Name: easy manipulation of architectures in protein-based hydrogels for cell culture applications
Uploaded: 2017-08-04T11:00:00
Description: Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

作者要感谢巴登-符腾堡基金会的财政支持，"仿生材料合成"框架 (BioMatS-14)。

1.水凝胶制备
<ol>
	<li>用去离子 H2O 打造 20%(w/v) BSA 股票 （股票解决方案 A） 1 毫升混合 200 毫克的牛血清白蛋白。</li>
	<li>用去离子水，创建 THPC 4.835 毫升混合 165 微升 THPC 解决方案 （134 毫克/毫升） 的股票 （股票解决方案 B） 解。</li>
	<li>体重 1 毫克的 KCSSGKSRGDS (1,111.1 g/mol) 肽 （或等效的细胞粘附肽） 和稀释在 100 μ L 的无菌 H2O 获得 10 毫克/毫升的解决方案 （股票 C）。 注：...

<ol>
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The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of th...

水凝胶是水的形成不溶性的 3D 网络能够结合大量的材料。这种材料可以提供优良的环境条件，活细胞。目前，那里越来越感兴趣，在三维水凝胶结构的生成和发展的过程来定制他们的化学和物理特性。一旦完成，该细胞的生长和操纵细胞行为的模板可以生成的1，2，，34。这些 3D 结构不仅创造一个更为自然和现实的环境，比传统的二维方法，但他们也揭示新的可能性，为干细胞的生长或肿瘤模型5。不同的材料拥有一系列的特性主要取决于孔隙大小的凝胶6。毛孔发挥关键作用，细胞培养应用程序、 组织工程和干细胞的定向的生长。例如，通过矩阵，弥漫的氧气和营养物质和适当数额必须能够达到细胞7。另一方面，必须尽快，去除有害代谢物和足够的空间用于细胞生长必须可用7。因此，材料特性和，因此孔隙大小，严重影响矩阵的潜在利益和可能的应用程序。取决于材料的性能，不同的细胞生长过程可以发生在三维细胞培养，包括形成的神经元结构;生长和分化的皮肤或骨细胞;和特殊的干细胞，如肝细胞或成纤维细胞2，3，8，9，，1011定向的生长。影响材料的可能应用的另一个关键点是对外部刺激12其稳定性。例如，水凝胶必须保持其在细胞培养基或人体的机械完整性。
近年来，研究三维单元文化水凝胶加剧，并进行了许多研究解决系统13的 3D 建筑。由化学合成的成分组成的水凝胶最常用因为他们可以很容易合成和化学改性和他们表现出较高的稳定性进行了研究 （见朱等人，2011 年为审查）5。然而，蛋白质有许多有益的特性： 如所谓的"精密聚合物"，他们都是生物相容性;他们有一个已定义的长度;它们是相对容易的修改;和他们有大量的目标站点14，15。在这方面，特异性高、 创新结构可以生成在许多领域中的应用。在此研究中，基于蛋白质的凝胶16用于演示的既定方法影响材料的 3D 架构的能力。此外，还研究了适用于孔隙水的产生和能力。
有许多的不同技术，可用来修改三维结构，包括方法简单、 复杂、 高度专业化的技术，从不同科学领域的材料。广泛的技术是利用静电纺丝技术来生成定义良好的结构17。带电的纤维由电场拉扯从解决方案，然后凝固暴露在氧气。这种方式，可以产生几个纳米到几个微米范围内的纤维。额外的技术调整大小、 结构和分布的毛孔内矩阵是软光刻技术、 光刻、 水动力聚焦、 电喷涂、 和生物印刷18，，1920。这些技术的一个重大缺点是他们对具体的、 昂贵的设备和特殊的化学物质或材料的依赖。此外，这些技术的经验往往不是直接转移到蛋白质为基础的材料，和许多的化学品和方法不是兼容的单元格。
另一方面，许多技术不要依赖特殊的设备，使他们更容易和便宜适用，并重现。结构操作的普遍做法是溶液浇铸21，，2223。粒子在聚合反应之前添加和均匀分布，饱和溶液。在聚合反应后, 变化的条件，如稀释或 ph 值的变化，导致溶剂化物的粒子，而毛孔留在材料内。在这些技术，如盐、 糖、 石蜡、 明胶和粉笔，所使用的化学品是廉价和容易获得。在冷冻干燥，肿水凝胶被冻结。真空条件下液相的后续升华是然后执行23，，2425。从网络的水升华是足够温和，保持材料的特定三维结构。在气体发泡，解决方案是用气体分流聚合反应发生，留下毛孔内凝胶21时。取决于气体流可以调整的大小和分布的毛孔。
形成蛋白质凝胶，与四羟甲基氯化磷 (THPC) 在曼尼希反应，以便形成共价键伯胺和链接器四武装分子26的羟基之间的反应是牛血清白蛋白。反应发生后通过过度清洗的材料移除可能有害的中间体。
这项研究表明治疗不同的技术来操纵和裁缝毛孔的大小基于 BSA 的材料的可能性。每个技术可以用作在任何实验室全世界，没有特别的设备是必要的。此外，不同的参数，如溶胀率、 酶促降解性、 pH 稳定性和温度敏感性、 被检测，并比较彼此，尤其是尊重对不同技术的影响上的 3D 架构生成。最后，材料是羧基与细胞粘附肽调查材料对细胞培养中的可能应用。使用了两种不同的模型细胞系： A549 和 MCF7。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:253px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">10010023</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (high glucose)</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Nonessential Amino Acid Solution</td>
			<td>Sigma Aldrich</td>
			<td>M7145-100ML</td>
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			<td height="20" style="height:20px;">Trypsin EDTA 0.05 % Phenol Red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300062</td>
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			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>&#214;lfabrik Schmidt</td>
			<td>2133</td>
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			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Carl Roth&#160;</td>
			<td>9265.1</td>
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			<td height="20" style="height:20px;">Albumin Fraction V</td>
			<td>Carl Roth&#160;</td>
			<td>3854.2</td>
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		<tr height="20">
			<td height="20" style="height:20px;">THPC</td>
			<td>Sigma Aldrich</td>
			<td>404861-100ML</td>
			<td>Toxic</td>
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			<td height="20" style="height:20px;">0.1 % Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td>Slightly toxic</td>
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			<td height="20" style="height:20px;">Phalloidin-rhodamine&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>R415</td>
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			<td height="20" style="height:20px;">3.7 % Formaldehyde&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>F8775-25ML</td>
			<td>Toxic</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Rhodamine B</td>
			<td>Sigma Aldrich</td>
			<td>81-88-9</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Filtropur S 0.2,&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>2 83.1826.001&#160;&#160;&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">&#181; slide 8 well</td>
			<td>Ibidi GmbH</td>
			<td>80826</td>
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		<tr height="20">
			<td height="20" style="height:20px;">KCSSGKSRGDS peptide</td>
			<td>UPEP Ulm</td>
			<td>Custom sysnthesis</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>Carl Roth&#160;</td>
			<td>K928.5</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Falcon 5 ml Polysterene Round-Bottom Tube&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tubes 50 ml&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tubes 1,5 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,690,001</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tubes 2 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,691</td>
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		<tr height="20">
			<td height="20" style="height:20px;">CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td>
			<td>Greiner</td>
			<td>655073</td>
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		<tr height="20">
			<td height="20" style="height:20px;">FreezeDryer Epsilon 1-6D,&#160;</td>
			<td>Christ, Osterode am Harz, Germany</td>
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			<td>Carl Zeiss AG, Oberkochen, Germany</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484</td>
			<td>Carl Zeiss AG, Oberkochen, Germany</td>
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			<td height="20" style="height:20px;">GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7</td>
			<td>GSA GmbH</td>
			<td></td>
			<td></td>
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</table>

easy manipulation of architectures in protein-based hydrogels for cell culture applications

水凝胶被公认的有前途的材料，因为它们能够提供高度水合的细胞环境细胞文化申请。领域的 3D 模板正上升由于潜在的相似之处的那些材料到天然细胞外基质。基于蛋白质的凝胶是特别有前途，因为他们很容易能官能化，可以实现定义的结构可调的理化性质。然而，大孔 3D 模板单元格文化应用程序使用天然材料的生产，常常受到其较弱的力学性能相比，这些合成材料。在这里，不同的方法进行计算以产生大孔牛血清白蛋白 (BSA)-基于水凝胶系统，与在半径 10 至 70 微米范围内可调的孔径大小。此外，建立了一种方法来生成渠道在这几个几百微米长的蛋白质基础材料。不同的方法来产生毛孔，以及孔隙大小对材料的性能如膨胀比、 ph 值、 温度稳定性和酶降解行为的影响进行分析。利用共焦激光扫描显微镜对凝胶本机、 肿州考察了孔径大小。细胞培养应用的可行性进行了评价使用蛋白系统和两个模型细胞株的细胞粘附 RGD 多肽修饰： 人类乳腺癌细胞 (A549) 和 adenocarcinomic 肺泡基底上皮细胞 (MCF7)。

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron micr...

Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

不同的方法来操作基于蛋白质的凝胶中的三维结构对材料性能在这里评价。大孔网络一种细胞粘附肽，羧基化，它们在细胞培养的可行性评估使用两种不同的模型细胞株。

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Research

JoVE Journal

Biochemistry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

定量应用同质MALDI样品的制备

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

科研

生物化学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...