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 och FK främjades av ett EPSRC Följ på fonden. BL-V och DS var och stöds av MRC doktorandtjänster. KC stöddes genom finansiering från Storbritannien regenerativ medicin Platform.

1. Syntes av PHNGAD (Poly [1,6-hexanodiol / neopentylglykol / di (etylenglykol) -alt-adipinsyra] diol) <img alt="Schema 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Schema 1: Syntes av PHNAGD Schematisk representation av syntesen av PHNAGD.. PHNAGD framställdes genom reaktion av 1,6-Hexanodiol, dietylenglykol, neoppentyl glykol och adipinsyra. PHNAGD, Poly [1,6-hexanodiol / neopentylglykol / di (etylenglykol) -alt-adipinsyra] diol....

<ol>
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Många av de nuvarande metoder som används för att generera leverceller från stamceller beroende av odefinierade matriser av animaliskt ursprung. Dessa substrat kan vara kostsamt och mycket varierande, vilket påverkar cellernas funktion och stabilitet, vilket innebär ett betydande hinder för ansökan. Därför genomförde vi en skärm för syntetiska material som stöder kulturen i stamcells härledda hepatocyter. Vi har identifierat, en enkel polyuretan (PU134), som bildas genom att polymerisera PHNGAD, MDI och e...

Biologiska material har använts i stor utsträckning i underhållet och differentiering av pluripotenta stamceller 1. Samtidigt som dessa biologiska substrat ofta innehåller en myriad av odefinierade komponenter. Matrigel är ett vanligt använt substrat för stamcellkultur och differentiering. Tyvärr påverkar dess varierande sammansättning cellfunktion och fenotyp. Även om en rad olika alternativ, mer definierade biologiska matriser använts 2-7, deras animaliskt ursprung eller dålig skalbarhet gör dem olämpliga kandidater för industriell tillverkning. Därför identifiering av syntetiska alternativ, med definierad sammansättning och tillförlitlig prestanda, är viktiga mål i stamcellsforskningen. I ett försök att övervinna begränsningarna i odefinierade cellodling, har tvärvetenskapliga samarbeten mellan kemi och biologi identifierade syntetiska material med förmåga att stödja cellfenotyp. SynthEtic substrat är skalbar, kostnadseffektiv, och kan tillverkas i komplexa 3D-strukturer, imitera in vivo miljön. På grund av dessa egenskaper syntetiska substrat har ofta används för att stödja och driva differentiering av många celltyper 8-10. Avancerade och hög genomströmning analyser har möjliggjort en snabb screening av syntetiska material, från stora bibliotek, och levereras nya material med flexibla fastigheter med breda tillämpningar inom biomedicinsk forskning och utveckling 11-13. Använda hög genomströmning, polymermikro array screening-teknik, vi snabbt identifierat en enkel polyuretan (PU134), lämplig för underhåll av mänskliga stamcells härledda hepatocyter. Denna polymer befanns vara överlägsna djur härledda substrat med avseende på hepatocyte differentiering och funktion 14-16. Vi har därefter optimerat beläggningsförhållanden, topografi och steriliseringsprocess för att få tillgång effekterpå polymer prestanda stabilisera hepatocyte funktion och livslängd. Detta har stor betydelse när det gäller att förstå grunderna i hepatocyte biologi för cellbaserad modellering och regenerativ medicin applikationer. Den teknik som beskrivs här representerar ett exempel på hur ytan av en syntetisk polymer kan optimeras för att bevara cellfenotyp. Vi tror att kombinationen av denna teknik med en effektiv serumfritt hepatocyte differentiering protokollet har potential att ge en skalbar produktion av leverceller som används vid in vitro-modellering och regenerativ medicin.

<table border="0" cellpadding="0" cellspacing="0">
	<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.

Polymer lösningsmedel påverkar topografin av polymeren belagd yta Polyuretan 134 löstes i kloroform, antingen ensamt eller i kombination med toluen eller tetrahydrofuran eller diklormetan och glas spinn beläggs med olika formuleringar. Svepelektronmikroskopi (SEM) och atomkraftsmikroskopi (AFM) användes för att karakterisera de fysikaliska egenskaperna hos polymerbeläggningarna (figur 1). Beläggningen erhålles med användning av toluen eller klorofo...

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Denna artikel kommer att fokusera på att utveckla polymerbelagda ytor för långsiktig, stabil kultur av stamceller härstammar humana hepatocyter.

Stabiliserande Hepatocellulär Fenotyp Använda Optimerade Syntetiska Ytor

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

Kovalent Bindning av BMP-2 på ytorna med en själv monterade monolager Approach

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 ...

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

Mot Biomimicking Wood: Fabricated Fristående Filmer av nanocellulosa, lignin och en syntetisk polykatjon

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 och IR-spektroskopi väldefinierade ytor förbehandlade av Soft Landing av Mass-utvalda Joner

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

Syntetisk Metodik för Asymmetric Ferrocen Härledda Bio-konjugerade system via Solid Phase Harts-metodik

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

Optimering av syntetiska proteiner: Namnet på Interpositional beroenden Indikerings strukturellt och / eller funktionellt knutna Stoder

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

En optimerat protokoll för effektiv Radiomärkning av guld nanopartiklar med hjälp av en<sup&gt; 125</sup&gt; I-märkt Azid prostetisk grupp

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

Färg Spot Test som presumtiva verktyg för snabb påvisande av syntetiska katinoner

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

Karakterisering av syntetiska polymerer via Matrix assisterad Laser Desorption jonisering tid för flygning (MALDI-TOF) masspektrometri

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

Ljus-driven molekylära motorer på ytor för enstaka molekylär avbildning

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

En lättköpt syntetiska metoden att få Bismuth Oxyiodide mikrosfärer högfunktionella fotokatalytisk procedurer för vatten rening

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