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 og FK blev støttet af et EPSRC Følg på fonden. BL-V og DS blev understøttet af hver MRC ph.d.-stipendier. KC blev støttet af midler fra britiske regenerativ medicin Platform.

1. Syntese af PHNGAD (poly [1,6-hexanodiol / neopentylglycol / di (ethylenglycol) -alt-adipinsyre] diol) <img alt="Skema 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Skema 1: Syntese af PHNAGD Skematisk repræsentation af syntesen af PHNAGD.. PHNAGD blev fremstillet ved omsætning af 1,6-Hexanodiol, diethylenglycol, neoppentyl glykol og adipinsyre. PHNAGD, Poly [1,6-hexanodiol / neopentylglycol / di (ethylenglycol) -alt-adipinsyre] ...

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Mange af de nuværende metoder, der anvendes til at frembringe hepatocytter fra stamceller afhængige udefinerede matrixer af animalsk oprindelse. Disse substrater kan være dyrt og meget varierende, der påvirker celle funktion og stabilitet, hvilket er en væsentlig hindring for ansøgningen. Derfor udførte vi en skærm til syntetiske materialer, der støtter kulturen i stamceller afledt hepatocytter. Vi har identificeret en simpel polyurethan (PU134), der dannes ved at polymerisere PHNGAD, MDI og et strækkemiddel, ...

Biologiske materialer er ofte blevet brugt i vedligeholdelse og differentiering af pluripotente stamceller 1. Samtidig med at disse biologiske substrater ofte indeholder et utal af udefinerede komponenter. Matrigel er en almindeligt anvendt substrat for stamcelle kultur og differentiering. Desværre, dets varierende sammensætning påvirker celle funktion og fænotype. Selv om en bred vifte af alternative, mere definerede biologiske matricer er blevet brugt 2-7, deres animalsk oprindelse eller dårlig skalerbarhed gør dem uegnede kandidater til industriel fremstilling. Derfor identifikation af syntetiske alternativer, med defineret sammensætning og pålidelige resultater, er centrale mål i stamcelleforskning. I et forsøg på at overvinde de begrænsninger af udefinerede celledyrkningssubstrater har tværgående samarbejder mellem kemi og biologi identificeret syntetiske materialer med kapacitet til at støtte cellefænotype. SynthETIC substrater er skalerbar, omkostningseffektiv, og kan fremstilles i komplekse 3D strukturer, efterligne in vivo miljø. På grund af disse egenskaber syntetiske substrater er ofte blevet brugt til at understøtte og drive differentiering af mange celletyper 8-10. Avancerede og analyser med høj kapacitet har lettet hurtig screening af syntetiske materialer, fra store biblioteker, og leveres nye materialer med fleksible egenskaber med brede applikationer inden for biomedicinsk forskning og udvikling 11-13. Ved hjælp af high throughput, polymer microarray screening teknologi, vi hurtigt kan identificere en simpel polyurethan (PU134), velegnet til vedligeholdelse af humane stamceller afledt hepatocytter. Denne polymer viste sig at være overlegen i forhold til animalsk afledte substrater med hensyn til hepatocyt differentiering og funktion 14-16. Vi har efterfølgende optimeret belægning, topografi og sterilisering proces at få adgang til effekteron polymer ydeevne stabilisere hepatocyt funktion og levetid. Dette har betydelige konsekvenser med hensyn til forståelsen af ​​grundlaget for hepatocyt biologi til cellebaserede modellering og regenerativ medicin applikationer. Teknologien er beskrevet her, er et eksempel på, hvordan overfladen af ​​en syntetisk polymer, kan optimeres for at bevare cellens fænotype. Vi mener, at kombinationen af denne teknologi med en effektiv serum fri hepatocyt differentiering protokol har potentialet til at levere en skalerbar produktion af hepatocytter til brug som in vitro-modellering og 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 opløsningsmiddel påvirker topografien af polymeren overflade Polyurethan 134 blev solubiliseret i chloroform, enten alene eller i kombination med toluen eller tetrahydrofuran eller dichlormethan, og de objektglas spin-belagt med de forskellige formuleringer. Scanning elektronmikroskopi (SEM) og atomic force mikroskopi (AFM) blev anvendt til at karakterisere de fysiske egenskaber af de polymere overtræk (figur 1). Belægningen opnås ved anvendelse...

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Denne artikel vil fokusere på udvikling af polymer overflader for langsigtet, stabil kultur stamceller afledt humane hepatocytter.

Stabiliserende Hepatocellulære Fænotype Brug optimerede syntetiske overflader

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 binding af BMP-2 på overflader med en selv-samlet monolag 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

Mod Biomimicking Wood: Færdige Fritstående Film af Nanocellulose, Lignin, og en syntetisk 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 og IR-spektroskopi veldefinerede Overflader Udarbejdet af Soft Landing af Mass udvalgte ioner

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 Metode til Asymmetrisk Ferrocen Afledte Bio-konjugat systemer via Solid Phase Resin-metode

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 af syntetiske Proteiner: Identifikation af Interpositional Afhængigheder Angivelse Strukturelt og / eller funktionelt knyttet Rester

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 optimeret protokollen om Effektiv Radioaktiv mærkning af Gold Nanopartikler ved hjælp af en<sup&gt; 125</sup&gt; I-mærket Azide prostetiske 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

Farve Spot Test som en Præsumptive værktøj til hurtig påvisning af syntetiske Cathinones

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 af syntetiske polymerer via Matrix Assisted Laser Desorption ionisering tid for flyvning (MALDI-TOF) massespektrometri

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

Lys-drevet molekylære motorer på overflader for enkelt molekylær billeddannelse

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 letkøbt syntetiske metode til at opnå Bismuth Oxyiodide mikrokugler yderst funktionelt for fotokatalytiske processer af vand hastige

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