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 Ge...

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

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 ...

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 ...

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 ...

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 ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Bau eines vielschichtigen mesenchymale Stammzellen-Blatt mit einem 3D dynamische Kultursystem

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Eine Kulturmethode zur Erhaltung von quiescent Human Hematopoietic Stammzellen

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Herstellung und Charakterisierung von Graphen-basierter 3D-Biohybrid-Hydrogel-Biotinte für das periphere Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...