Name: easy manipulation of architectures in protein-based hydrogels for cell culture applications
Uploaded: 2017-08-04T11:00:00.000+00:00
Duration: 8 min 50 s
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: 此步骤是可选的只需要包括如果水凝胶是基于细胞培养中的应用。</li>
	<li>底部的 96 孔板中删除并替换可移动的塑料包装。 注意: 对于板用在这里，底部可以很容易被删除所到的每口井; 底部施加压力薄塑料底只会掉出来。</li>
	<li>牛血清白蛋白原液的混合 100 微升 （A） 和 100 μ L THPC 的股票解 (B) (可选: 4 微升股票解 C 官能化、 细胞粘附凝胶的) 在 96 孔板获得 200 微升的水凝胶。吹打向上和向下至少 5 次，保证均匀的凝胶聚合后混合组件。</li>
	<li>96 孔板置于室温 (RT) 约 10 分钟，直到所有水凝胶进行了正确的聚合。</li>
	<li>仔细地，慢慢地从板的底部去除塑料包装。</li>
	<li>按出使用一张小邮票 96 孔板水凝胶并将它们转移到无菌的 PBS，pH 7.4 1.5 毫升管。 注: 水凝胶具有圆柱状，直径约 4 毫米和 8 毫米的高度。</li>
	<li>存储在磷酸盐缓冲液 (PBS) 在 4 ° C 到几个月的水凝胶。</li>
</ol>
2.冷冻水凝胶
<ol>
	<li>1.5 毫升管装满 500 µ L 的无菌去离子 H2O 和取消上限。将水凝胶转移到 1.5 毫升反应管使用一把铲子。</li>
	<li>包好 1.5 毫升管与石蜡膜在至少三种层的每个小瓶紧密相关。使用一根针穿透小孔的膜，使管中的气体释放。</li>
	<li>请继续执行以下步骤之一:
	<ol>
		<li>将小瓶转移到液态氮解决方案 5 分钟，以保证水和水凝胶完全冻结。从液氮中取出后立即, 将小瓶转移到冷冻干燥机，防止材料解冻。 注意: 此过程导致毛孔大约 10-15 微米的半径。</li>
		<li>把玻璃瓶放在-20 ° C 隔夜慢慢冻结的水凝胶。立即删除后从-20 ° C，将小瓶转移到冷冻干燥机，防止材料解冻。 注意: 此过程导致毛孔大约半径 50-60 微米。</li>
	</ol>
	</li>
	<li>后 24 小时 （和完全蒸发的水和周围的水凝胶），解冻材料由从冷冻干燥机中移除它。 注: 孔隙大小可以用共焦激光扫描显微镜 （步骤 5，"水凝胶可视化"） 分析。</li>
</ol>
3.颗粒浸出
<ol>
	<li>准备水凝胶在 1.1-1.5 的步骤中所述。</li>
	<li>直接混合组件后, 添加 NaCl 直到饱和发生 （36 毫克/毫升）。添加盐，直到盐的晶体可以看作在溶液中是白色沉淀。</li>
	<li>转移到振动筛的 96 孔板，摇动直到聚合发生 （约 10 分钟）。水凝胶从删除板，1.7-1.8 步骤中所述。</li>
	<li>孵育水凝胶在室温 (20 rpm) 钻井液振动筛的无菌水中洗脱所有盐从水凝胶模板至少 24 小时。直到几个月在 PBS 为存储在 4 ° C 的水凝胶。</li>
</ol>
4.通道形成
<ol>
	<li>在步骤 1 中所述制备水凝胶。使用一把钳子的解决方案中移除水凝胶、 删除多余的水与高度吸水纸，和放在一块干冰。</li>
	<li>冻结的水凝胶为 30 s 和仔细地从块中删除它。不损坏水凝胶;仔细地用一把铲子刮掉块。转移到 1.5 毫升管中的水凝胶和干它一夜之间在 37 ° c。</li>
</ol>
5.水凝胶可视化
<ol>
	<li>备罗丹明 B 原液稀释 1 毫克的罗丹明 B 在 10 毫升的 PBS。直到达到 0.001 毫克/毫升浓度稀释在 PBS 中的罗丹明原液法制备连续稀释 (稀释因子: 100)</li>
	<li>从存储解决方案 （例如，从步骤 2.4.） 中移除水凝胶和转移到 1.5 毫升管与 1 毫升的 0.01 毫克/毫升罗丹明 B 解决方案。染色水凝胶一夜之间在罗丹明 B 溶液在室温下。</li>
	<li>第二天，转移至 10 毫升的 PBS (pH 7.4) 和至少 3 小时洗要可视化的水凝胶。</li>
	<li>好转移到 µ 幻灯片 8 水凝胶和用 PBS 盖住它。</li>
	<li>小切出的水凝胶，并在使用刀片 （约 0.5 毫米的高度）。</li>
	<li>使用激光共聚焦显微镜，可视化的水凝胶在波长为 514 nm (目的: 教统会计划-Neofluar 40 x / 1.30 油 DIC M27，平面扫描模式: 514 nm 和缩放: 1 X)。</li>
</ol>
6.细胞文化可行性
<ol>
	<li>无菌过滤所有股票解决方案 （A、 B 和 C） 0.45 微米的过滤层。</li>
	<li>制备水凝胶在 1.1-1.4，包括细胞粘附肽在水凝胶聚合之前的步骤中所述。</li>
	<li>上述各组分混合后立即移液器混合物 µ 幻灯片 8 成很顺利，直到底部完全覆盖。 注: 此步骤必须执行快速、 直接混合组件后, 作为聚合在幻灯片中的几分钟内发生。</li>
	<li>粘附细胞培养及通道获得单个细胞悬浮液中的标准细胞培养技术。转移细胞提前预热、 无菌 DMEM 细胞培养液中辅以胎牛血清 (FBS，10%(w/v))、 青霉素-链霉素 （1%(w/v)) 和非必需氨基酸溶液 (MEM，1%(w/v))。</li>
	<li>与 Neubauer 计数室和仔细吸管 200 微升细胞 (2 x 105细胞/厘米2) 水凝胶表面所需数目的单元格进行计数。</li>
	<li>涵盖 µ 幻灯片 8 井盖，把它转移到孵化器 （37 ° C，5%CO2）。孵育至少 4 h 在 37 ° c。</li>
	<li>后的细胞附着至少 4 小时，洗两次带 200 µ L 无菌细胞培养 PBS 的单元格。</li>
	<li>与 200 微升的 3.7%(v/v) 在室温下 10 分钟的甲醛固定的细胞和两次用 PBS 洗 （~ 200 μ）。 注: 穿适当的个人防护设备时处理甲醛。</li>
	<li>通透细胞带 200 微升 0.1%Triton X 5 分钟洗两次用 PBS 的单元格。</li>
	<li>与鬼笔罗丹细胞染色由 195 µ L 的 PBS 混合 5 µ L 的甲醇股票并将其添加到单元格在室温染色细胞 20 分钟在黑暗中。两次用 PBS 洗涤。</li>
	<li>探讨细胞粘附特性利用共聚焦显微镜在 514 nm (目的: 教统会计划-Neofluar 40 x / 1.30 油 DIC M27，平面扫描模式: 514 nm 和缩放: 1 X)。使用适当的软件图像进行分析 （见表的材料）。</li>
</ol>
7.水凝胶性质
<ol>
	<li>膨胀率。
	<ol>
		<li>至少一天完全干凝胶在 37 ° C。</li>
		<li>衡量每个水凝胶，并注意确切的重量。</li>
		<li>2 毫升反应管装满 1.5 毫升的 PBS。</li>
		<li>转移到此 2 毫升反应管的水凝胶，并让它完全沉浸在 PBS。</li>
		<li>在 PBS 在室温下达到平衡与 PBS 中至少两天离开水凝胶。</li>
		<li>三天后，从解决方案中移除水凝胶和干燥用纸巾要从水凝胶表面除去多余的水分。</li>
		<li>权衡的水凝胶。</li>
		<li>计算的溶胀比使用下面的公式: 
		<img alt="Equation" src="/files/ftp_upload/55813/55813eq1.jpg"> 干凝胶 （第 7.1.2 步。） 重量和 Ws Wd在哪里是湿凝胶 （步骤 7.1.7） 的重量。乘以 100 要膨胀的比率 %。</li>
	</ol>
	</li>
	<li>ph 值和温度稳定性。
	<ol>
		<li>转移到 15 毫升反应管中 5 毫升的 PBS。</li>
		<li>用氢氧化钠和盐酸.带至适当的温度 （例如， RT，37 ° C 或 80 ° C） 解决方案调整到适当的 ph 值 (例如， pH 2，7 或 10) 解决方案。</li>
		<li>转让是要追究到适当的解决方案及其稳定性的水凝胶。如有必要，请调整 ph 值。 注: 所有水凝胶应该完全肿起来了在这一点 （见的溶胀比），防止由于肿胀的材料重量的正确解释。</li>
		<li>在特定的时间间隔后, 从解决方案 （例如，每个小时至 2 天） 中移除水凝胶，用高吸水的纸巾，除去多余的水分，并权衡它干。</li>
	</ol>
	</li>
	<li>酶促降解。
	<ol>
		<li>准备股票酶溶液 300 U 胰蛋白酶、 胃蛋白酶，按照制造商的说明。</li>
		<li>转移到适当的解决方案 （例如，从步骤 1.8） 水凝胶。 注: 所有水凝胶应该完全肿起来了在这一点上，防止体重的不正确解释。</li>
		<li>经过一定的时间间隔，从解决方案 （例如，每个小时） 中移除水凝胶，用高吸水的纸巾，除去多余的水分，并权衡它干。</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrakis(hydroxymethyl)+phosphonium+chloride+as+a+covalent+cross-linking+agent+for+cell+encapsulation+within+protein-based+hydrogels.">Tetrakis(hydroxymethyl) phosphonium chloride as a covalent cross-linking agent for cell encapsulation within protein-based hydrogels.</a> Biomacromolecules. 13 (12), 3912-3916 (2008).</li><li>Cal&#243;, E., Khutoryanskiy, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+hydrogels:+A+review+of+patents+and+commercial+products.">Biomedical applications of hydrogels: A review of patents and commercial products.</a> Eur Polym J. 65, 252-267 (2015).</li><li>Huang, H., Herrera, A. I., Luo, Z., Prakash, O., Sun, X. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Athrombogenic+hydrogel+coatings+for+medical+devices--Examination+of+biological+properties.">Athrombogenic hydrogel coatings for medical devices--Examination of biological properties.</a> Colloid Surface B. 130, 192-198 (2015).</li><li>L&#252;, S., Li, B., Ni, B., Sun, Z., Liu, M., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+injectable+hydrogel+for+three-dimensional+cell+culture:+chondroitin+sulfate+bioconjugated+with+poly(N-isopropylacrylamide)+synthesized+by+RAFT+polymerization.">Thermoresponsive injectable hydrogel for three-dimensional cell culture: chondroitin sulfate bioconjugated with poly(N-isopropylacrylamide) synthesized by RAFT polymerization.</a> Soft Matter. 7 (22), 10763 (2011).</li><li>Ruoslahti, E., Pierschbacher, M. 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作者宣称，他们有没有经济利益的竞争。

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 the network, thus rendering the material useless or simply inapplicable for the desired application12. Proteins possess a known potential for application in hydrogels due to their defined structure, their intrinsic biocompatibility (because they are derived from natural sources), and their many available modification sites (e.g., reactive amino-, hydroxyl- or carboxyl- groups). However, many protein-based hydrogels lack sufficient mechanical stability compared to their synthetic counterparts16. Because the introduction of large 3D architecture may lead to the complete structural disintegration of a hydrogel, the feasibility of methods to tune 3D structures must be critically evaluated to obtain the desired material properties and to achieve the full potential of the protein material. This was one reason for the evaluation of the freeze-drying and particle leaching processes and of the effect those procedures had on the resulting properties of the protein hydrogels in this study.
A critical point while working with hydrogels is the visualization of the materials on the macro-scale. Quite often, electron microscopy-based methods are used to examine the 3D structure of a hydrogel28. While doing this, it is important to consider that many procedures used to prepare the sample for electron microscopy might interact with the material and significantly alter its properties and integrity (e.g., the freezing of the sample). To overcome this problem, confocal laser scanning microscopy was used here to evaluate the produced 3D architectures and the size and distribution of the pores within the hydrogels. As opposed to electron microscopy, only a simple staining step in the native hydrated environment of the swollen state of the material is necessary to analyze the micro-scale structure of the matrix.
Freeze-drying is a very well-established method in soft matter research and biochemistry. It allows for the production of pores of a certain size and regular distribution throughout the matrix due to ice crystal formation in the network. By applying very low pressure (i.e., ~0.05 mbar) and temperatures below 0 &#176;C, the ice sublimates from the material, leaving pores behind without further disintegrating the surrounding network. In the swollen state, pores of about 10 to 70 &#181;m were detected in the template (Figure 1), with a narrow size distribution for smaller pores and a wider distribution for larger pores. Those sizes are within a suitable range for most cell culture-related applications, like hematopoietic stem cell incorporation3 and the ingrowth of osteoids and fibroblasts21. However, this method also has certain limitations. For some applications, the creation of larger pores might be beneficial22. The size limit that could be achieved with freeze drying was approximately 70 &#181;m in this material, but larger pores have already been developed by different groups21.
The dried hydrogels must be handled with care; due to their fragile structure, they tend to break apart when slight pressure is applied to the dried material. The critical step in the preparation of the material is the polymerization. Both components must be mixed properly and quickly to guarantee the production of a uniform hydrogel. If the hydrogels are not mixed immediately, a partial polymerization might occur, leading to an uneven pore distribution within the matrix.
Another technique that is well-established for the modification of synthetic polymer materials is the so-called particle leaching. Solid but dispersible particles are incorporated into the material prior to the gel-sol transition and are later eliminated from the polymerized material by changing external conditions, leading to the solubilization of the particles13,29. Paraffin and gelatin can be melted by increasing the temperature29, while crystalline particles, like sugars and salts, are dissolved by changing the buffer composition or dilution13. For this protein-based material, the radii of the pores could be changed 10-fold within the 3D matrix. Untreated salt crystals created the largest pores, with radii about 50 to 70 &#181;m (Figure 2). One limitation of this method is the maximum size of the pores: above 70 &#181;m, a considerable population of the pores exhibited fractures in their backbones, thus destabilizing the matrix. To overcome this problem, salt crystals were ground with a mortar and pestle, and significantly smaller pores, without damaged structural backbones, were produced. To improve the method, the crystals can be sieved to achieve a specific size3. Another critical step is the saturation of the solution: the hydrogels must be completely liquid when adding the salt. If the hydrogel is already partly polymerized, uneven pores will appear in the mesh, or the gel will break. If too little salt is added, the solution might be too diluted, causing no pores appear. To overcome this problem, a test series must be made prior to the experiment to find the exact amount of salt that is necessary for saturation.
Channels within a material can be useful for certain applications that require scaffold material (e.g., the ingrowth of axons into a template)11, but they are often difficult to produce in soft materials due to the lack of stability and rigidity. We established a process that includes the use of a block of solid carbon dioxide to produce linear-oriented channels within the protein hydrogel, with a length of several hundred microns and a radius of approximately 20 &#181;m (Figure 3). During their formation, the time that the hydrogel remains on the block of dry ice is essential and must be evaluated individually for each material. Short freezing times do not lead to the formation of structures, while longer freezing times break the network. When removing the material, damage to the hydrogel can be avoided by pressing a spatula against the block of ice and scratching some of the ice off the block along with the hydrogel.
All procedures influence the structure of the material and may thus severely reduce the beneficial properties of the material. To address this effect, different basic gel parameters were evaluated. The swelling ratio is one of the most important features with respect to the use of a hydrogel in cell culture, as it directly correlates with the free water content, an essential factor for the growth and survival of cells within a 3D template. All treated hydrogels were dried and brought to swelling equilibrium prior to the measurements. The swelling ratios for the different methods varied from 534% to 1,153% (Table 1). The free water content is crucial to prevent the accumulation of harmful intermediates in the environment of the cell. At the same time, nutrients, growth factors, and oxygen should be able to diffuse rapidly throughout the gel to fulfill the demands of a growing and dividing cell7. With both freeze-drying and salt leaching, a high water content could be achieved for the protein-based macroporous hydrogels. Furthermore, the swelling ratio has a significant influence on the differentiation of stem cells within a matrix and can be further improved by incorporating PEG into the material8. This is easy to do in protein hydrogels with a variety of target sites.
Many hydrogels are intended for use in biotechnological fields, such as cell culture2, tissue engineering30, or transplantation medicine31. The material must deal with harsh external conditions, including temperature or pH changes and the presence of degradation agents, like proteases or catalytic active substances. Especially in materials intended for use in the human body, such as coatings for medical devices32 or injectable hydrogels33, the systems might mutually interact with the host system, thus influencing their stability and possibly limiting their use. As a variety of methods to tune 3D architecture severely influence the material stability, all procedures used in this study were investigated for their influence on the mechanical and chemical properties of the hydrogel. The reaction of the BSA protein hydrogel mainly depends upon the applied stimuli. For example, a temperature increase from RT to 37 &#176;C or a pH shift into the acidic range did not alter the protein stability, while a shift to basic conditions reduced the residual weight after a week (Table 1). More importantly, none of the procedures used here influenced the stability of the matrix in comparison to one another (Table 1).
For many applications, a cleavable backbone is desirable. Most protein hydrogels are biodegradable. However, many hydrogels require extended decomposing times, as agents must often work from the outside to the inside of the material. This further leads to uneven degradation patterns, resulting in the complete breakdown of the network after a certain, undefined time. Untreated BSA hydrogel was degraded after several days, with the complete breakdown and disintegration of the network after a certain time. Hydrogels treated with freeze-drying or particle leaching approaches degraded in a predictable manner within hours when in the presence of enzymes, as shown in Figure 4. For macroporous hydrogels, enzymes can freely diffuse throughout the matrix and cleave the backbone of the whole template, leading to an degradation and predictable degradation times. The degradation time was found to be dependent upon the sizes of the pores within the material. For example, hydrogels frozen at -20 &#176;C, with an average pore radius of 60 &#181;m, could be degraded to half of their weight within 3.2 h using 300 U of pepsin. Hydrogels frozen at -196 &#176;C, with a significantly smaller average pore radius of 10 &#181;m, had a half-life of 6.5 h at the same pepsin concentration (Table 1).
A major concern for cell culture applications is the adhesion potential of the matrix. Many materials facilitate adhesion due to their inherent adhesion properties, while other systems must be provided with proper cellular adhesion through the incorporation of cell-adhesive structures. One prominent example of guaranteed cell adhesion is a cell-adhesive peptide, with RGD one of the most prominent and well-characterized34. Protein-based materials are normally easy to modify due to the vast amount of accessible target sites within the template. To prove the general feasibility of this BSA-based protein hydrogel for cell culture applications, the material was functionalized with a cell-adhesive RGD peptide. To facilitate the process, a specific KCSSGKSRGDS peptide was designed with a primary amine in the lysine moiety, which can be targeted by the crosslinking agent during polymerization. A549 and MCF7 cells were grown on the surface of the protein hydrogel and showed distinctive adhesion after 24 h of growth, with high viability16. The growth of the cells and the strong adhesion, which could not be reversed by washing with PBS, show the viability of the cells on the matrix and the general feasibility of the produced material for cell culture application. The direct incorporation of the peptide is both an advantage and a limitation of this system: on the one hand, it simplifies the procedure and eliminates the need for an additional reaction step. On the other hand, many different cell-adhesive peptides have been developed and used over the last decade5, and some of them bear primary amines in their structure. Those amines will readily react with the hydroxy groups of the linker molecule, thus potentially disabling the adhesion properties of the peptide. The peptide must be chosen according to this system. Another option is to add the peptide of choice after the polymerization. This way, it can be linked to different moieties in the protein backbone, whichever is best for the desired application.
For further use in 3D cell culture, the system must be optimized and adapted for each specific cell line. By changing the protein or crosslinker concentration, the properties can already be tuned, especially the mechanical properties of the material. The presented methods offer a range of tools for the manipulation of hydrogels, with no need for special equipment and with low costs. The techniques maintain the general properties of the protein-based hydrogel, making them useful for the initial evaluation of 3D cell culture applications.

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。

<table><tbody><tr><td>Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (high glucose)</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>11140-050</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>10270-106</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>MEM Nonessential Amino Acid Solution</td><td>Sigma Aldrich</td><td>M7145-100ML</td><td></td></tr><tr><td>Trypsin EDTA 0.05% Phenol Red</td><td>Thermo Fisher Scientific</td><td>25300062</td><td></td></tr><tr><td>Ethanol 99.8%, verg&amp;auml;llt</td><td>&amp;Ouml;lfabrik Schmidt</td><td>2133</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Carl Roth&amp;nbsp;</td><td>9265.1</td><td></td></tr><tr><td>Albumin Fraction V</td><td>Carl Roth&amp;nbsp;</td><td>3854.2</td><td></td></tr><tr><td>THPC</td><td>Sigma Aldrich</td><td>404861-100ML</td><td>Toxic</td></tr><tr><td>0.1% Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td>Slightly toxic</td></tr><tr><td>Phalloidin-rhodamine&amp;nbsp;</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>R415</td><td></td></tr><tr><td>3.7% Formaldehyde&amp;nbsp;</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>F8775-25ML</td><td>Toxic</td></tr><tr><td>Rhodamine B</td><td>Sigma Aldrich</td><td>81-88-9</td><td></td></tr><tr><td>Filtropur S 0.2&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>2 83.1826.001&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&amp;micro; slide 8 well</td><td>Ibidi GmbH</td><td>80826</td><td></td></tr><tr><td>KCSSGKSRGDS peptide</td><td>UPEP Ulm</td><td>Custom sysnthesis</td><td></td></tr><tr><td>Ethanol 99.8%, verg&amp;auml;llt</td><td>Carl Roth&amp;nbsp;</td><td>K928.5</td><td></td></tr><tr><td>Falcon 5 ml Polysterene Round-Bottom Tube&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>62.547.254&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Tubes 50 ml&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>62.547.254&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Tubes 1.5 ml&amp;nbsp;&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>72,690,001</td><td></td></tr><tr><td>Tubes 2 ml&amp;nbsp;&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>72,691</td><td></td></tr><tr><td>CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td><td>Greiner</td><td>655073</td><td></td></tr><tr><td>FreezeDryer Epsilon 1-6D,&amp;nbsp;</td><td>Christ, Osterode am Harz, Germany</td><td></td><td></td></tr><tr><td>Confocal Laser Scanning Microscope&amp;nbsp;</td><td>Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484</td><td>Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7</td><td>GSA GmbH</td><td></td><td></td></tr></tbody></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 microscopy. This is because the sample preparation (i.e., drying) process often changes the hydrogel properties. To overcome this problem, the material samples here were analyzed using confocal microscopy, a method that allows for the characterization of the material in its native, water-swollen state. Rhodamine B can bind to the hydrogel backbone via electrostatic interaction and facilitates the visualization of the material. To investigate the feasibility of a freeze-drying approach to modify this protein-based system, hydrogels were subsequently polymerized and frozen at 196 &#176;C in liquid nitrogen and at -20 &#176;C. The results show a clear influence of the freezing temperature on the resulting pore sizes. At -196 &#176;C, small pores with a radius of about 10 &#181;m and a narrow size distribution were produced. On the other hand, freezing at higher temperatures leads to the production of materials with much larger pores, whose radii are between 50 and 70 &#181;m, as seen in Figure 1. The slow freezing process leads to the formation of larger ice crystals, which results in bigger pores within the material after the sublimation of the ice.
Another technique for pore generation, which is well-described for synthetic materials, is particle leaching. Solid particles like paraffin, gelatin, salt, or another solid-state material, are incorporated into the hydrogel prior to solidification. They are then eluted from the solid hydrogel by changing external conditions, such as temperature, Ph, or buffer composition by dilution. The BSA-based hydrogel was polymerized in the presence of high salt concentrations. After the sol-to-gel transition, salt crystals were removed from the material by adding an excess of water to dilute the crystals. This was followed by the confocal analysis of the resulting materials. The size of the salt crystals was changed by grinding NaCl with a mortar and pestle. As shown in Figure 2, the pore size can be tremendously changed using this method. Depending upon the crystals used (untreated or ground for a certain time), pore sizes from 10 to 70 &#181;m could be produced. Appropriately sized pores are essential for many applications that feature the incorporation of cells into a hydrogel. However, more sophisticated structures might be needed when the creation of more complex structures is desired (e.g., to provide structural guidance for neuronal cells along a gradient). Complex architectures can often only be produced with special equipment (e.g., spin coating, electrospinning, lithographic, or bioprinting techniques). However, the production of linear channels can be realized with a simple freeze-drying approach and the use of dry ice. The channels produced have radii of about 20-30 &#181;m and lengths of several hundreds of micrometers, as shown in Figure 3.
Another crucial feature of hydrogels is the biodegradability of the template matrix, both in vivo and in vitro1. Protein hydrogels offer the advantage of being biodegradable. However, many systems lack fast degradation due to the restricted target sites within the material. Proteolytic agents must work their way from the outside of a material to the inside, resulting in very slow degradation times. In contrast, macroporous hydrogels are normally well-diffusible. This allows enzymes to target protein structures throughout the whole template simultaneously, which greatly reduces degradation times. For the presented protein-based hydrogel, the proteolytic degradation time depends mainly on the pore sizes within the matrix. A representative degradation kinetic is shown in Figure 4. Pore generation reduces the time from several days to a few hours for trypsin and pepsin degradation, as shown in Table 1.
The main benefit of hydrogels is the high water content within the materials, which is desirable to mimic the extracellular matrix27. The swelling ratio represents the amount of water a gel can absorb and hold after drying and is a valid indicator of the free water content of a macroporous hydrogel. Furthermore, diffusion depends strongly upon the free water content and is required for the transport of nutrients towards the cells and the removal of toxic metabolites. The swelling ratio correlates directly with the pore size within the material, as shown in Table 1. By changing the pore size using the demonstrated methods, it is possible to alter the diffusion behavior in the template and thus to influence cell fate by manipulating their feed.
The stability of a hydrogel to external stimuli, such as pH and temperature changes, severely limits or expands the possible applications of a system. Particularly for application in a cell culture-related area, resistance to changes in the external parameters is essential, as cells and cell culture matrix mutually influence their properties and behavior (e.g., the acidification of the medium might lead to hydrogel degradation, and hydrogel degradation leads to cell release or death)22. To demonstrate the feasibility of the methods described here for pore generation, all hydrogel stabilities were determined for increasing temperatures (37 &#176;C and 80 &#176;C) and changing pH values (pH 2, 7, and 10). The residual weights of the hydrogels, which were treated using different methods, are summarized in Table 1. In conclusion, the presented protein-based hydrogels are stable over a wide range of conditions, while the residual weights of the hydrogels at high temperatures only reduce gradually. For the swelling ratio, it is important to consider that a partial unfolding might occur at higher temperatures, even if most BSA molecules should be held in place by the four-armed linker.
Whenever a macroporous hydrogel is intended for use in cell culture, the adhesion properties of the material play a crucial role in the individual application. Some materials bear inherent adhesion properties (e.g., heparin, short, extracellular matrix-derived peptide structures, or fibronectin)5. For those that do not have these features, the possibility to efficiently modify the material is desirable in order to introduce cell adhesion properties. One major advantage of protein hydrogels is the presence of a variety of accessible functional groups on the surface, which can be targeted by specific reactive linker molecules. Another option is the incorporation of cell adhesive peptides during polymerization, which can further facilitate the handling of the material and the production process, as only a single step is necessary to produce cell-adhesive materials. By using a four-armed, amine-reactive crosslinking agent26, the cell adhesion mediating peptide (in this case, RGD) can be directly co-polymerized during hydrogel formation. This procedure results in the proper adhesion of the cells, as shown in Figure 5. Two model cell lines, human breast cancer cells (A549, CCL-185) and adenocarcinomic human alveolar basal epithelial cells (MCF 7, HTB-22) were used to investigate the general feasibility of using this modified hydrogel in 3D cell culture. Both cell lines showed very good adhesion potentials on the modified hydrogels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55813/55813fig1.jpg" /> 
Figure 1: Freeze-drying the hydrogels. After hydrogel polymerization, the gels were transferred to -196 &#176;C (liquid nitrogen) or -20 &#176;C. The material was stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The size and distribution of the pores within the material were analyzed with imaging software and were plotted for the different temperatures. On the right side, representative pictures of the materials frozen at (A) -196 &#176;C and (B) -20 &#176;C are shown. The error bars represent the standard deviation. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55813/55813fig2.jpg" /> 
Figure 2: Hydrogel particle leaching. Salt crystals are ground with a mortar and pestle for (A) 10 min, (B) 2 min, (C) 1 min, and (D) 0 min. Both hydrogel components are mixed, and the solution is saturated with salt crystals. After the polymerization of the materials, salt is eluted from the material by diluting the template in large amounts of deionized water. The material is stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The size and distribution of the pores within the material were analyzed with imaging software and plotted for the different grinding times. Images A to D show the pores within representative hydrogels, where salt crystals of varying sizes were used. The error bars represent the standard deviation. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55813/55813fig3.jpg" /> 
Figure 3: Channel formation. The polymerized hydrogels were transferred to a block of dry ice for 30 s. This was followed by the sublimation of the ice crystals from the structure at 0.05 mbar and -85 &#176;C. The material was stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The channels were several hundred microns long and had diameters of about 20 &#181;m. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55813/55813fig4.jpg" /> 
Figure 4: Enzymatic degradation. Representative degradation pattern of a macroporous hydrogel treated using a salt-leaching approach with ground salt crystals (10 min). Degradation took place in solution with 300 U trypsin and pepsin and was compared to an untreated hydrogel in PBS by measuring the residual weight of the material every hour. The error bars represent the standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55813/55813fig5.jpg" /> 
Figure 5: Cellular behavior on hydrogels. To investigate the potential of the material in cell culture, hydrogels were functionalized with a cell-adhesive RGD peptide. The hydrogels were polymerized into a &#181;-slide 8 well and seeded with 2 x 105/cm2 A549 and MCF7 cells. The cells were allowed to grow and adhere to the material for 24 h. After fixation with 3.7% formaldehyde and permeabilization with 0.1% Triton X-100, the cells were stained with cell-specific phalloidin-rhodamine and were visualized with inverted confocal laser scanning microscopy at a wavelength of 514 nm. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Salt leaching</td>
			<td rowspan="2">Salt leaching (grinded salt, 10 min)</td>
			<td>Frozen at&#160;</td>
			<td>Frozen at&#160;</td>
			<td rowspan="2">Gradient freezing</td>
			<td rowspan="2">Non-porous hydrogels</td>
		</tr>
		<tr>
			<td>-20&#176;C</td>
			<td>-196&#176;C</td>
		</tr>
		<tr>
			<td>pH 2 [%]</td>
			<td>95.3 &#177; 3.8</td>
			<td>96.2 &#177; 3.3</td>
			<td>94.4 &#177; 3.4</td>
			<td>96.2 &#177; 5.2</td>
			<td>89.7 &#177; 4.3</td>
			<td>69.5 &#177; 4.4</td>
		</tr>
		<tr>
			<td>pH 7 [%]</td>
			<td>95.6 &#177; 2.3</td>
			<td>94.4 &#177; 4.2</td>
			<td>95.3 &#177; 5.6</td>
			<td>94.2 &#177; 3.2</td>
			<td>94.1 &#177; 3.2</td>
			<td>98.2 &#177; 1.6</td>
		</tr>
		<tr>
			<td>pH 10 [%]</td>
			<td>82.1 &#177; 4.4</td>
			<td>86.1 &#177; 3.2</td>
			<td>76.3 &#177; 5.5</td>
			<td>83.2 &#177; 4.3</td>
			<td>84.2 &#177; 4.5</td>
			<td>42.3 &#177; 4.1</td>
		</tr>
		<tr>
			<td>RT [%]</td>
			<td>97.4 &#177; 4.4</td>
			<td>95.4 &#177; 0.42</td>
			<td>91.3 &#177; 2.2</td>
			<td>94.3 &#177; 4.1</td>
			<td>97.1 &#177; 1.9</td>
			<td>99.1 &#177; 2.2</td>
		</tr>
		<tr>
			<td>37&#176;C [%]</td>
			<td>95.3 &#177; 4.2</td>
			<td>97.4 &#177; 0.4</td>
			<td>93.2 &#177; 3.3</td>
			<td>96.2 &#177; 1.9</td>
			<td>95.3 &#177; 4.3</td>
			<td>98.3 &#177; 3.4</td>
		</tr>
		<tr>
			<td>80&#176;C [%]</td>
			<td>81.2 &#177; 4.4</td>
			<td>83.6 &#177; 4.5</td>
			<td>84.2 &#177; 4.9</td>
			<td>83.5 &#177; 3.4</td>
			<td>91.4 &#177; 8.1</td>
			<td>70.2 &#177; 6.2</td>
		</tr>
		<tr>
			<td>Swelling ratio [%]</td>
			<td>1153 &#177; 110</td>
			<td>534 &#177; 45</td>
			<td>1312&#160; &#177; 91</td>
			<td>834 &#177; 78&#160;</td>
			<td>823 &#177; 163</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin [h]</td>
			<td>4.5 &#177; 0.23</td>
			<td>7.4 &#177; 0.29</td>
			<td>3.2 &#177; 0.21</td>
			<td>6.5 &#177; 0.13</td>
			<td>4.2 &#177; 0.13</td>
			<td>55.0 &#177; 2.48</td>
		</tr>
		<tr>
			<td>Pepsin [h]</td>
			<td>3.1 &#177; 0.19</td>
			<td>4.0 &#177; 0.22</td>
			<td>2.4 &#177; 0.13</td>
			<td>3.8 &#177; 0.14</td>
			<td>3.5 &#177; 0.19</td>
			<td>46.5 &#177; 3.02</td>
		</tr>
	</tbody>
</table>
Table 1: Hydrogel properties. To investigate the influence of the pore formation methods used here (top line), different types of hydrogels were investigated for their pH and temperature stabilities, swelling ratio, and enzymatic degradation pattern (left column). For pH values of 2, 7, and 10 and temperatures of RT, 37 &#176;C, and 80 &#176;C, the average residual weights of the hydrogels after 7 days are displayed as percentages. For enzymatic degradation, the half-lives (h) of the materials in 300 U trypsin and pepsin are shown. For the swelling ratios, the water uptakes based upon the dried gels are shown are percentages.

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 ...

easy-manipulation-architectures-protein-based-hydrogels-for-cell

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.7K 次观看.  Ulm University. 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。

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

去耦力学传递线索的影响制备羟基聚丙烯酰胺水凝胶的

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...