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>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Trypsin EDTA 0.05 % Phenol Red</td>
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			<td>25300062</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>&#214;lfabrik Schmidt</td>
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		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
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			<td>3854.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">THPC</td>
			<td>Sigma Aldrich</td>
			<td>404861-100ML</td>
			<td>Toxic</td>
		</tr>
		<tr height="20">
			<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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		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin-rhodamine&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr height="20">
			<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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			<td height="20" style="height:20px;">Rhodamine B</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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#181; slide 8 well</td>
			<td>Ibidi GmbH</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCSSGKSRGDS peptide</td>
			<td>UPEP Ulm</td>
			<td>Custom sysnthesis</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td>
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			<td>655073</td>
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			<td height="20" style="height:20px;">FreezeDryer Epsilon 1-6D,&#160;</td>
			<td>Christ, Osterode am Harz, Germany</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Confocal Laser Scanning Microscope&#160;</td>
			<td>Carl Zeiss AG, Oberkochen, Germany</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>GSA GmbH</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 ...

The overall goal of this methodology is to create macroporous hydrogels with varying pore sizes for cell culture applications. This method can help answer key questions in the field of 3D cell culture such as the principle feasibility of a novel hydrogel material for 3D cell culture applications. The main advantage of this methodology is that it offers a range of techniques to optimize and adjust a novel material to the requirements of different types of cells.To begin, dilute one milligram of the RGD peptide or an equivalent cell adhesive peptide in 100 microliters of sterile water to obtain the 10 milligram per milliliter solution. Next, remove the bottom of a 96 well plate and replace it with removal plastic wrap. Mix 100 microliters of BSA stock solution and 100 microliters of THPC stock solution in the 96 well plate to obtain 200 microliters of the hydrogel.Mix the components by pipetting up and down at least five times to guarantee a uniform hydrogel after polymerization. Place the 96 well plate at room temperature for about 10 minutes until all hydrogels are properly polymerized. Following polymerization, carefully and slowly remove the plastic wrap from the bottom of the plate.Press the hydrogels out of the 96 well plate using a small stab and transfer them to 1.5 milliliter tubes with sterile PBS pH 7.4. Store the hydrogels in phosphate buffered saline at four degrees Celsius for up to several months. To freeze-dry the hydrogels, first fill 1.5 milliliter tubes with 500 microliters of sterile deionized water and remove the caps.Transfer the hydrogels to the 1.5 milliliter reaction tubes using forceps. Wrap the 1.5 milliliter tubes tightly with paraffin film using at least three layers for each vial. Use a needle to pierce small holes in the film to enable gas release from the tubes.To guarantee that the water and hydrogel completely freeze, transfer the vial to liquid nitrogen solution for five minutes. Immediately after removal from the liquid nitrogen, transfer the vials to the freeze-dryer to prevent the material from thawing. As an alternative freezing step, keep the vials at minus 20 degrees Celsius overnight to slowly freeze the hydrogels.Immediately after removal from minus 20 degrees Celsius, transfer the vials to the freeze-dryer to prevent the material from thawing. After 24 hours and the complete evaporation of the water in and around the hydrogel, thaw the material by removing it from the freeze-dryer. For particle leaching, mix the components of the hydrogel as before.Following mixing, immediately add sodium chloride until saturation occurs. Add salt until salt crystals can be seen in the solution as a white precipitate. Pore sizes can be altered by grinding the salt crystals prior to use.Transfer the 96 well plate to a shaker and shake until polymerization takes place. After about 10 minutes, remove the hydrogels from the plate as before. Incubate the hydrogels for at least 24 hours at room temperature in sterile water on a rotator to elute all salt from the hydrogel template.Store the hydrogels at four degrees Celsius in PBS for up to several months. For gentle formation, prepare hydrogels as described. Then remove a hydrogel from solution using a pincer.Remove the excess water with highly absorbent paper and place it on top of a block of dry ice. Freeze the hydrogel for 30 seconds and carefully remove it from the block. Do not damage the hydrogel.Carefully use a spatula to scrape it off the block. Transfer the hydrogel to a 1.5 milliliter tube and dry it overnight at 37 degrees Celsius. Prepare a Rhodamine B stock solution as well as serial dilutions as described in the text protocol.Remove the hydrogel from the stored solution and transfer it to a 1.5 milliliter tube with one milliliter of 0.01 milligram per milliliter Rhodamine B solution. Stain the hydrogel overnight in Rhodamine B solution at room temperature. The next day, transfer the hydrogel that is to be visualized to 10 milliliters of PBS and wash for at least three hours.Cut small slices out of the hydrogel using a blade. Then transfer the hydrogel onto an eight well microscopic slide and cover it with PBS. Using a confocal laser scanning microscope, visualize the hydrogel at a wavelength of 514 nanometers.Sterile filter all stock solutions with a 0.45 micron filter. Begin preparation of the hydrogel and include the cell adhesive peptide prior to hydrogel polymerization. After mixing the components, immediately pipette the mixture into an eight well microscopic slide until the bottom is completely covered.Transfer the cells into pre-warmed sterile DMEM cell culture medium supplemented with fetal bovine serum, penicillin streptomycin, and nonessential amino acid solution. After counting the cells with a Neubauer counting chamber, carefully pipette 200 microliters of the desired number of cells onto the hydrogel surface. Cover the eight well microscopic slide with a lid and transfer it to an incubator.After at least four hours of cellular attachment, wash the cells twice with 200 microliters of sterile cell culture PBS. Next, fix the cells with 200 microliters of 3.7%formaldehyde for 10 minutes at room temperature and wash twice with PBS. Permeabilize the cells with 200 microliters of 0.1%Triton X for five minutes before washing twice more with PBS.Stain the cells with Toluidine Rhodamine by mixing five microliters of methanolic stock with 195 microliters of PBS and adding it to the cells at room temperature. Stain the cells for 20 minutes in the dark before washing twice with PBS. Investigate the cell adhesion properties using a confocal microscope at 514 nanometers.Analyze the hydrogel properties as described in the text protocol. As shown here, the polymerized and stained hydrogels have no distinctive architectures. After freeze-drying the hydrogels at different temperatures, the average size of the pores within the hydrogel can be extended up to 60 microns.Salt leaching results in uniform networks with pore sizes of about 10 microns. The size can be further adjusted by grinding salt crystals prior to use. The use of a gradient freezing approach on a block of dry ice leads to the formation of channels with a diameter of about 20 microns and a length of several hundreds of microns.Prior to functionalization with the cell adhesive moiety, cells cannot adhere to the surface of the hydrogel and show a round morphology. Only after providing the material with the cell adhesive peptide, the cells find focal adhesion points which enable a proper adhesion of the cell onto a material. This is crucial for use of novel materials in 3D cell culture applications.After watching this video, you should have a good understanding of how to manipulate a hydrogel's architecture with simple methods. This method can be applied for most novel hydrogel systems to learn about the use in cell culture and to optimize the hydrogel's architecture depending on the intended use.

Research

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