und einer Härterlösung mit einem Verhältnis von 1: 5 (das heißt, 12,5 g PDMS und 2,5 g Härter). </li><li> Mischen Sie die 15 g der Lösung gründlich, entgast die Blasen in einer Vakuumkammer, und dann verbreiten die gemischte Lösung auf einen mikrostrukturierten Oberfläche der Silizium-Wafer gleichmäßig innerhalb einer Foliengießen Gericht. </li><li> Setzen Sie den Silizium-Wafer auf eine 65 ° C erhitzt Platte für 90 min auf eine ebene Fläche, um die PDMS zu heilen. </li><li> Entfernen Sie die ausgehärtete PDMS aus der Gussschale und dem Silizium-Wafer. </li><li> Schneiden Sie die Kanten der PDMS und legen Sie sie auf eine Petrischale von 100 mm Durchmesser mitdie mikro Seite nach oben. </li><li> Auf der Petrischale unter Verwendung von 70% Ethanol und destilliertes Wasser für die primäre Sterilisations Waschen der Mikromuster gehärtet PDMS. Dann trocknet sie vollständig für 10 min in einem Ofen bei 65 ° C. </li><li> Auflösen und mischen O / N 3 g eines pulverförmigen nichtionischen Tensid in 100 ml destilliertem Wasser gelöst, wodurch eine 3% (w / v) Beschichtungslösung. </li><li> Platzieren der mikro PDMS Formen in einer Plasmareiniger und reinigen sie für 1 min (85 W, 0,73 mbar), um eine hydrophile Oberfläche zu schaffen, um die Zugabe von wässrigen Flüssigkeiten zu erleichtern. Dann Beschichten der Oberfläche der PDMS mit 100 ml Tensidlösung für mindestens 3 hr (ORO / N) unter Verwendung eines Labor Wippe. </li><li> Waschen Sie die Tensidlösung aus PDMS Formen und trocknet sie vollständig in einem Ofen bei 65 ° C für 10 min. Dann sterilisiert jede mikrostrukturierte Form durch Belichtung mit ultravioletter Strahlung (UV) über 30 min. </li></ol> 2. Bereiten Sie die Zellsuspension in einem Hydrogel-Vorläufer <ol><li> ZuHydrogel-Vorläufer herzustellen, werden in 10 ml phosphatgepufferter Salzlösung (PBS) Man löst 0,1 g Natrium-Alginat Pulver, wodurch eine 1% (w / v) Alginat Vorläufers. Um das Pulver vollständig aufzulösen, Inkubation und mischen sie O / N. </li><li> Die Lösung wird durch ein 0,22 um-Filter in eine 1 ml Spritze verbunden. </li><li> Kultur die HepG2-Zellen in Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum und 1% Penicillin-Streptomycin auf einer herkömmlichen Gewebekulturschale bis 70% ergänzt - 80% Konfluenz in einer 5% CO 2 befeuchteten Inkubator bei 37 ° C . </li><li> Die Zellen einmal mit PBS zu waschen, und dann trypsinize sie durch Zugabe von 1 ml 0,05% Trypsin-EDTA für 4 min in einem 5% CO 2 befeuchteten Inkubator bei 37 ° C. Zentrifugation der Zellen von der Kulturschale für 3 min und resuspendieren mit 1 ml PBS nach der Entfernung des Überstands bei 250 × g geerntet. </li><li> Zählen die Anzahl von einzelnen verteilten Zellen in PBS unter Verwendung eines automatisierten ZellZähler. </li><li> Nach der Zentrifugation bei 250 × g für 3 min und PBS Entfernung, 1 ml der 1% (w / v) Natriumalginatlösung zu der verbleibende Zellpellet und mischen sie sanft mit Pipettieren. 10 7 Zellen / ml - die endgültige Saatdichte der Zellen sollte 5 x 10 6 ist. Inkubieren der Zell / Hydrogel-Suspension in einem 5% CO 2 befeuchteten Inkubator bei 37 ° C. </li></ol> 3. Laden und Vernetzung von Cell / Hydrogel Federung <ol><li> Aufzulösen 1,47 g Calciumchlorid zu dehydratisieren in 100 ml ddH 2 O zu einer vernetzenden Reagens (dh 100 mM CaCl 2 · 2H 2 O in ddH 2 O). </li><li> Spülen Sie das Innere eines Luftbefeuchter mit Ultraschall-Wandler mit 70% Ethanol, und füllen Sie es mit 200 ml Vernetzungsreagenz. Der Befeuchter ist 110 mm breit, 300 mm lang und 170 mm tief (dh 110 mm x 300 mm x 170 mm (B x H x T)). </li><li> Legen Sie die mikrostrukturierten PDMSFormen in einem Plasmafilter und reinigen sie für 1 min bei 85 W, um eine hydrophile Oberfläche zu schaffen. </li><li> 7,2 ul des gut vermischten Zell / Hydrogel-Suspension kontinuierlich geladen am Rand des Mikromusters auf der Form. Das gezeigte Beispiel verwendet eine Leberläppchens artigen Maschenmuster (1). Das Volumen der Suspension ist abhängig von der topographischen Muster. </li><li> Um die Gelierung der Zelle / Hydrogel-Suspension zu erreichen, schalten Sie den Luftbefeuchter und überprüfen, dass der Luftbefeuchter erzeugt einen Nebel des Vernetzungsreagens. Spray das Vernetzungsreagenz auf das Hydrogel-Vorläufer für 5 min, für den topographischen Oberfläche des PDMS Formen in einem Bereich von 5 cm. Hinweis: Entfernungen länger und kürzer als 5 cm konnte unvollständige und ungleichmäßige Gelierung zu bewirken sind. Sicherzustellen, dass der Befeuchter mit einer Sprührate von 250 ml / h, wodurch 20 ml Nebel des Vernetzungsreagens in 5 min. </li><li> Nach der Vernetzungsprozess, schalten Sie den Luftbefeuchter und füllen Sie die PDMS-molds mit PBS. </li></ol> 4. Handhabung des Einzel Modular Hydrogel Blätter <ol><li> Nehmen Sie jede gehärtete Hydrogelfolie von den mikrostrukturierten Formen über Pipettieren PBS sanft um das Hydrogel Blatt unter Verwendung eines 200 ul Pipettenspitze. </li><li> Pick up jede schwimm Hydrogelfolie mit einer End-zu schneiden 1.000 ul Pipettenspitze Ende. Jedes Leberläppchens förmige Netz Hydrogelfolie hat Abmessungen von 8 mm x 8,7 mm und ist 100 bis 200 &amp; mgr; m dick. </li><li> Übertragen Sie eine einzelne Schicht des Hydrogels Blatt in 1 ml DMEM in einer 12-Well-Platte, und die Kultur der Zellen in vitro in einer schwimmenden Weise mit der Hydrogel-Konstrukt als eine Einheit Komponente im 12-Well-Platte über eine Woche in einem 5 % CO 2 befeuchteten Inkubator bei 37 ° C. Tauschen Sie jeden zweiten Tag des Kulturmediums. </li></ol> 5. Montage von mehrschichtigen Hydrogel Blätter <ol><li> Die Schritte 1.1 bis 1.5, ein PDMS-Rahmen mit Abmessungen von 18 mm x 18 mm x 4 mm zu erzeugen (W xH x T), und die 170 &amp; mgr; m hohen Säulenstrukturen an der unteren Fläche enthält. Verwenden von 42 g der Mischung von PDMS und Härter, mit dem gleichen Verhältnis, wie in Schritt 1.2. Legen Sie eine spezielle Polycarbonatform auf dem Siliziumwafer für die PDMS-Rahmen, um eine Innenrahmen mit Abmessungen von 8 mm x 9 mm x 2 mm zu erstellen (B x H x T). </li><li> Sterilisieren der PDMS Rahmen und 180 &amp; mgr; m Porennylonfilterpapiere in destilliertem Wasser und einer Pinzette in einem Autoklaven unter Wasser für 15 min bei 121 ° C. </li><li> Legen Sie die sterilisiert PDMS Rahmen auf ein Viertel der ein Stück Nylon-Filterpapier (mit einem Durchmesser von 5 cm) in einem 60-mm-Petrischale. </li><li> Übertragen einer modularen Hydrogelfolie in das Innere des PDMS-Rahmen unter Verwendung einer End-zu schneiden 1.000 &amp; mgr; l-Pipettenspitze. Die Hydrogelfolien zur Montage verwendet werden, sollten für mindestens einen Tag, nachdem sie hergestellt wurden, kultiviert werden. </li><li> Richten Sie die Kante jeder modularen Hydrogelfolie mit dem PDMS-Rahmen mit einem leeren 200 ul Pipettenspitze. </li><li> Wiederholen Sie die Schritte 5.4 und 5.5 unter Verwendung modularer Hydrogelfolien um einen Stapel von 4 bilden - 6 Schichten. </li><li> Entfernen des Kulturmediums durch Fließen ihn durch die Säulenstrukturen am Boden des PDMS-Rahmen. Es werden nun 2 ul Alginatlösung (2% w / v) an einer Ecke des Mehrschichtkonstrukts. </li><li> Sprühen eines Nebels des Vernetzungsreagenz für 30 s auf dem Mehrschichtkonstrukt, um die Kanten der einzelnen Schichten mit denen einer anderen zu befestigen. Ein 2-ml Nebel des Vernetzungsreagenz (mit einer Sprührate von 250 ml / h). </li><li> Spülen Sie die mehrschichtige Aufbau vorsichtig mit 400 ul DMEM und entfernen Sie die PDMS-Rahmen mit einer Pinzette. </li><li> Nehmen Sie das mehrschichtige Aufbau aus dem Filterpapier durch vorsichtiges Abwischen mit einem Zellheber nach der Zugabe von 4 ml DMEM. </li><li> Übertragen Sie die mehrschichtige Aufbau zu einer 6-Well-Platte, die 3 ml DMEM mit Filterpapier, und Kultur die Zellen in vitro in einer schwimmenden Weise mit dem Hydrogel-Konstrukt, wieeine Multiskalenzellgerüst in 6-Well-Platte über eine Woche in einem 5% CO 2 befeuchteten Inkubator bei 37 ° C. Tauschen Sie jeden zweiten Tag des Kulturmediums. </li></ol>

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The authors have nothing to disclose.

Dieses Protokoll liefert ein einfaches Verfahren zur Herstellung von modularen Hydrogelfolien und der Zusammensetzung und der 3D-Zellgerüste bilden. Um eindeutige gemustert Alginat Strukturen in kurzer Zeit zu bauen, sollten wir eine Vernetzung Prozess, der ausreichend steif Strukturen erstellen können, die komplexen Mikrostrukturen aus der Form zu halten, sowie Aufrechterhaltung der Lebensfähigkeit der Zellen und den Stoffwechsel zu identifizieren. Wir haben ein Vernetzungsverfahren entw...

Hydrogele sind besonders vielversprechender Biomaterialien und sollen in grundlegende Biologie, pharmakologischen Tests und der Medizin wichtig. 1 Biofabrication von Hydrogel-basierten zellularen Konstrukte wurde vorgeschlagen, um die Verwendung von Tierversuchen zu reduzieren, 2,3 ersetzen transplantierbaren Geweben, 4 und verbessern zellbasierte Assays. 5,6 Wasser enthaltende (hydro-) viskoelastische Materialien (Gele) ermöglichen eine große Anzahl von Zellen, die eingekapselt und in einer Gerüststruktur, die 3D-zelluläre Mikroumgebung steuern aufrechterhalten werden. In Verbindung mit der Führung des mikrofluidischen oder Mikrostrukturierungstechniken kann die Geometrie der Hydrogel-Konstrukte genau auf zellulärer Ebene gesteuert werden. Bis heute sind eine Vielzahl von Formen von Hydrogelen, einschließlich Teilchen, 7-9 Fasern, 10 - 12 und Blatt, von 13 bis 15 wurden als Bausteine ​​in von unten nach oben ange verwendetSchmerzen auf die Herstellung von Makromaßstab mehrzelligen Architekturen. Beide Hydrogel-basierte Partikel und Fasern sind für Anwendungen wie Mikromaßstab zellulären Umgebungen war leicht und schnell hergestellt, mit fluidischen Steuerungen mit mikrofluidischen Vorrichtungen. Da jedoch die Grundeinheiten von technischen Geweben, würde es schwierig sein, um sie neu anzuordnen und um es zu vergrößern ihr Volumen als Makromaßstab Konstrukte. 16 Es ist schwieriger makro skaliert Konstrukte zu erreichen, als mikrometergroße Grundmodule zu produzieren. Blattartigen Einheiten Hydrogelbasis Konstrukte können verwendet werden, um das Volumen der Gerüste durch einen einfachen Montageprozess zu erhöhen. Folgerichtig gestapelten Schichten Hydrogelfolien bieten nicht nur eine Volumenvergrößerung, sondern auch eine geometrische Ausdehnung in einem 3D-Raum. Wir haben bereits berichtet, ein Verfahren zur Herstellung mikro Hydrogelfolien, 13 - 15 zusammen mit ihrer Anordnung in mehreren LegeEred Zellarchitekturen. Die Technik ermöglicht komplexe Mikrostrukturierung und der modulare Aufbau von Zellkonstrukten über einen Stapelvorgang von mehrschichtigen Strukturen. Durch die Herstellung von gestapelten modularen Hydrogelfolien, die mikrostrukturiert sind, kann ein 3D-Zellkultursystem mit einer gesteuerten Makromaßstab zelluläre Mikroumgebung realisiert werden. Dieses Video-Protokoll beschreibt ein einfaches, aber leistungsfähiges Herstellungsverfahren, das verwendet werden kann, um modulare Hydrogelfolien konstruieren, auf der Grundlage der menschlichen Leberzelllinie (HepG2). Wir zeigen hier einfache Manipulation dieser gemusterten modularen Hydrogelfolien und deren Anordnung in eine Mehrschichtstruktur.

<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

Hydrogele können auf der Mikrowaage unter Verwendung von Mikrofluid oder Mikrostrukturierungstechniken, um eine in vivo-ähnliche dreidimensionale (3D) Gewebegeometrie bereitzustellen strukturiert werden. Die erhaltenen 3D Hydrogelbasis zellulären Konstrukte wurden als Alternative zu Tierversuchen zur weitgehenden biologischen Studien, pharmakologischen Tests und Organtransplantationsanwendungen eingeführt. Obwohl auf Hydrogel-Basis und Fasern können leicht hergestellt werden, ist es schwierig, sie zum Wiederaufbau von Gewebe zu manipulieren. In diesem Video, beschreiben wir ein Herstellungsverfahren für mikro Alginat-Hydrogel Blättern, zusammen mit deren Montage ein Makro angelegte 3D-Zellkultursystem mit einem kontrollierten zellulären Mikroumgebung bilden. 200 um und mit präzisen Mikro - unter Verwendung einer Nebelform des Calcium-Geliermittel werden dünne Hydrogelfolien leicht mit einer Dicke im Bereich von 100 erzeugt. Zellen können dann mit der geometrischen Führung der Hydrogelfolien in gezüchtet werdenfreistehenden Bedingungen. Weiterhin kann die Hydrogelfolien leicht manipuliert werden mit einer Mikropipette mit einem End-Schnittspitze und kann durch Stapeln unter Verwendung einer gemusterten Polydimethylsiloxan (PDMS) Rahmen in mehrschichtigen Strukturen aufgebaut werden. Diese modularen Hydrogelfolien, die mit einem einfachen Verfahren hergestellt werden kann, gibt es Anwendungsmöglichkeiten der in vitro-Wirkstoffassays und biologische Untersuchungen, einschließlich funktionelle Studien von Mikro- und Makrostruktur und Geweberekonstruktion.

Wir haben die Herstellung und Manipulation von freistehenden Mobil Hydrogel Blättern beschrieben. Wie in Figur 1 gezeigt, hergestellt wir mikro PDMS Schimmelpilze und zellhaltige Hydrogel wurde auf die hydrophile Oberfläche dieser Formen und geladen vernetzt mit einem Luftbefeuchter, um eine aerosolierte Nebel von Geliermittel zu erzeugen. Nach der Freigabe aus den Formen wurden HepG2-Zellen in freistehender Hydrogelfolien mit verschiedenen Mustern (Abbildung</...

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.

Bau von Modular Hydrogel Blätter für mikrostrukturierten Makro skaliert 3D Cellular Architektur

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

construction-modular-hydrogel-sheets-for-micropatterned-macro-scaled

Research

JoVE Journal

Bioengineering

10.2K Aufrufe.  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. 

Serie: 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.

Herstellung von mikrostrukturierten Hydrogele für Neural Kultur-Systeme mit Dynamic Mask Projektion Photolithographie

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

Forschung

Biotechnik

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.

Einfache Handhabung von Architekturen in Protein-basierte Hydrogele für Zelle Kultur Anwendungen

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

Biochemie

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.

Drucken Thermoresponsive Reverse-Formen für die Erstellung von Gemusterte Zweikomponenten-Hydrogele für 3D Zellkultur

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

Immunologie und Infektion

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.

Gradient Stamm Chip für anregende zellulären Verhaltensweisen in Zelle beladenen Hydrogel

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.

Licht-vermittelte Bildung und Musterung von Hydrogelen für Zellkulturanwendungen

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.

Eine vielseitige Methode von Patterning Proteinen und Zellen

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.

Zusatzstoff Herstellungstechnik für die einfache und schnelle Herstellung von Hydrogel-basierte Micromachines mit magnetisch reagieren Komponenten

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.

Erste 3D Cluster Zellkontrolle in einem Hybrid Gel Cube Gerät für wiederholbare Muster Formationen

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.

Protokolle des 3D-Bioprintings von Gelatine Methacryloyl Hydrogel-basierten 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.

Vernetzte makroporöse 3D-Gerüste aus Mikrogelstäben

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