Wir möchten, dass Smithfield Farmen zu danken für die intakte Schweinelungengewebe zu spenden. Wir möchten auch Dr. Hu Yang, Dr. Christina Tang und die VCU Abteilung für Plastische Chirurgie für die Erlaubnis, zu nutzen, um ihre Ausrüstung zu danken. Hydrogele und Gewebeproben wurden für SEM bei der VCU Abteilung für Anatomie vorbereitet und Neurobiologie Mikroskopie Einrichtung unterstützt, teilweise durch die Finanzierung von NIH-NINDS-Center Core-Grants 5 P30 NS047463 und zum Teil durch Finanzierungsform NIH-NCI Cancer Center Support Grants P30 CA016059. REM-Aufnahmen von Proben, die bei der VCU Nanotechnologie Kern Charakterisierung Facility (NCC). Diese Arbeit wurde von der National Science Foundation, CMMI 1351162 finanziert.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Lösung </td><td> Sterile Filter </td><td> Richtungen </td></tr><tr><td> DiH 2 O </td><td> ja </td><td> DiH 2 O; steril filtriert </td></tr><tr><td> 0,1% Triton X-100-Lösung </td><td> ja </td><td> Unter Abzugshaube mit 100 &amp; mgr; l Triton-X 100 - Lösung auf 100 ml DiH 2 O und rühren , bis sie gelöst; Sterilfilter. </td></tr><tr><td> 2% Desoxycholat-Lösung </td><td> ja </td><td> Unter Laborabzug 2 g Natriumdesoxycholat-Lösung pro 100 ml DiH 2 O und rühren , bis sie gelöst; Sterilfilter </td></tr><tr><td> 1 M NaCl </td><td> ja </td><td> Hinzufügen , 58,44 g NaCl zu 1 L DiH 2 O; agitieren, bis sie gelöst; Sterilfilter </td></tr><tr><td> DNase-Lösung </td><td> ja </td><td> In 12.000Einheiten DNase auf 1 L DiH 2 O; In 0.156 g MgSO 4 (Wasserfrei) und 0,222 g CaCl 2; agitieren, bis sie gelöst; Sterilfilter </td></tr><tr><td> PBS </td><td> ja </td><td> Kombinieren 27,2 g Na 2 HPO 4 · 7H 2 O (dibasisches Heptahydrat), 80 g NaCl, 2 g KCl und mit 10 L DiH 2 O; agitieren, bis sie gelöst; pH-Wert auf 7,4; Sterilfilter </td></tr></tbody></table> Tabelle 1: Lösungen für Tissue Dezellularisierung Erforderlich. Nehmen Sie die oben genannten Lösungen für die Dezellularisierung Prozess. Lagerung bei 4 ° C. Etwa 2,5 L für eine Schweine Lunge benötigt werden. 1. Hydrogelierung <ol><li> Porcine Lung Dezellularisierung (angepasst von Referenzen 9,10) <ol><li> Erhalten, ein en bloc normalen Schweine Lunge, aus einem Schlachthof, mit Herz und vasculature intakt. </li><li> Verwenden Gewebe Schere oder einem Skalpell, das Herz zu sezieren entfernt Vaskulatur so nahe wie möglich an das Herz schneiden, werden verbleibende Vaskulatur in späteren Schritten für die Perfusion verwendet werden. </li><li> sezieren vorsichtig das Bindegewebe rund um die Luftröhre, Bronchien und Gefäßen mit einem Skalpell oder einer Schere entfernt. HINWEIS: Ermitteln Sie die beste Lunge für den Rest des Verfahrens zu verwenden, indem eine Lunge ohne große Schnitte oder Einstiche durch die Pleura und große Mengen an Atelektase oder Gefäßverschluss wählen, die die Wirksamkeit des Dezellularisierung Prozess behindern können. </li><li> Schneiden Sie das suboptimal Lunge verlässt, so viel Bronchus zur Trachea wie möglich angebracht entfernt (Herz, Bindegewebe und den Lappen der Lunge entfernt entsorgt werden kann). Es sollte eine Lunge zur Trachea und Vaskulatur befestigt bleibt sein. HINWEIS: Bewahren Sie Abschnitte für die Histologie, falls gewünscht. 9 </li><li> Schließen Sie nicht angeschlossen Bronchien mit Klammern oder Naht prevent Überschuss Rückstau. </li></ol><ol start="6"><li> Bereiten Sie die Dezellularisierung Lösungen und im Kühlschrank lagern bei 4 ° C , bis sie benötigt (Tabelle 1). </li><li> Mit Hilfe einer Handpumpe eine Kanüle eingeführt, die Lungenarterie zu passen, perfuse sowohl das Lungengewebe 3-mal mit DI-Wasser durch Lungenarterie und der Trachea. Perfuse Gefäß erste jedes Mal. Beginnen Sie mit ca. 1 L in Gefäßen und 1,5 l in der Trachea für jede Perfusion, aber als Zelltrümmer mehr Flüssigkeit entfernt wird, kann durch das System durchblutet werden. </li><li> Perfundieren sowohl Vaskulatur und der Trachea mit Triton X-100-Lösung. </li><li> Unterzutauchen Lungengewebe in Triton X-100-Lösung für 24 h bei 4 ° C. </li><li> Perfuse Gefäßsystem und der Trachea 3 mal mit VE-Wasser zu spülen. </li><li> Perfuse beide Gefäße und der Trachea mit Desoxycholat-Lösung. </li><li> Tauchen Sie die Gewebeschnitte in Desoxycholat für 24 h bei 4 ° C. </li><li> Perfuse Gefäßsystem und der Trachea 3 mal mit VE-Wasser zu spülen. </li><li>Perfuse beide Gefäße und der Trachea mit NaCl-Lösung. </li><li> Unterzutauchen Gewebe in filtriert NaCl-Lösung für 1 h bei 4 ° C. </li><li> Perfuse Gefäßsystem und der Trachea 3 mal mit VE-Wasser zu spülen. </li><li> Perfuse beide Gefäße und der Trachea mit DNase-Lösung. </li><li> Unterzutauchen Gewebe in filtriert DNase-Lösung für 1 Stunde bei 4 ° C. </li><li> Perfuse beide Gefäße und der Trachea 5-mal mit PBS. </li><li> Sezieren weg bemerkbar Knorpelgewebe, Luftröhre und alle Tubuli 2 mm oder größer im Durchmesser (in erster Linie um den Hilus und medialen Abschnitte der Lunge) aus Atemwege leitet, so dass nur Atmungszonen (in erster Linie die Randbereiche). </li><li> Präparieren Gewebe in 1-Zoll-Abschnitte oder kleiner. Ausrichtung des Gewebes keine Rolle spielt für diesen Schritt. </li><li> überschüssige Flüssigkeit und Stelle Gewebe in 50 ml konischen Röhrchen abgießen. Frieren Sie das Gewebe bei -80 ° C. HINWEIS: Bewahren Abschnitte für die Histologie Entfernung von Zellen und Zell debri zu gewährleistens, wenn gewünscht. Wir verwenden H &amp; E, α-Galactosidase, PicoGreen, Hydroxyprolin, Ninhydrin, Alcianblau, SDS-PAGE und Massenspektrometrie charakterisiert werden. 9 </li></ol></li><li> Lung Verarbeitung <ol><li> Entfernen Sie Deckel aus gefrorenen Röhrchen dezellularisierter Lungengewebe enthält. </li><li> Legen Sie Filterpapier über die Rohröffnung einsetzen und mit Gummiband. HINWEIS: Rohr und der Inhalt sollte noch sonst in -80 ° C platzieren eingefroren werden, bis gefroren. </li><li> Lyophilisieren das Gewebe, bis das gesamte überschüssige Flüssigkeit weg ist, einen Gefriertrockner mit nach Hersteller Richtungen. Bei -80 ° C bis zum fertig zu fräsen. </li><li> Vor flüssigem Stickstoff zu Gefrierschrank Mühle beginnt hinzufügen Isolierung zu kühlen und interne Komponenten auf die Arbeitsbedingungen. </li><li> Entfernen Sie magnetische Mühle bar aus Mühle Rohr und fügen Gewebe Boden zu bedecken. </li><li> Ersetzen Mühle bar und fügen Sie locker gepackten Gewebe. Die Mühle Bar sollte noch frei bewegen. </li><li> Schließen Sie die Mühle Rohr und place in Gefrierschrank Mühle. </li><li> Füllen von flüssigem Stickstoff bis max Fülllinie. </li><li> Gefrierschrank Mühle alle Gewebe in ein feines Pulver (ca. 5 min, Impaktion Rate von ~ 600 min -1), in einem Polycarbonat - Zylinder mit einem Edelstahl - Impaktor sowie Edelstahl Endstopfen mit der Gefriermühle nach den Herstelleranweisungen. Bei -80 ° C bis zur Verwendung. </li></ol></li><li> Mikroporöse Gelbildung (8 mg / ml) ( in Anlehnung an Referenzen 9,11) <ol><li> 1% (w / v) des dezellularisierten Pulver und 0,1% (w / v) des Pepsins auf 0,01 M HCl, unter konstantem Rühren (sollte in der Lage sein Strömung auf der obersten Ebene der Flüssigkeit zu sehen), bei Raumtemperatur, 48 Std. HINWEIS: Das Pulver statisch aufgeladen ist, so das HCl-Zugabe, nachdem das Pulver die Möglichkeit bietet den Überschuss aus den Rohrwänden zu waschen. </li><li> Nach der Verdauung 48 h, legen Sie die Lösung und Reagenzien auf Eis für 5 min. </li><li> Verwendung gekühlter 10% (v / v) 0,1 M NaOH, eind 11.11% (v / v) 10 x PBS (die gesamte Lösung zu bringen Konzentration 1x), bringen die verdauten Proteinlösung auf physiologischen pH von 7,4. HINWEIS: Die Lösung kann jetzt bis zu einer Woche bei 4 ° C gelagert werden. Führen Sie Gelieren Kinetik mit rheometry 9 , falls gewünscht. </li></ol></li></ol> 2. Vernetzungs Hydrogele mechanische Festigkeit zu verbessern <ol><li> Lösungen von 1%, 0,1% und 0,01% w / v Genipin Vernetzungslösung wurden durch Lösen der erforderlichen Menge Genipin Pulver in 10% DMSO hergestellt. </li><li> Vortex, um die Lösung alle 15 min für 1 Stunde. </li><li> In Genipin Lösung ECM Hydrogele zu decken (100 &amp; mgr; l für jede Vertiefung einer 96-Well-Platte), die zuvor in Teil 1.3.4 zusammengestellt. </li><li> Verlassen jedes ECM Hydrogel für 24 h bei 37 ° C zu vernetzen. </li><li> Spülen Sie das ECM-Hydrogel 3-mal mit PBS, bis die Waschlösung nicht mehr blau. Die vernetzten Hydrogele werden dann bereit sein, für die weitere Charakterisierung und cell Kulturstudien. </li></ol> 3. Zellkultur mit mikroporöser Gel <ol><li> Zweidimensionale Beschichtungs <ol><li> Verwendung der Lösung aus 1.3.3 werden 20 ul Pregel-Lösung in jede Vertiefung einer 96-well nicht behandelten Gewebekulturplatte. </li><li> Kühlen Sie über Nacht bei 4 ° C Proteinadsorption an der Platte zu ermöglichen. </li><li> Absaugen Vorgelats Lösung und Spülen mit PBS. </li><li> Passage Zellen 9. </li><li> In 10.000 Zellen / cm 2 in die Vertiefungen. </li><li> Erhöhen Medien zu 100 ul pro Vertiefung und Inkubation bei 37 ° C. </li></ol></li><li> Dreidimensionale Zellkultur 9 <ol><li> Verwendung Lösung aus 1.3.3 resuspendieren pelletierten Zellen auf eine Konzentration von 1 Million Zellen / ml Vorgellösung. </li><li> Schnell 16 ul Pregel-Zellsuspension in jede Vertiefung einer 96-Well-Platte verzichtet werden kann, ein ~ 500 &amp; mgr; m dicke Hydrogel für 3D-Zellkultur zu bilden. </li><li> bei 37 ° C inkubieren 30min für Gel zu bilden. </li><li> 100 l Medien an die Spitze der gebildeten Hydrogele und bei 37 ° C inkubiert. </li></ol></li><li> Dreidimensionale Zellkultur auf variabler Steifigkeit Gele <ol><li> Mit Lösung von 1.3.3, Pipette 100 ul Pregel-Lösung in jede Vertiefung einer 96-Well-Platte. </li><li> bei 37 ° C 30 Minuten inkubieren für Gel zu bilden. Hinweis: Die Vernetzung Schritte von Teil 2 kann hier verwendet werden. </li><li> Passage Zellen 9. </li><li> In 10.000 Zellen / cm 2 in die Vertiefungen. </li><li> Erhöhen Medien zu 100 ul pro Vertiefung und Inkubation bei 37 ° C. </li></ol></li></ol>

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Die Autoren haben nichts zu offenbaren.

Einer der integralen Aspekte der Biologie ist die Selbstorganisation der Moleküle in hierarchischer Strukturen, die eine bestimmte Aufgabe auszuführen. 13 Im Labor Selbstorganisation hängt von zahlreichen Faktoren wie Salzkonzentration, pH, und die Verdauung Dauer. Wie gezeigt ist, selbstorganisierend Hydrogel bildet sich, wenn solubilisiert Proteine ​​Rückkehr zu einer physiologischen Temperatur. Das Hydrogel gebildet ist in der Lage zur Förderung der Zellanhaftung und Proliferation in vitro.</e...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>Rheometer</td><td>Discovery</td><td>HR-2</td><td>Use gloves and eye protection.</td></tr></tbody></table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Hier stellen wir ein Verfahren zur Mehrkomponenten - Zellkultur - Hydrogele für in - vitro - Lungen - Zellkultur zu etablieren. Beginnend mit gesunden en bloc Lungengewebe von Schweinen, Ratte oder Maus wird das Gewebe durchblutet und unter Wasser in nachfolgenden chemischen Reinigungsmittel , die Zelltrümmer zu entfernen. Histologische Vergleich des Gewebes vor und nach der Verarbeitung bestätigt Entfernung von mehr als 95% der Doppelstrang-DNA und alpha-Galactosidase-Färbung schlägt die Mehrheit der Zelltrümmer entfernt wird. Nach Dezellularisierung wird das Gewebe lyophilisiert und dann in ein Pulver cryomilled. Das Matrixpulver wird 48 h in einer sauren Pepsinverdau Lösung verdaut und dann die Pregel-Lösung neutralisiert zu bilden. Gelieren der Pregel Lösung kann durch Inkubation bei 37 ° C induziert werden und kann unmittelbar nach der Neutralisation oder bei 4 ° C gelagert werden für bis zu zwei Wochen. Beschichtungen können unter Verwendung der Vorgellösung auf einer nicht behandelten Platte für c gebildet werdenell Befestigung. Zellen können in dem Vorgel vor Selbstmontage ausgesetzt werden, um eine 3D-Kultur zu erreichen, die auf der Oberfläche eines geformten Gels plattiert, aus dem die Zellen durch das Stützgerüst migrieren können oder auf den Beschichtungen überzogen. Änderungen der Strategie vorgestellt kann Gelierungstemperatur, Stärke oder Protein Fragmentgrößen auswirken. Jenseits Hydrogelbildung kann das Hydrogel Steifigkeit erhöht werden Genipin verwenden.

Unter Verwendung dieses Verfahrens haben wir Hydrogele aus normalen Schwein, Ratte produziert und Mauslungen (Abbildung 1). Verarbeitet Lungen liefern schätzungsweise 5 mg, 40 mg und 10 g ECM-Pulver sind. Ein Überblick über das Verfahren ist in Abbildung 2 dargestellt. Key-Visualisierungen während des Prozesses sind: weißes Aussehen der Lunge nach deoxycholate Spülen; nach der Bildung Pregel sollte die Lösung opak sein, und die Lösung sollte für...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Dies ist eine Methode, um eine 3-dimensionale Zellkultur Gerüst von pulmonaler extrazellulären Matrix zu schaffen. Intact Lunge in Hydrogelen verarbeitet, die das Wachstum von Zellen in drei Dimensionen zu unterstützen.

Tunable Hydrogele aus Pulmonary Extrazellulärmatrix für 3D-Zellkultur

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

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.6K Aufrufe.  Virginia Commonwealth University.  Dies ist eine Methode, um eine 3-dimensionale Zellkultur Gerüst von pulmonaler extrazellulären Matrix zu schaffen. Intact Lunge in Hydrogelen verarbeitet, die das Wachstum von Zellen in drei Dimensionen zu unterstützen. 

Serie: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

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 ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting cellularized Konstruiert unter Verwendung eines gewebespezifischen Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues für injizierbare Regenerative Therapie und Hochdurchsatz-Drogen-Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Ultradünne derzei-elastischen Hydrogele als eine biomimetische Basalmembran für Dual Zellkultur

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

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

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

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

Produktion von Elastin-Like Protein Hydrogele für Kapselung und Immunostaining Zellen in 3D

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

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Kontrollierte Dehnung von 3D-Hydrogelen unter Live-Mikroskopie-Bildgebung

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Hydrogel-Arrays ermöglichen erhöhten Durchsatz für Screening-Effekte von Matrixkomponenten und Therapeutika in 3D-Tumormodellen

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Photoabstimmbare biogedruckte Hydrogele zur Untersuchung zellulärer Reaktionen auf ECM-Versteifung

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Autor im Rampenlicht: Chronische Lungenerkrankungen mit 3D-gedruckten phototunablen Hydrogelen verstehen 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

3D-Bioprinting von phototunbaren Hydrogelen zur Untersuchung der Fibroblastenaktivierung

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...