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 и FK при поддержке одного EPSRC следовать по фонда. BL-V и DS друг были поддержаны MRC PhD studentships. KC поддержали финансирование из Великобритании регенеративной медицины платформы.

1. Синтез PHNGAD (поли [1,6-hexanodiol / неопентил гликоль / ди (этиленгликоль)--alt адипиновой кислоты] диол) <img alt="Схема 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Схема 1: Синтез PHNAGD Схематическое представление синтеза PHNAGD.. PHNAGD готовили по реакции 1,6-Hexanodiol, диэтиленглик?...

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Многие из существующих методов, используемых для генерации гепатоциты из стволовых клеток зависят от неопределенных матриц животного происхождения. Эти субстраты могут быть дорогостоящими и весьма неоднородно, затрагивая функции клеток и стабильности, что представляет собой значит...

Биологические материалы широко используются в поддержании и дифференциации плюрипотентных стволовых клеток 1. В то время как позволяет, эти биологические субстраты часто содержат множество неопределенных компонентов. Матригель является широко используемым субстратом для культуры и дифференциации стволовых клеток. К сожалению, его переменный состав влияет функцию клеток и фенотип. Хотя разнообразие альтернативных, более определенные биологические матриц были использованы 2-7, их животного происхождения или плохой масштабируемости делает их непригодными кандидатов для промышленного производства. Поэтому идентификация синтетические альтернативы, с определенным составом и надежной работы, являются ключевыми целями в исследовании стволовых клеток. В попытке преодолеть ограничения неопределенных культивирования клеток субстратов, междисциплинарное сотрудничество между химией и биологией определили синтетические материалы со способностью поддерживать фенотип клеток. SynthEtic субстраты являются масштабируемыми, экономически эффективным, и могут быть изготовлены в сложных 3D структур, имитируя в естественных условиях окружающей среды. Благодаря этим свойствам синтетические субстраты были широко используются для поддержки и привод дифференцировку многих типах клеток 8-10. Расширенный и высокой пропускной анализы способствовали оперативно провести проверку синтетических материалов, из крупных библиотек, и доставлены новые материалы с гибкими свойствами с широкого применения в биомедицинских исследованиях и разработках 11-13. Используя высокую пропускную способность, полимерной технологии скрининга микро-массив, мы быстро определили простой полиуретан (PU134), подходит для поддержания человека стволовых клеток, полученных гепатоцитов. Этот полимер был найден, чтобы быть выше на животных, полученных подложек в отношении дифференцировки гепатоцитов и функции 14-16. Мы впоследствии оптимизировать процесс при нанесении покрытий, топографии и стерилизации для доступа эффектына полимерной производительности в стабилизации функцию гепатоцитов и продолжительность жизни. Это имеет важные последствия в отношении понимания основ гепатоцитов биологии для основе моделирования клеток и регенеративной медицины приложений. Технология здесь описано представляет собой пример того, как поверхность из синтетического полимера могут быть оптимизированы, чтобы сохранить фенотип клеток. Мы считаем, что сочетание этой технологии с протоколом дифференцировки бесплатно гепатоцитов эффективность сыворотки имеет потенциал, чтобы обеспечить масштабируемое производство гепатоцитов для использования в моделировании в пробирке и регенеративной медицины.

<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.

Растворителя полимера влияет на топографию поверхности с полимерным покрытием Полиуретан 134 солюбилизировали в хлороформе, либо по отдельности, либо в сочетании с толуол или тетрагидрофуран или дихлорметан, и предметные стекла с покрытием по спину с различны?...

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Эта статья будет сосредоточена на разработке полимерных покрытием поверхностей для долгосрочного, стабильного культура стволовых клеток получены человеческие гепатоциты.

Стабилизация Гепатоцеллюлярной Фенотип используя оптимизированные синтетические покрытия

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

Ковалентного связывания БМП-2 на поверхностях Использование самоорганизующихся монослоя подход

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

К Biomimicking Вуд: сварная свободно стоящие фильмы Nanocellulose, лигнин, и синтетический поликатиона

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

На месте SIMS и ИК спектроскопии Ну определенных поверхность, подготовленную мягкой посадки Масса выбранных ионов

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

Синтетическая методология для асимметричных Ферроцен, полученных Bio-сопряженных систем через Solid методологии фазы смолу на основе

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

Оптимизация синтетических белков: Идентификация Interpositional зависимостей индикации Конструктивно и / или функционально связанных остатков

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

Оптимизированного протокола для эффективного радиоактивной наночастиц золота с помощью<sup&gt; 125</sup&gt; I-меченый Azide простетической группы

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

Цветовой тест месте как предполагаемого инструмент для быстрого обнаружения синтетических 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

Характеризации синтетических полимеров через матрицы помогает лазерная десорбция ионизации время полета (MALDI-TOF) масс-спектрометрии

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

Свет driven молекулярные моторы на поверхностях для одного Молекулярное воображение

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

Снисходительный синтетический метод для получения висмута Oxyiodide микросферы высокофункциональная фотокаталитический процессов очистки воды

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