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とFKは基金に関するEPSRCフォローによってサポートされていました。 BL-VとDSがそれぞれのMRC博士studentshipsによってサポートされていました。 KCは、英国再生医療プラットフォームからの資金によってサポートされていました。

PHNGAD（ポリ[1,6-hexanodiol /ネオペンチルグリコール/ジ（エチレングリコール）-alt - アジピン酸]ジオール）の合成<img alt="スキーム1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> スキーム1：PHNAGDの合成 PHNAGDの合成の模式図。。 PHNAGDは、1,6 - Hexanodiol、ジエチレングリコール、neoppentylグリコールとアジピン酸との反応により調製した。 PHNAGD?...

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幹細胞から肝細胞を生成するために使用される現在の方法の多くは、動物由来の未定義のマトリックスに依存している。これらの基板は、アプリケーションに重大な障壁を表す、細胞の機能および安定性に影響を与える、費用がかかり、非常に可変であることができる。そこで、幹細胞由来肝細胞の培養を支持する合成材料のためのスクリーンを行った。私たちは、同定した、堅牢な肝細胞?...

生物学的材料は、広く、多能性幹細胞1の維持および分化に使用されている。可能にしながら、これらの生物学的基質は、多くの場合、未定義の構成要素の無数が含まれています。マトリゲルは、幹細胞の培養および分化のための一般的に使用される基板である。残念ながら、その可変組成は、細胞の機能および表現型に影響を与える。代替、複数の定義された生物学的マトリックスの多様2-7使用されてきたが、それらの動物由来または貧弱な拡張性は、彼らに工業的な製造には適さない候補にする。したがって、定義された組成と信頼できる性能を有する合成代替物の同定は、幹細胞研究の重要な目標である。 未定義の細胞培養基質の制限を克服する試みにおいて、化学と生物学との間の学際的コラボレーションは、細胞表現型をサポートする能力を有する合成材料を同定した。シンセエティックな基板は、スケーラブルであり、費用対効果、および複雑な3次元構造に製造することができ、 生体内環境を模倣する。によるこれらの特性に合成基質は、広く多くの細胞型8-10の分化を支持し、駆動するために使用されてきた。 Advancedおよびハイスループットアッセイは、大規模なライブラリーから、合成材料の迅速なスクリーニングを容易にし、生物医学の研究開発11-13の幅広いアプリケーションとの柔軟な特性を有する新規材料を提供してきました。高いスループットを利用して、高分子マイクロアレイスクリーニング技術は、急速にヒト幹細胞由来の肝細胞の維持に適した単純なポリウレタン（PU134）を同定した。このポリマーは、肝細胞の分化および機能14-16に関して動物由来の基質よりも優れていることが判明した。私たちは、その後の効果をアクセスするための塗布条件、地形や滅菌プロセスを最適化した肝細胞機能と寿命を安定化するポリマーの性能に。これは、セルベースの​​モデリングと再生医療アプリケーションのための肝細胞生物学の基礎を理解することに関して重要な意味を持っている。 ここで説明する技術は、合成ポリマーの表面が細胞表現型を維持するために最適化することができる方法の例を示している。私たちは、効率的な無血清肝細胞分化プロトコルでこの技術の組み合わせは、 インビトロモデリングや再生医療で使用するための肝細胞のスケーラブルな生産を提供する可能性を持っていることを信じています。

<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.

ポリマー溶媒は、ポリマーコーティングされた表面の形状に影響を与える ポリウレタン134は、トルエンまたはテトラヒドロフランまたはジクロロメタンとの単独または組み合わせのいずれかで、クロロホルムに溶解し、ガラススライドは、異なる製剤でスピンコートした。走査電子顕微鏡（SEM）および原子間力顕微鏡（AFM）は、ポリマーコーティング（ <str...

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

この記事では、ヒト肝細胞を由来幹細胞の長期、安定的な培養のためにポリマーコーティングされた表面の開発に注力していきます。

最適化された合成表面を使用した肝細胞表現型の安定化

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

共有結合は、自己組織化単分子膜の手法を用いて表面上のBMP-2の結合

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

Nanocellulose、リグニンから製造自立フィルム、合成ポリカチオン：Biomimickingウッドに向けて

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

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

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

固相樹脂系の方法論を経由して、非対称フェロセン派生バイオコンジュゲートシステムのための合成方法論

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

合成タンパク質の最適化：中間物挿入依存関係の同定構造的インジケートおよび/または機能的に連結された残基

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

使用した金ナノ粒子の効率的な放射性標識のために最適化されたプロトコル<sup&gt; 125</sup&gt; I標識アジド補綴グループ

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

合成 Cathinones の急速な検出のための推定ツールとして色スポット テスト

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

マトリクスによる合成高分子のキャラクタリゼーション支援レーザー脱離イオン化時間 (MALDI Tof) 質量分析装置

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

単一分子イメージングの表面上の光駆動の分子モーター

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

水洗水の光触媒プロセスのビスマス Oxyiodide 微粒子高機能を取得する簡便な合成法

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