这项工作是由美国国家科学基金会研究生研究奖学金SK和国家保健授予R01EB008722研究所的支持。

A.材料的制备和灭菌<ol><li>之前封装材料的准备，准备一个光引发剂Irgacure 2959磷酸盐（I2959）0.5％的溶液缓冲液（PBS），经搅拌和孵化数天在37 ° C该解决方案，然后可以过滤消毒和存放在室温下长期使用的注射器。 </li><li>根据所需成分的合成的凝胶，聚合物和交联剂的重量计算（使用一个电子表格程序，如Microsoft Excel）需要。由于必须在一个确定的总量来实现所需的浓度在水凝胶，交联剂和聚合物溶液的总凝胶体积的部分溶解的聚合物重量也必须使用电子表格。应作出类似的计算，如果任何挂件基团（如粘合剂RGD或YGSR含有寡肽）交联前纳入。 </li><li> Eppendorf管中，称取所需的聚合物和交联剂的数量和地点。相应调整计算电子表格，得到的聚合物的实际重量。 （ 注 ：卷可以改变，以弥补实际中得到的聚合物和交联剂质量）。 </li><li>准备使用刀片削减独立包装的无菌1毫升一次性注射器顶部凝胶模具。确保平砍photopatterned凝胶使用的模具。 </li><li>将注射器模具，Eppendorf管（瓶盖打开）和任何其他必要的材料下杀菌光源（通常是生物安全柜内置），消毒30​​分钟。 </li></ol> B.细胞制备<ol><li>经杀菌，添加适当的无菌缓冲液（每个电子表格计算;而选择的缓冲区可以有所不同，PBS和三乙醇胺，如弱碱缓冲通常用于自由基和迈克尔型交联，分别）聚合物的Eppendorf和（可选）被列入挂件基团（例如，粘合剂肽）和涡解散（如果震荡以外的无菌罩包装用封口膜Eppendorf公司）。 </li><li>挂件基团的解决方案中添加适当体积的聚合物溶液和包装用封口膜密封。简要涡和孵化，在37 ° C反应（如果可能的话，顶上旋转搅拌板）在细胞的准备。这涉及到一种肽巯基的反应（通过半胱氨酸组）丙烯酸酯或甲基丙烯酸甲酯，使肽是作为在最后的水凝胶的挂件组。 </li><li> Trypsinize和计数细胞，并为每个凝胶含有适当数量的部分分开。例如，对于在10万细胞/毫升，独立成一个单独的锥形管150万细胞的密度设置了三个50μL凝胶。这是建议，准备在一个单独的设置由于凝胶的时间（注：如果凝胶合成多套，单Eppendorf公司所有集合含有足量的聚合物溶液可以制备）不超过4凝胶。 </li><li>离心机的细胞。 </li><li>虽然细胞是停转<ol class="lalpha"><li> 光引发自由基交联 ：添加0.5％I2959的解决方案，以平等的聚合物溶液的总凝胶设置体积的10％（最终引发剂浓度为0.05 wt％的）。 </li><li> 迈克尔型（除）交联 ：添加交联剂的缓冲区，达到相应的浓度解决方案。 </li><li> 对于连续交联：这些凝胶结合了两种类型的交联，并要求前面的步骤。 </li></ol></li></ol> C.水凝胶的形成<ol><li>吸上清液离心细胞部分。为液体，等待最初的愿望和重新吸后定居，不破坏细胞沉淀中删除尽可能多的媒体。 </li><li>与聚合物溶液重悬细胞沉淀，直至细胞是均匀分布的。尽量减少气泡的形成，慢慢地移液的解决方案和向下（约10个周期应该足够分发细胞）。 </li><li>交联水凝胶<ol><li> 光引发自由基交联 ：分装到注射器尖模具的适当的音量。揭露自由基交联注射器（例如，10分钟暴露于365 nm的紫外线照射，4 mW/cm2的紫外线是为丙烯酸酯官能聚合物常见）。这一步，​​应快速进行，以尽量减少紫外线照射细胞之前解决。 </li><li> 迈克尔型（此外）交联 ：使用宽口枪头，加入交联剂溶液适当体积的聚合物/电池解决方案，设置所需的个人凝胶体积移液器，吸液管的解决方案和向下同质化，并分装到注射器尖端模具。这一步，​​应快速进行，以确保细胞均匀分布。 10-30分钟（取决于各个系统）允许交联的文化引擎盖孵化。 </li><li> 连续交联 ：这涉及到迈克尔型加成反应第一，然后暴露在光线下的第二个交联。只有一小部分的理论交联应在第一次交联反应，然后无菌口罩应直接适用于凝胶表面的光线照射，用消毒镊子前。一个功能化染料（例如，methacrylated罗丹明（MeRho）在终浓度为20纳米凝胶），也可能被添加到最初的解决方案，以可视化的图案，因为它会优先纳入彻底交联的凝胶地区（图2 ）。 </li></ol></li></ol> D.细胞培养及分析<ol><li>以下交联成一个组织培养板，孵化和分析媒体井（见下面的步骤）的暴跌凝胶。 </li><li>细胞可形态使用光学显微镜（图3，通常情况下，合成的凝胶是透明的）的可视化和活力，用荧光活/死染色试剂盒（图3和4）。细胞肌动蛋白（例如，罗丹明或荧光标记的鬼笔环肽）和细胞核染色（例如，4&#39; - 6 - Diamidino - 2 -苯基吲哚（DAPI）），可以执行下列程序使用标准的固定和通透的程序（图4）。 <ol class="laplha"><li> 5分钟，用PBS冲洗结构3X。 </li><li>修复凝胶1 mL 4％的甲醛在室温下30分钟的潜伏期。 </li><li>阻塞含3％（W / V）BSA和0.5％（W / V）吐温的PBS溶液冲洗结构3X。 </li><li> 0.25％（W / V）X阻断20分钟的解决方案，海卫一的通透膜。 </li><li>阻断溶液冲洗结构3X。 </li><li> F -肌动蛋白染色0.66微克/毫升FITC或罗丹明鬼笔环肽在阻塞在室温两小时的解决方案。 </li><li>封闭液冲洗3倍。 </li><li>使用20分钟0.4μL/ mL的PBS中的DAPI染色细胞的细胞核。 </li><li>用PBS冲洗3倍。 </li><li>使用啶或共聚焦显微镜可视化细胞。 </li></ol></li><li>凝胶内的细胞的扩散通常使用市售的检测（例如，总DNA或生化内容）评估。 PicoGreen是一个常用和敏感的荧光核酸定量双链DNA染色（Singer 等人）。细胞也算归初步控制数量，使用步骤2中所述的可视化方法。 </li><li>组织学切片和染色，也可用于可视化细胞，并形成矩阵可能被用来获取载玻片上的横截面切片的三维水凝胶的脱水和切片以下协议： <ol class="lalpha"><li> 固定和脱水 <ol class="lroman"><li>在PBS冲洗标本3X。 </li><li>修复5 - 30分钟，在4％多聚甲醛。 </li><li>在PBS冲洗标本3X。 </li><li>孵育样品中的乙醇在25℃，1小时以下的乙醇（V / V）在水中的浓度（按顺序）：50％，70％，95％，95％，100％（在这一点上，样品可存放在25℃过夜）。 </li></ol></li><li> 结算和渗透 <ol class="lroman"><li>转移到100％Citrisolv，为1小时25 ° C一小时65 ° C。 </li><li>样品在培养皿等截面样品暴露的刀片切成两半。 </li><li>传输Citrisolv /微切割石蜡为1小时，以1:1混合，在65 ° C。 </li><li>传送1小时的100％微切割石蜡在65 ° C。 </li><li>一夜之间转移到100％的微切割石蜡在65 ° C。 </li><li> 1-2小时，以100％的微切割石蜡转移在65 ° C。 </li></ol></li><li> 嵌入 <ol class="lroman"><li>位置剥离模具100％石蜡标本。 </li><li>将少量的蜡模床，并插入样品的两个半断面朝下。 </li><li>申请顶级的过滤片<ol><li>添加额外的蜡淹没顶件〜10分钟，并允许变硬。 </li></ol></li><li>将插入到4 ° C冰箱，让标本变硬过夜。 </li><li>标本可能是部分ED翌日，通常使用市售切片机，每片5-10微米的厚度。剥离模具通常垂直安装，对切片机阶段，手动控制用于设置样品与刀片的切片厚度，切片和对齐。 </li></ol></li><li> RNA的提取 基因的表达，也可以定量评估（例如，通过实时聚合酶链反应（PCR））。 RNA提取三维凝胶，下面给出的例子，协议是从简单的2D情况下完全不同。 <ol class="lroman"><li>每个三维凝胶标签认证的核糖核酸酶自由Eppendorf管，每管加500μL的Trizol ®试剂。 </li><li>使用杵（组织磨床），直到获得一个明确的解决办法是手工研磨凝胶。确保不同的磨床用于每个条件。 </li><li>涡每个Eppendorf公司为20秒均质样品。 </li><li>在室温下孵育五分钟的样本，以允许完整的核蛋白复合物的分解。 </li><li> 20分钟冰酷的样本</li><li>添加200μL（每毫升Trizol法）氯仿每个样品和旋涡。 </li><li>五分钟，在室温下孵育样本。 </li><li>在12000 XG离心样品在4 ° C 15分钟。 </li><li>水相（明确的顶层）转移到新的Eppendorf管（避免界面的地区和较低的红，酚 - 氯仿相）。 RNA保持在清晰的水相。 </li><li> RNA分离和储存常用的技术，现在可以使用。 </li><li>下面的RNA提取，逆转录酶和实时PCR市售包经常被用来与了一些修改（Elisseff等。Strehin等 ）。 。 </li></ol></li></ol></li></ol>代表性的成果： 请参阅图2-4（参考上述协议）为代表photopatterned水凝胶，凝胶内的细胞封装的可视化结果。 <img src="/files/ftp_upload/1590/1590fig1_2.jpg" alt="图1" /> 图1。3D封装过程的概述。图的步骤逐步协议中的章节标题。 <img src="/files/ftp_upload/1590/1590fig2.jpg" alt="图2" /> 图2。Photopatterned水凝胶使用顺序迈克尔型除了当时的自由基聚合。 （一）具有部分交联的水凝胶的照片活性染料纳入photopatterning示意图。 （二）圈或（C）条纹透明胶片面具图案的透明质酸凝胶。 Methacrylated罗丹明（MeRho）注册成立，只有在被暴露在光线下的地区的凝胶。比例尺= 100微米。 <img src="/files/ftp_upload/1590/1590fig3.jpg" alt="图3" /> 图3。细胞在三维凝胶可视化。可视化的干细胞通过（一）光镜或（二）生活（绿色，钙黄绿素AM）和死（红色，乙锭二聚体- 1）染色24小时为1万细胞/ mL封装在一个通过光交联透明质酸凝胶发起自由基机理。比例尺= 100微米。 <img src="/files/ftp_upload/1590/1590fig4.jpg" alt="图4" /> 图4。细胞在三维凝胶可视化。 （一）活细胞（钙黄绿素AM）或（b）细胞肌动蛋白（罗丹明鬼笔环肽）和细胞核（DAPI）的5天以下封装在一个通过迈克尔型机制的交联透明质酸凝胶在1万细胞/ mL的hMSCs染色（使用挂件的胶粘剂的肽和双功能蛋白质降解的肽交联剂）。比例尺= 100微米。

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Anal Biochem. 249, 228-238 (1997).</li><li>Lutolf, M. P., Raeber, G. P., Zisch, A. H., Tirelli, N., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Responsive+Synthetic+Hydrogels.">Cell-Responsive Synthetic Hydrogels.</a> Adv Mater. 15, 888-892 (2003).</li><li>Elisseeff, J. H., Ruffner, M., Kim, T. G., Williams, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Photoencapsulation+in+Hydrogels.">Cellular Photoencapsulation in Hydrogels.</a> Culture of Cells for Tissue Engineering. , (2006).</li><li>Chung, C., Mesa, J., Miller, G. J., Randolph, M. A., Gill, T. J., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+auricular+chondrocyte+expansion+on+neocartilage+formation+in+photocrosslinked+hyaluronic+acid+networks.">Effects of auricular chondrocyte expansion on neocartilage formation in photocrosslinked hyaluronic acid networks.</a> Tissue Eng. 12, 2665-2773 (2006).</li><li>Burdick, J. A., Vunjak-Novakovic, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+microenvironments+for+controlled+stem+cell+differentiation.">Engineered microenvironments for controlled stem cell differentiation.</a> Tissue Eng. Part A. 15, 205-219 (2008).</li><li>Salinas, C. N., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+Mode+Thiol-Acrylate+Photopolymerization+for+the+Synthesis+of+PEG-Peptide+Hydrogels.">Mixed Mode Thiol-Acrylate Photopolymerization for the Synthesis of PEG-Peptide Hydrogels.</a> Macromolecules. 41, 6019-6026 (2008).</li><li>Kraehenbuehl, T. P., Ferreira, L. S., Zammaretti, P., Hubbell, J. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-responsive+hydrogel+for+encapsulation+of+vascular+cells.">Cell-responsive hydrogel for encapsulation of vascular cells.</a> Biomaterials. 30, 4318-4324 (2009).</li><li>Strehin, I. A., Elisseeff, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+ECM+Production+by+Cells+Encapsulated+in+Hydrogels.">Characterizing ECM Production by Cells Encapsulated in Hydrogels.</a> Methods Mol Biol. 522, 1-14 (2009).</li><li>Khetan, S., Katz, J. S., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+crosslinking+to+control+cellular+spreading+in+3-dimensional+hydrogels.">Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels.</a> Soft Matter. 5, 1601-1606 (2009).</li></ol>

所描述的协议代表一个简单而强大的的的方法，封装在cytocompatible地细胞内的水凝胶支架。这种技术是特别重要，因为终末分化，干细胞可以表现出明显不同的行为，在2D与3D的微环境。即使涉及植入材料（如原位自由基聚合），细胞封装和分析的可行性和分化的治疗方法，可以帮助在材料的发展过程中。描述的连续过程，可用于空间处理的水凝胶环境，因此细胞的行为（ 见Khetan等。 ）。 总体而言，最关键的因素所描述的协议是所有使用的解决方案的同质性。特别是，应采取特殊照顾，以确保细胞是均匀地分布在预聚物溶液，并通过吸管，再悬浮的解决方案是充分混合，加入交联剂的解决方案。未能确保这一点，可能会导致聚集在凝胶结构和肿胀的细胞或局部变化。 这里介绍的协议主要集中在封装细胞的过程，重要的是还注意到，封装可能需要一些调整，以分析，通常用于2D播种的书面协议。几个例子说明，可视化和评估水凝胶中的细胞。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>.2 M Triethanolamine Buffer</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T0449</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14040-117</td>
<td>1x, sterile liquid</td>
</tr>
<tr>
<td>Live/Dead staining kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Includes live (Calcein AM) and dead (ethidium homodimer-1) probes</td>
</tr>
<tr>
<td>Phalloidin-FITC</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5282</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine Phalloidin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>R415</td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D9542</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxyethyl thiocarbamoyl rhodamine B (MeRho)</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Citrisolv</td>
<td>Reagent</td>
<td>Fisher</td>
<td>22-143975</td>
<td>&nbsp;</td>
<tr>
<td>Micro-cut paraffin</td>
<td>Reagent</td>
<td>Polysciences, Inc.</td>
<td>24202</td>
<td>&nbsp;</td>
<tr>
<td>Trizol reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>0.22 &mu;m pore size nylon syringe filters</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>09-719C</td>
<td>&nbsp;</td>
<tr>
<td>1 mL disposable syringes</td>
<td>Equipment</td>
<td>BD biosciences</td>
<td>309602</td>
<td>Cut syringes at consistent height for UV exposure</td>
</tr>
<tr>
<td>Wide orifice pipette tips</td>
<td>Equipment</td>
<td>Sigma</td>
<td>P6800</td>
<td>&nbsp;</td>
<tr>
<td>Omnicure UV Spot Cure System with 365 nm filter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>254 nm germicidal UV light source</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Built into most biological safety cabinets</td>
</tr>		</tbody>
		</table>
		</div>

cellular encapsulation in 3d hydrogels for tissue engineering 

细胞内的水凝胶的3D封装代表一个日益重要和流行的技术，对培养细胞和组织工程结构的发展。这种环境中更好地模拟细胞在体内的观察 ，比较标准的组织培养，由于组织的性质和3D环境。合成聚合物水凝胶，水肿，可设计是稳定的，或通过水解或水解降解，作为新的组织沉积是由封装细胞网络。这些应用程序，如聚乙二醇（PEG）和透明质酸的聚合物的种类繁多，已探索。最常见的聚合物功能化与活性基团，如甲基丙烯酸或丙烯酸酯通过各种机制进行交联的能力。在过去的十年中，已经取得了很大的进展，工程这些微环境-例如，通过物理或高架共价纳入生化线索-以提高生存能力和直接的细胞表型，包括封装的干细胞的分化（伯迪克等）。 细胞的三维封装下面的方法进行了优化，在我们和其他实验室，以最大限度地提高细胞相容性和减少凝胶处理步骤。在以下协议（见程序的说明图1），它是假定能够发生交联官能聚合物已手;优良的高分子化学应用于组织工程领域的评论可能会发现其他地方（伯迪克等 ），这些方法都与一系列聚合物类型兼容。此外，迈克尔型除了（见Lutolf 等。）和光发起自由基（见Elisseeff 等）。机制集中在这里只占报告交联技术的一小部分。混合模式下的交联，反应性基团，其中一个部分是第一所消耗的除交联，由一个激进的机制，是常用的另一个导演（Khetan等，萨利纳斯等。）封装细胞的表型和强大的范例。

Watch this Scientific Journal Video about Cellular Encapsulation in 3D Hydrogels for Tissue Engineering at JoVE.com

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

我们目前合成水凝胶内的细胞的三维（3D）封装的协议。封装过程概述了交联（迈克尔型除了和光发起的自由基机制）两种常用的方法，以及评估封装细胞行为的一些技巧。

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

cellular-encapsulation-in-3d-hydrogels-for-tissue-engineering

Research

JoVE Journal

Biology

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

36.7K 次观看.  University of Pennsylvania. 我们目前合成水凝胶内的细胞的三维（3D）封装的协议。封装过程概述了交联（迈克尔型除了和光发起的自由基机制）两种常用的方法，以及评估封装细胞行为的一些技巧。

视频: Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Experimental Approaches to Tissue Engineering

组织工程的实验方法

科研

生物学

Title Cell Encapsulation by Droplets

通过飞沫标题细胞封装

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

PuraMatrix Encapsulation of Cancer Cells

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these models utilize reconstituted basement membrane derived from animals which contain a variable amount of growth factors and cytokines that can influence the growth of these cell culture models. Here, we describe in detail the preparation and use of PuraMatrix, a commercially available self assembling peptide gel that is devoid of animal-derived material and pathogens to encapsulate and propagate the ovarian cancer cell line, OVCAR-5. We begin by describing how to prepare the PuraMatrix prior to use. Next, we demonstrate how to properly mix the PuraMatrix and cell suspension to encapsulate the cells in the hydrogel. Upon the addition of cell culture media or injection into a physiological environment, the peptide component of PuraMatrix rapidly self assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 5-200 nm1. In addition, we demonstrate how to propagate cultures grown in encapsulated PuraMatrix. When encapsulated in PuraMatrix, OVCAR-5 cells assemble into three dimensional acinar structures that more closely resemble the morphology of micrometastatic nodules observed in the clinic than monolayer in vitro models. Using confocal microscopy we illustrate the appearance of representative OVCAR-5 cells encapsulated in PuraMatrix on day 1, 3, 5, and 7 post plating. The use of PuraMatrix to culture cancer cells should improve our understanding of the disease and allow us to assess treatment response in more clinically predictive model systems.

This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.

PuraMatrix封装癌细胞

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

机械性刺激软骨细胞琼脂糖凝胶

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA交联聚丙烯酰胺水凝胶的制备

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...