Name: stabilizing hepatocellular phenotype using optimized synthetic surfaces
Uploaded: 2014-09-26T14:00:00
Description: Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

DCH, MB und FK wurden von einem EPSRC Folgen auf Fonds unterstützt. BL-V und DS wurden jeweils durch MRC PhD student unterstützt. KC wurde durch Mittel aus dem UK Regenerative Medizin-Plattform unterstützt.

1. Synthese von PHNGAD (Poly [1,6-hexanodiol / Neopentylglykol / Di (ethylenglykol)--alt Adipinsäure] diol) <img alt="Schema 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Schema 1: Synthese von PHNAGD Schematische Darstellung der Synthese von PHNAGD.. PHNAGD wurde durch Umsetzung von 1,6-Hexanodiol, Diethylenglykol, neoppentyl Glykol und Adipinsäure hergestellt. PHNAGD, Poly [1,6-hexanodiol / Neopentylglykol / Di (ethylenglykol)--a...

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Viele der verwendeten Hepatozyten aus Stammzellen erzeugen aktuellen Methoden beruhen auf undefinierte Matrizen tierischen Ursprungs. Diese Substrate können kostspielig und sehr variabel sein und somit die Zellfunktion und der Stabilität, die ein großes Hindernis für Anwendung. Daher führten wir einen Bildschirm für synthetische Materialien, die die Kultur der Stammzell abgeleitet Hepatozyten zu unterstützen. Wir haben festgestellt, eine einfache Polyurethan (PU134) durch Polymerisation PHNGAD, MDI und ein Streck...

Biologische Materialien sind weit verbreitet bei der Aufrechterhaltung und Differenzierung von pluripotenten Stammzellen 1 verwendet. Und ermöglichen, enthalten diese biologischen Substraten oft eine Vielzahl von unbestimmten Komponenten. Matrigel ist eine häufig verwendete Substrat für die Stammzellkultur und Differenzierung. Leider beeinflusst die variable Zusammensetzung der Zellfunktion und Phänotyp. Obwohl eine Vielzahl von alternativen, definierten biologischen Matrices wurden verwendet, 2-7, ihre tierischen Ursprungs oder schlechte Skalierbarkeit macht sie ungeeignete Kandidaten für die industrielle Fertigung. Daher ist die Identifizierung von synthetischen Alternativen, mit definierter Zusammensetzung und zuverlässige Leistung, sind wichtige Ziele in der Stammzellforschung. In einem Versuch, die Grenzen der undefinierten Zellkultursubstraten zu überwinden, sind interdisziplinäre Kooperationen zwischen Chemie und Biologie synthetischen Materialien mit der Fähigkeit, Zell-Phänotyp unterstützt identifiziert. SynthWDV-Substrate sind skalierbar, kostengünstig und kann in komplexe 3D-Strukturen hergestellt werden, imitiert die in vivo-Umgebung. Aufgrund dieser Eigenschaften synthetischen Substraten sind weit verbreitet zu unterstützen und zu fahren Differenzierung vieler Zelltypen 8-10. Erweiterte und Hochdurchsatz-Assays haben die schnelle Screening von synthetischen Materialien erleichtert, von großen Bibliotheken, und lieferte neuartige Materialien mit flexiblen Eigenschaften mit breiten Anwendungen in der biomedizinischen Forschung und Entwicklung 11-13. Verwendung von Hochdurchsatz-, Polymer-Mikroarray-Screening-Technologie, haben wir schnell eine einfache Polyurethan (PU134), geeignet für die Instandhaltung von menschlichen Stammzellen abgeleiteten Hepatozyten identifiziert. Dieses Polymer wurde zu überlegen Tieren stamm Substrate hinsichtlich Hepatozyten-Differenzierung und-Funktion 14-16 sein. Wir haben anschließend die Beschichtungsbedingungen, Topographie und Sterilisationsverfahren, Effekte zugreifen optimiertauf Polymer Leistung bei der Stabilisierung Hepatozyten Funktion und Lebensdauer. Dies hat erhebliche Auswirkungen in Bezug auf das Verständnis der Biologie Grundlagen der Hepatozyten für Zell-basierte Modellierung und Anwendungen der regenerativen Medizin. Die hier beschriebene Technologie ist ein Beispiel, wie die Oberfläche eines synthetischen Polymers kann optimiert werden, um Zell-Phänotyp zu bewahren. Wir glauben, dass die Kombination dieser Technologie mit einer effizienten serumfreien Hepatozyten Differenzierungsprotokoll hat das Potenzial, eine skalierbare Herstellung von Hepatozyten zur Verwendung in in vitro-Modellierung und regenerative Medizin bereitzustellen.

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	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmun B&#252;hler</td>
			<td>KS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>CIS Biointernational</td>
			<td>IBL 637&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating System</td>
			<td>&#160;P-6708</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope&#160;</td>
			<td>Philips</td>
			<td>XL30CPSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope</td>
			<td>DimensionV Nanoscope, VEECO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p4-GLO CYP3A4</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>UV bulb</td>
			<td>ESCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoScope analysis software</td>
			<td>VEECO</td>
			<td></td>
			<td>version 1.20</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>TH45200</td>
			<td>Use Volocity 4 Software</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Corning, UK&#160;</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Scientific Laboratory Supplies</td>
			<td>MIC3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylene glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>93171</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;1,6-hexanediol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>240117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neopentyl glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>408255</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrous N,N-Dimethylformamide</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>676845</td>
			<td></td>
		</tr>
		<tr>
			<td>titanium (IV) butoxide&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>244112</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-butanediol&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>493732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#8217;-Methylenebis(phenyl isocyanate)</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>101688</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofurane</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Bal-Tec SCD 050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Inmunostaining</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (-MgCl2, -CaCl2)</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>&#160;Store at room temperature</td>
		</tr>
		<tr>
			<td>PBST, PBS made up with 0.1% TWEEN 20&#160;</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>EC607&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>CHE5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich, UK</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>MOWIOL 488 DAPI</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>Made up in Tris HCL and glycerol as per manufacturers instructions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell culture and Functional assay</td>
		</tr>
		<tr>
			<td>CYP3A activity pGLO kit</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatozyme</td>
			<td>Gibco</td>
			<td>17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>TryLE express</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/12604013">12604013</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Polymerlösungsmittel beeinflusst die Topographie des mit Polymer beschichteten Oberfläche Polyurethan-134 wurde in Chloroform gelöst, entweder allein oder in Kombination mit Toluol oder Tetrahydrofuran oder Dichlormethan, und die Objektträger aus Glas mit den verschiedenen Formulierungen schleudert. Rasterelektronenmikroskopie (SEM) und Rasterkraftmikroskopie (AFM) wurden zur Charakterisierung der physikalischen Eigenschaften der Polymerschichten (Abbildung 1).</...

Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Dieser Artikel wird auf die Entwicklung von Polymer-beschichteten Oberflächen für die langfristige Ausrichtung, stabile Kultur der Stammzell abgeleitet humanen Hepatozyten.

Stabilisierung Leberzell Phänotyp mit optimierten Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

Research

JoVE Journal

Chemistry

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Die kovalente Bindung von BMP-2 auf Oberflächen mit einem selbstorganisierten Monoschicht-Ansatz

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Forschung

Chemie

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Gegen biomimetischen Holz: Hergestellt Freistehende Filme aus Nanocellulose, Lignin und eines synthetischen Polykation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

In-situ-SIMS und IR-Spektroskopie von gut definierten Oberflächen durch weiche Landung der Messe-Ionen ausgewählt Vorbereitet

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Synthesemethoden für Asymmetric Ferrocen abgeleitete Bio-Konjugat-Systemen über Festphasen-Resin-basierte Methodik

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Optimierung der synthetische Proteine: Bezeichnung interpositional Abhängigkeiten Anzeige- strukturell und / oder funktionell verknüpft Rückstände

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Ein optimiertes Protokoll für die effiziente radioaktive Markierung von Gold-Nanopartikeln durch eine Verwendung von<sup&gt; 125</sup&gt; I-markiertem Azide prosthetische Gruppe

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Vor Ort Farbtest als mutmaßlichen Werkzeug für die schnelle Erkennung von synthetischen Cathinone

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

Charakterisierung von synthetischen Polymeren Matrix Assisted Laser Desorption Ionisation Zeit der Massenspektrometrie Flug (MALDI-TOF)

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

Lichtgetriebenen molekularen Motoren auf Oberflächen für einzelne molekulare Bildgebung

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Ein Facile synthetische Methode, Wismut Oxyiodide Mikrosphären hochfunktionelle der photokatalytischen Prozesse der Wasser Depuration zu erhalten

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...