Die Autoren danken dem NSERC Discovery Grant 402462 &quot;Tissue Engineering Scaffolds zur Steuerung von induzierten pluripotenten Stammzellen-Verhalten&quot; zu bestätigen.

Hinweise vor dem Start Protokoll: Fibrinogen ist ein Protein, Blut gewonnen und damit entsprechende Sicherheitsschulungen müssen vor Behandlung abgeschlossen sein. Dieses Protokoll erfordert 2 Tage Zeit in Anspruch, so die gewünschten Stammzellen Kulturen angemessen zu gewährleisten, sind sie bereit für die Aussaat. Im Hinblick auf die Berechnung, wie viel Fibrinogen abwiegen, drei 35-mm-Petrischalen von Tris-gepufferter Kochsalzlösung (TBS, pH 7,4), die 110-130 mg Fibrinogen in 3 ml TBS gelöst wird ausreichend sein, um 1 24-Well-Platte mit 400 zu erzeugen ul Fibrin Gerüste in jede Vertiefung. 1. Day One: Making Fibrinogen-Lösungen und Dialyse über Nacht <ol><li> Nehmen lyophilisiert Fibrinogen aus dem Kühlschrank und lassen Sie ihn 20 Minuten lang sitzen, damit es auf Raumtemperatur kommen. </li><li> In 3 ml Tris-gepufferter Salzlösung (TBS) zu jedem 35 mm Petrischale. Jede Platte von Fibrinogen erhält man 2 bis 3 ml Fibrinogen-Lösung in einem Konzentrationsbereich von 12 bis 20mg / ml und die Gesamtmenge von Fibrinogen benötigt werden, können auf der Grundlage dieser Näherungen berechnet werden. </li><li> Abwiegen ca. 100-130 mg Fibrinogen (Fg) und streuen auf die Oberfläche des TBS, die in jedem Gericht. Warten Sie 5 Minuten für das Fibrinogen zu beginnen, in Lösung gehen. Cover Petri-Schale mit Deckel. </li><li> Petrischalen inkubieren bei 37 ° C für 2 Stunden, damit das Fibrinogen vollständig in die TBS-Lösung gehen. </li><li> Wet Dialyseschlauch (7000 MW abgeschnitten) mit TBS. Falten unteren Ende des Rohrs und Klemme Ende mit einer Dialyse Klemme geschlossen. </li><li> Pipettieren Sie die Fibrinogen-Lösungen von Geschirr in Dialyseschlauch. Falten Sie über oberen Ende und mit einer Klammer verschlossen Dialyse zu klemmen. </li><li> Platz Dialyseschlauch mit Fibrinogen-Lösung in einem 4 Liter-Behälter mit TBS gewaschen. Mix-Lösung mit Rührplatte setzen auf die niedrige Drehzahl (100 rpm). </li><li> Dialysieren Lösung über Nacht rühren auf Platte für mindestens 12 Stunden. Die TBS-Lösung muss nicht über diesen Zeitraum geändert werden. </li></ol&gt; 2. Tag 2: Polymerisation von Fibrin Gerüste und Aussaat von Stammzellen in Gerüste Alle Schritte für diesen Prozess beschrieben werden, sollten in einem sterilen Gewebekultur Abzug durchgeführt werden, da diese Gerüste mit Stammzell-Kulturen ausgesät werden. <ol><li> Entfernen Sie die Fibrinogen-Lösung aus Dialyseschlauch und in einen geeigneten, großen konischen Rohr (entweder 15 ml oder 50 ml). </li><li> Filter Fibrinogen-Lösung mit einer 5,0 um Spritze zur Aufnahme größerer Verunreinigungen in der Lösung zu entfernen. Abhängig von dem Volumen der Lösung kann die Verwendung von mehreren Filtern erforderlich sein, die gesamte Lösung zu verarbeiten. </li><li> Sterilfilter der Fibrinogen-Lösung mit einem 0,22 um Filter in einem 50 ml konischen Röhrchen. Unter Verwendung eines geeigneten Größe Pipette bestimmen das Volumen des gefilterten Fibrinogen-Lösung für eine spätere Verwendung, wenn dabei Berechnungen. Abhängig von dem Volumen der Lösung kann die Verwendung von mehreren Filtern erforderlich sein, die ent verarbeitenIRE-Lösung. </li><li> Wird die Extinktion der Fibrinogen-Lösung bei 280 nm mit dem Spektralphotometer. Eine Verdünnung von 50 ist oft notwendig, eine Absorption unter 1 zu erhalten. </li><li> Berechnen Sie die Konzentration des Proteins in der Fibrinogen-Lösung mit der folgenden Gleichung: [Fibrinogen in mg / ml] = (A 280 × Verdünnungsfaktor) 1,55 ÷ 1,55, wo ist der Extinktionskoeffizient bei A 280 für humanes Fibrinogen 13. Weiter berechnet die Menge an TBS erforderlich, um die Konzentration der Lösung auf 11,1 mg / ml Fibrinogen auf das ursprüngliche Volumen bezogen verdünnen. Die Endkonzentration des Proteins in der Fibrin-Gerüste werden mit 10 mg / ml nach der Polymerisation sein. </li><li> Erhalten sterilen Thrombin und Calciumchlorid-Lösungen. Fügen Sie zunächst 15 ul Thrombin-Lösung (Konzentration: 40 U / ml - Endkonzentration im Gerüst wird 2 U / ml) auf der rechten Seite jeder Vertiefung in der ersten Reihe der 24-Well-Platte. Als Nächstes fügen Sie 15 ul 50 mM Kalzium chlrid-Lösung auf der linken Seite eines jeden gut. Schließlich, fügen Sie 270 ul der Fibrinogen-Lösung auf die Platte. Schütteln Platte von Seite zu Seite durch Schütteln von hinten nach vorne, um sicherzustellen, Mischen von Lösungen an. </li><li> Lassen Sie die Zeile mit der Mischungen für 5 Minuten bei Raumtemperatur polymerisieren. Die Gerüste werden mehr undurchsichtig, da sie zu polymerisieren. </li><li> Wiederholen Sie die Schritte 2.6 und 2.7 bis die gewünschte Anzahl von Fibrin Gerüsten polymerisiert worden. </li><li> Nach der letzten 5-minütigen Inkubation, stellen die Platten in einer 37 ° C-Inkubator, um die vollständige Polymerisation von Gerüsten zu ermöglichen. </li><li> Nach der einstündigen Inkubation abgeschlossen ist, ist der nächste Schritt, um das Gerüst mit Samen Embryoidkörpern. Mit einer 20 ul pipettemen, wählen Sie eine individuelle Embryoidkörper und Platz in der Mitte des Fibrin Schafott. Prüfen Sie mit Mikroskop, dass jeder auch ein einzelnes Embryoidkörper enthält. </li><li> 5 l der Thrombin-Lösung (Konzentration: 40 U / ml) und 5 ul 50 mM CalciumchloridLösung auf der jeweils Embryoidkörper von 90 &amp;mgr; l Fibrinogen-Lösung in jede Vertiefung gefolgt. Die Lösung sollte eine Blase bilden Kapselung der Embryoidkörpern. </li><li> Legen Sie die Platte wieder in 37 ° C Inkubator für eine weitere Stunde, um die Polymerisation zu gewährleisten. </li><li> Nach der Inkubation 1 ml der entsprechenden Zellkulturmedien in jede Vertiefung und Platz Platte wieder in 37 ° C Inkubator. </li></ol> 3. Repräsentative Ergebnisse 1 zeigt eine schematische Darstellung der Seitenansicht einer einzelnen Vertiefung, die das 3D-Gerüst Fibrin Kultursystem mit einer Embryoidkörper. 2A zeigt repräsentative Bilder ausgesät von Maus-induzierte pluripotente Stammzellen die auf embryonalen Maus-Fibroblasten-Feeder-Schichten kultiviert. Diese Zellen werden dann veranlaßt, Embryoid Bodies, Aggregate von Zellen, die neuralen Vorläuferzellen, unter Verwendung eines 8 Tage Retinsäure Behandlungsprotokoll (2B) als previousl bildeny beschrieben 14, während Abbildung 3 zeigt das Aussehen eines iPS-abgeleitet Embryoidkörper nach 3 Tagen der Kultur innerhalb der 3D-Fibrin-Gerüsten. Ähnliche Ergebnisse wurden zuvor mit embryonalen Stammzellen der Maus 7 erhalten wird. Zusätzlich wurde diese Methode verwendet, um iPS-Kultur abgeleitet Embryoidkörpern hergestellt unter Verwendung eines anderen Protokolls mit Retinsäure und purmorphamine 15 innerhalb der 3D-Fibrin-Gerüste, demonstriert die Vielseitigkeit dieser Kultur-System. <img alt="Abbildung 1." src="/files/ftp_upload/3641/3641fig1.jpg" /> Abbildung 1. Schematische Darstellung der Seitenansicht eines einzelnen gut mit einem 3D-Gerüst entkernt Fibrin mit einem Embryoidkörper. <img alt="Abbildung 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> Abbildung 2. Maus induzierte pluripotente Stammzellen (iPS) Zellkultur und Differenzierung. Um diese Zellen zu differenzieren neural Phänotypen sind die iPS-Zellen in Suspension kultiviert, um Aggregate von Zellen, die sogenannten Embryoidkörpern (EVG) zu produzieren. Diese werden dann mit EBs Retinsäure behandelt, um neuronale Differenzierung induzieren und das Protokoll wurde bereits verwendet, um neuronale Differenzierung von embryonalen Stammzellen zu induzieren. A) Nicht differenzierte iPS-Zellen der Maus Kolonie auf einer embryonalen Maus-Fibroblasten-Feeder-Schicht kultiviert. B) Embryoidkörpern Maus aus iPS-Zellen in Suspensionskultur abgeleiteten am Tag 8 nach der Behandlung mit Retinsäure ergriffen werden, um neuronale Differenzierung zu induzieren. Maßstabsbalken ist 100 um. <img alt="Abbildung 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> 3. Beispiel Ergebnisse einer Maus iPS Embryoidkörper nach 3 Tagen Kultur innerhalb eines 3D-Gerüst Fibrin. Die Zellen haben begonnen, zu migrieren und innerhalb des Fibrin Gerüst unterscheiden. Maßstabsbalken ist 500 um.

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Die Autoren haben nichts zu offenbaren.

Dieses Protokoll oben genannten ein Verfahren zur Erzeugung von 3D Fibrin Gerüste für pluripotente Stammzellen in Kultur, die speziell für embryonalen und induzierten pluripotenten Stammzellen. Diese 3D-basierte Biomaterial Kultursystem genauer imitiert die Stammzellnische in vivo gefunden und als Ergebnis kann verwendet werden, um biologische Signale zu screenen, um ihre Wirkung auf Differenzierung der Stammzellen zu bestimmen 6 werden. Unsere Beobachtungen haben gezeigt, dass diese Gerüste, wenn sie mit Stammzellen abgeleitet Embryoidkörpern bleiben für 2 Wochen in vitro, bevor er vollständig abgebaut ausgesät. Zur weiteren Erhöhung der Stabilität dieser Gerüste, kann Aprotinin (eine Protease-Inhibitor) verwendet, um den Abbau durch Addition an den Zellkulturmedien zu verlangsamen. 7,16 Diese Gerüste wurden auch erfolgreich für neuronale Tissue Engineering-Anwendungen, insbesondere die Erzeugung von Gewebe verwendet ähnlich der in das zentrale Nervensystem 17 fanden. Während andere groups haben iPS-Zellen mit Fibrinkleber zur Behandlung ischämischer Schlaganfall 18 kombiniert, stellt diese Arbeit die erste bekannte Bericht der Kombination von iPS-Zellen mit Fibrin Gerüste für neuronale Tissue Engineering-Anwendungen. In dieser Arbeit wurde als Ausgangspunkt für die Kombination einer Reihe von Stammzelltypen mit Fibrin Gerüsten für andere Anwendungen in der Gewebezüchtung serviert.

Benötigte Ausrüstung: Analysenwaage pH-Meter Zellkultur-Inkubator (37 ° C, 5% CO 2) Rührplatte Spektrophotometer Sterile Gewebekulturabzug Tris-gepufferter Kochsalzlösung (pH 7,4) (L Need 4 plus genug zum Lösen Fibrinogen) 50 mM Calciumchlorid-Lösung Sterile konische Röhrchen (15 bzw. 50 ml) 35 mm Petrischalen Dialyseschlauch (7.000 MW-Cutoff) Dialyse-Clips 5,0 um Spritzenfilter Einzeln verpackte sterile Spritze 0,22 um-Filter Spritze 24-Well-Gewebekulturplatten <table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Fibrinogen (human) </td><td> CalbiocSaum </td><td> 341578 </td><td></td></tr><tr><td> Thrombin (human) </td><td> Sigma </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

Die Stammzellen werden in natürlich vorkommenden 3D Mikroumgebungen in vivo, was oft als Stammzellnische 1 sind gemäß vorhanden. Kultivierung von Stammzellen in der 3D-Gerüste Biomaterial bietet eine Möglichkeit, genau nachahmen diese Mikroumgebungen, was einen Vorteil gegenüber herkömmlichen 2D-Kulturverfahren unter Verwendung von Polystyrol als auch ein Verfahren zur Engineering Ersatzgewebe 2. Während 2D Gewebekultur Polystyrol ist für die Mehrheit der Zellkultur-Experimente verwendet wurde, kann 3D Biomaterial Gerüsten näher replizieren Mikroumgebungen in vivo durch eine genauere strukturellen Zellpolarität in der Umwelt und besitzt biochemische und mechanische Eigenschaften ähnlich weichen Gewebe. 3 Eine Vielzahl von natürlich gewonnenen synthetischen Biomaterialien und Gerüste wurden als 3D-Umgebungen für die Unterstützung Stammzellwachstumsfaktoraktivität untersucht worden. Während synthetische Gerüste synthetisiert, um eine größere r haben werdenAnge von mechanischen und chemischen Eigenschaften und haben oft eine höhere Reproduzierbarkeit werden natürliche Biomaterialien oft von Proteinen und Polysacchariden in der extrazellulären Matrix und als Ergebnis enthalten Bindungsstellen für Zell-Adhäsion und bereitwillig unterstützen Zellkultur gefunden komponiert. Fibrin Gerüste, die durch Polymerisation von Fibrinogen aus Plasma erhalten hergestellt wurden in großem Umfang für eine Vielzahl von Anwendungen in der Gewebezüchtung in vitro und in vivo 4 untersucht. Solche Gerüste können modifiziert werden unter Verwendung einer Vielzahl von Methoden, um Systeme mit kontrollierter Freisetzung zur Abgabe von therapeutischen Faktoren 5 einzubauen. Frühere Arbeiten haben gezeigt, dass solche Gerüste, um erfolgreich Kultur embryonaler Stammzellen verwendet werden kann und das Grundgerüst-basierte Kultur kann verwendet werden, um die Wirkungen von verschiedenen Wachstumsfaktoren auf die Differenzierung der Stammzellen in 6,7 ausgesät zu screenen. Dieses Protokoll Details der Prozess der polymerizing Fibrin Gerüste aus Fibrinogen Lösungen mit der enzymatischen Aktivität von Thrombin. Der Prozess dauert 2 Tage in Anspruch, einschließlich einer nächtlichen Dialyse Schritt für die Fibrinogen-Lösung zu Citrate, die Polymerisation hemmen entfernen. Diese Methoden beruhen auf detaillierten Fibrinogenkonzentrationen bestimmt als optimal für die embryonale und induzierte pluripotente Stammzellen in Kultur. Andere Gruppen haben weitere Fibrin Gerüste für eine Vielzahl von Zelltypen und Anwendungen untersucht - die Vielseitigkeit dieses Ansatzes 12.08.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Diese Arbeit wird die Herstellung von 3D-Fibrin-Gerüste für die Kultivierung und Differenzierung von Stammzellen plutipotent. Solche Gerüste verwendet werden, um die Auswirkungen von verschiedenen biologischen Verbindungen zur Stammzellforschung Verhalten Bildschirm als auch modifiziert werden, um Drug Delivery-Systemen enthalten sein.

Erstellung von 3D-Fibrin-Gerüste für Stem Cell Culture Anwendungen

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

preparation-of-3d-fibrin-scaffolds-for-stem-cell-culture-applications

Nanyang Technological University

Research

JoVE Journal

Bioengineering

25.1K Aufrufe.  University of Victoria. Diese Arbeit wird die Herstellung von 3D-Fibrin-Gerüste für die Kultivierung und Differenzierung von Stammzellen plutipotent. Solche Gerüste verwendet werden, um die Auswirkungen von verschiedenen biologischen Verbindungen zur Stammzellforschung Verhalten Bildschirm als auch modifiziert werden, um Drug Delivery-Systemen enthalten sein.

Serie: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Selbstberichts Gerüste für 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Forschung

Biotechnik

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Probenvorbereitung Strategien für Massenspektrometrie Imaging von 3D-Zellkulturmodelle

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D-Hydrogel Scaffolds für Articular Chondrozyten-Kultur und Knorpel-Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Epithelialen Zellwiederbevölkerung und Herstellung von Nagetier extrazellulären Matrix Scaffolds für Nierengewebe Entwicklung

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Herstellung von mechanisch Tunable und Bioactive Metallgerüste für biomedizinische Anwendungen

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Mikrostrukturierung und Montage von 3D-Micro-Ader

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

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 ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Synthese von biokompatiblen Liquid Crystal Elastomerschäume als Zellgerüste für 3D Spatial Zellkulturen

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...