This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1，激活盖玻片的表面<ol><li>广场的圆形玻璃盖玻片（直径25毫米），在培养皿中，并涂抹上0.1 M的氢氧化钠溶液中5分钟（化学通风柜推荐）。 </li><li>除去NaOH溶液，充分浸泡盖玻片，用无菌DDH 2 O的20分钟，而在无菌培养罩轻轻摇晃在摇板。 </li><li>排水无菌DDH 2 O和重复步骤1.2。 </li><li>用无菌镊子取出盖玻片，并将其放置在一个新的培养皿激活面朝上。 </li><li>在高纯度氮气源源不断干盖玻片。 </li><li>在无菌培养罩，涂抹一薄层3（三甲氧基甲硅烷基）丙基丙烯酸酯（92％）在盖玻片的激活边搅拌1小时。 </li><li>广泛洗盖玻片，用无菌DDH 2 O 3清洗和浸泡他们在无菌DDH 2 O的一个新的培养皿。 </li><li>点击培养皿用封口膜，将它放在一个摇杆板下轻轻搅拌10分钟。 </li><li>删除DDH 2 O的盖玻片，用无菌镊子细技巧，并将其放置在一个新的培养皿激活面朝上。 </li><li>在室温下储存与铝箔干燥的地方，避免灰尘粘在盖玻片。 </li></ol> 2，制备羟基聚丙烯酰胺水凝胶的<ol><li>在1.5ml离心管中准备的65毫克N-羟乙基丙烯酰胺（HEA）的重量。它准备一个新鲜的HEA的解决方案是非常重要的。 </li><li>加入1毫升50毫米的HEPES缓冲HEA和混合使用涡旋直至完全HEA溶解。 </li><li>添加40％的400微升w / w的在HEPES丙烯酰胺溶液和2％的所需体积w / w的在HEPES双丙烯酰胺溶液（ 见表1），以达到所需的水凝胶的硬度。用50mM HEPES调整到5毫升的最终体积。 </li><li>混合使用涡旋和德加它的解决方案在为了降低该溶液中，以防止羟基聚丙烯酰胺聚合中的氧气浓度的真空室20分钟。 </li><li>在无菌罩，过滤用0.2微米孔径过滤脱气的解决方案，以消毒吧。 </li><li>在7分钟激活圆形玻璃盖玻片（直径22mm），在紫外线/臭氧清洁。 </li><li>制备100微升10％过硫酸铵（APS）溶液，即10毫克的APS于100μlDDH 2 O。它准备一个新鲜的APS解决方案是非常重要的。 </li><li>加入2.5微升四亚甲基二胺的（TEMED）和25微升的APS溶液的灭菌羟基聚丙烯酰胺溶液（步骤2.5），以引发聚合。混合溶液通过3个连续的移液操作而不引入气泡，在无菌条件下。 </li><li>在无菌罩，放置在25毫米盖玻片的羟基聚丙烯酰胺溶液的25微升滴（可从步骤1.9），并立即将22毫米GL屁股盖玻片（上制备步骤2.5）在熔滴上的顶端挤羟基聚丙烯酰胺溶液。中心无菌镊子22毫米的玻璃盖玻片和理顺任何气泡。 </li><li>允许羟基聚丙烯酰胺水凝胶的聚合在室温下进行15分钟。倒置手动剩余羟基的PAAm在Eppendorf管中的溶液，以跟随在完成聚合过程。 </li><li>完全沉浸在盖玻片，用无菌DDH 2 O和精心引进的22毫米的玻璃盖玻片和羟基聚丙烯酰胺水凝胶层之间的刀片边缘分离22毫米的玻璃盖玻片。 </li><li>用无菌PBS洗涤羟基-聚丙烯酰胺凝胶（3往来的PBS中），并让完全沉浸在无菌PBS中，以保持水合的凝胶。 </li><li>存储羟基-聚丙烯酰胺凝胶在无菌PBS中，在4℃下进行最多3天。 </li></ol> 3，聚二甲基硅氧烷（PDMS）Microstamp制造注:硅大师的制作前站需室温的PDMS microstamp制造。硅掌握此微细可以通过光刻技术，这需要专门的设备和培训来完成。合作与纳米加工设施，鼓励制造硅的主人。另外，联系公司，编造定制微硅主人需求。要注意的是该硅原型的制造中仅需要执行一次是很重要的。事实上，微硅的主人可以无限期使用，产生弹性邮票。 <ol><li>混合的PDMS和10固化剂:1的比例在塑料烧杯中，用吸管10分钟调匀。 </li><li>在真空下脱气PDMS混合物以除去在步骤3.1中形成气泡。 </li><li>放置在培养皿中的微芯片主，就可以投出10毫米厚的一层脱气PDMS混合物没有形成气泡。 </li><li>让固化的PDMS 2小时，在60℃的烤箱。 </li><li>在无尘的环境中，剥离的PDMS层和消费1厘米2 microstamps用手术刀。 </li><li>使用镊子，地方PDMS microstamps模式，在一个培养皿。 </li></ol> 4，微图案羟基聚丙烯酰胺水凝胶<ol><li>在乙醇/水的地方PDMS microstamps（50/50）溶液并超声处理15分钟。 </li><li>干燥该邮票用氮气流的流，并将其放置图案式中的UV /臭氧清洁器（λ&lt;200纳米）7分钟。 </li><li>在无菌罩，放置所需的蛋白质溶液（ 例如 ，100微克/毫升层粘连蛋白的PBS或25微克/毫升纤维连接蛋白的PBS）上的1厘米2的PDMS印章的微结构表面150微升下降。 </li><li>可选:修改的蛋白质溶液的浓度来调节细胞的配体密度。 </li><li>通过用移液管的无菌尖端朝向的每个角落移动它分布在印面的蛋白溶液邮票。 </li><li>离开的蛋白溶液在无菌罩，以吸附于PDMS压模60分钟。关掉灯，以避免蛋白质损坏。 </li><li>在无菌罩，转让羟基聚丙烯酰胺涂层的盖玻片（可从步骤2.13）到培养皿中。 </li><li>从羟基聚丙烯酰胺基质的表面，在无菌条件下，低氮流中取出多余的PBS。只要没有证据表明在凝胶表面积水的观察停止该程序。凝胶不应该在这个阶段彻底干燥。 </li><li>干仔细的PDMS结构表面具有高纯度氮气源源不断的印记。 </li><li>掌握了穿衣组织钳蛋白包被的邮票，将结构化表面与干凝胶表面接触。申请简要压力点与镊子对PDMS压模的顶部的前端，以保证邮票微特​​征和水凝胶表面之间的良好接触。 </li><li>离开日的PDMS印章ë水凝胶表面进行1小时，在室温。 </li><li>轻轻地从羟基聚丙烯酰胺水凝胶取出的PDMS印章用敷料组织钳，并按照4.1的步骤来清洁邮票。 </li><li> 3交流的PBS（pH值为7.4）在无菌条件下，每兑换10分钟广泛洗净加盖羟基聚丙烯酰胺水凝胶。 </li><li>可选:其它ECM蛋白的其他微图案可通过以下步骤4.5到4.11被添加到羟基的PAAm表面。 </li><li>钝化非印刷区与BSA的下一个温柔的搅动一摇板的无菌溶液，在5毫克/毫升的PBS在一个晚上，在4℃。 </li><li> 3交流的PBS（pH值为7.4）在无菌条件下，每兑换10分钟大量清洗。在这一阶段，冲压羟基聚丙烯酰胺水凝胶可以被存储在4℃下最多一周。 </li></ol>在微图案化羟基聚丙烯酰胺水凝胶5细胞沉积<ol><li>在孵育细胞介质盖玻片30-45分钟电镀前çELLS。 </li><li>在37℃下洗涤贴壁细胞在75cm 2的培养瓶中，用无菌PBS培养并用3ml胰蛋白酶-EDTA的或的Accutase 10分钟分离。 </li><li>传送的预温热的完全生长培养基中适当的所需量为您的细胞系为含有分离的细胞的烧瓶中，并离心细胞悬浮液3分钟，在650×g下。 </li><li>除去用微量上清，将细胞重悬于完全培养基中，在15-20,000个细胞/ ml。 </li><li>加入4毫升的细胞溶液到微图案盖玻片（来自步骤5.1）得到的，然后将细胞覆盖的盖玻片在培养箱中在37℃和5％的湿度下保持1-2小时。 </li><li>轻轻吸独立的单元，更换培养基。返回的贴壁细胞的培养器，并让他们传播完全（3-6小时，这取决于细胞类型）。 </li></ol>

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No conflicts of interest declared.

许多体外观测在现代细胞生物学已在刚性玻璃盖玻片上，常常涂上一层薄薄的细胞外基质蛋白或含RGD序列的合成肽进行的。然而，这样的基本培养基材不重述ECM的整个物理复杂性，因此不用于研究细胞的机械传导过程提供准确的模型。为了解决这个问题，我们提出了一个简单的替代功能化的二维凝胶与任何需要的量和细胞外基质蛋白的性质。此方法允许的重要机械传导线索，如矩阵刚度，细胞形态和禁闭或细胞的配体密度的独立控制。另外，羟基 - 聚丙烯酰胺凝胶克服当前官能化方法最大的局限性，这是无法固定，并控制一个以上的ECM蛋白的空间分布之一。由于水力的高亲和力XY-聚丙烯酰胺水凝胶的生物分子，可以控制各种ECM蛋白在同一表面上的空间分布，而与基质的刚性，通过使用顺序microprintings，它不需要任何附加的设备。 我们提出了一种基于羟基的丙烯酰胺凝胶中掺入到具有在成本或专长的最低要求超越其固有的非粘着性的新型的过程。在与对水凝胶的形成图案的蛋白质的现有技术相比，该羟基的PAAm方法避免使用刺激性的有毒化学品（ 如水合肼），昂贵的光反应性的交联剂（ 例如 ，磺基Sanpah），并在UV灯的功率和定位的依赖性功能化软凝胶。羟基-聚丙烯酰胺水凝胶保持稳定和活性持续数周，可以大量生产，容易缩微及其对生物分子的高亲和力使得调查的协同效应结合细胞外基质蛋白对细胞的功能。另外，羟基 - 聚丙烯酰胺水凝胶可以用来定量地调查在其周围的微环境所施加由细胞收缩力的量。事实上，我们以前曾表明1,20的荧光珠可以很容易地嵌入羟基聚丙烯酰胺水凝胶，以使用细胞牵引力显微镜（TFM）来测量位移场和估计由真核细胞产生的所得牵引场镀上二维微图案。 如许多其他缩印为基础的方法中，羟基聚丙烯酰胺水凝胶的主要限制是关于蛋白质微特征的空间分辨率。在硬凝胶（E&gt; 10千帕）时，空间分辨率约为千分尺和主要由邮票的分辨率，这取决于用来制造硅模具的光刻技术来确定。对较软的水凝胶，该空间分辨率变得部分地由第测定蛋白质转移，这成为主要的限制，达到微米的空间分辨率在E面变形。这里介绍的方法是目前只限于二维衬底。然而这种方法是高度可扩展的和进一步的研究目前正在进行中的实验室以这种方法扩展到三维结构。 我们希望，羟基聚丙烯酰胺水凝胶可以帮助破译那些密切相关的细胞微环境21,22的理化性质，包括胚胎发育，炎症反应，伤口愈合，神经突向外生长，成年组织复杂的细胞和组织处理的关键机制稳态，和疾病如纤维变性和癌症的发病机理。

局部细胞微环境的许多方面（ 例如 ，刚度，孔隙大小，蛋白质的性质，或细胞的配体密度）提供坐标设置监管线索控制的细 ​​胞过程，如运动性，细胞增殖，分化和基因表达的影响。的胞外环境中的物理化学性质的修改可以由细胞所感知，并导致不同的生理后果，包括细胞分化，迁移和分化的变形。然而，目前还不清楚，细胞如何转化流脑修改成细胞的生化信号。因此，具有重大意义工程师在体外微环境，可再生的细胞与微环境之间的相互作用来研究机械传导途径的控制，这是。为了解决这个问题，我们最近推出了一种新的方法1，称为羟基聚丙烯酰胺水凝胶，方便地生成两毛钱nsional软矩阵，允许独立控制重要的机械传导线索:刚度矩阵，单元的几何形状和约束，蛋白质和细胞配体浓度的本质。 ECM指示经由梯度的细胞过程中的形态发生素（趋化性），胶蛋白（趋触），和硬度（durotaxis）。在过去的几十年中，先进的体外平台已经开发了以分离这些细胞外的线索，以便弄清细胞如何能够翻译生物化学和生物物理特性为生理过程2-5。电子束6，光刻7，光化学固定8，或等离子体辅助的技术9已经开发了直接在微图案化基板的活细胞的生长。虽然这些技术已经取得了重要成果，其中大多数是不允许不同的线索对细胞行为的个人影响力之间的歧视他们需要的技术设备，很少有实验室能负担得起。在这些技术中，微接触印刷（μCP），已经成为一个强大的和可访问的方法创建细胞粘着微型岛屿10。最近，大量努力11-14已开发μCP上的水凝胶具有可调刚性以再现范围广的活组织中观察到刚性的。在这些作品，聚丙烯酰胺酰胺（PAAm）已成为流行15和已经是最常用的聚合物为基础的基质细胞的生物力学分析中的一个。 聚丙烯酰胺的表面通常被官能化的异双功能连接基N-磺基琥珀酰亚胺-6 - [4&#39;-叠氮-2&#39;-nitrophenylamido]（磺基SANPAH）和ECM蛋白通过紫外线活化的磺基 - SANPAH硝基苯的链接到表面叠氮基16。另一种技术包括在耦合肼到已严重氧化蛋白高碘17。海因德和他的同事介绍了图形化水凝胶仿生表面蛋白质和多肽的技术，需要在光聚合的丙烯酰基-链亲和素单体18的存在。最近，曾等人已报道基于聚丙烯酰胺的通过光学石英掩模深UV曝光的新图案化方法19，需要温育活化PA的凝胶用1 -乙基- 3 [3 -二甲基氨基丙基]碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）的水溶液之前添加的蛋白质。尽管这些技术来创建均匀和可再现的蛋白质微图案的能力，其中大多数遭受重大的局限性:长合成过程（ 例如 ，透析，冷冻干燥等），昂贵的化学化合物（ 例如 ，透明质酸，磺基SANPAH）或深紫外线照射。此外，这些技术不允许衬底的刚度，微图案的独立调制几何形状，细胞外基质蛋白的性质，以及细胞 - 配体密度。 考虑到这些限制考虑在内，我们已经开发了一种新的和简单的基于丙烯酰胺的方法，可以让各种软凝胶蛋白质和生物分子的固定化，并允许以破译其对细胞功能的作用，机械传导线索的独立调节。相反，治疗聚丙烯酰胺水凝胶与苛刻的化学化合物，我们引入聚丙烯酰胺聚合过程中的商业丙烯酰胺单体与羟基。这种简单的操作克服了聚丙烯酰胺水凝胶的固有抗粘附性，没有任何其它的技术要求。 羟基基团的存在导致的羟基聚丙烯酰胺水凝胶的形成氢键相互作用的蛋白质和生物分子的亲和性高。与μCP组合，羟基聚丙烯酰胺水凝胶能够迅速生成的二维培养平台与独立控制矩阵的刚性，ECM蛋白的类型，细胞配体密度和密闭的粘附性，其设想是一个强大的平台，学习机械传导。 该协议的目的是提供必要的信息，很容易使羟基聚丙烯酰胺水凝胶在没有材料科学的任何专业知识。最终的目标是提供一种手段，研究人员要求在细胞和组织水平，可能会导致更好的了解所涉及的病理生理机制，机械传导途径的生理有关的问题。

<table border="0" cellpadding="0" cellspacing="0" width="573">
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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
			<td style="width:71px;">Ultra-Violet Products</td>
			<td style="width:119px;">Model PR-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
			<td style="width:119px;">Model KS 130 Basic</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Vortexer</td>
			<td style="width:71px;">Scientific Industries</td>
			<td style="width:119px;">Model Vortex Genie2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
			<td style="width:71px;">Applied Vacuum Engineering</td>
			<td style="width:119px;">DP- 8-KIT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dressing tissue forceps</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F4392-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5731-500EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminium foil, thickness 0.5&#160;mm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">266574-3.4G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopore membrane filter (0,2 &#181;m pore size)</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">GTTP Filter code</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (25 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z114820</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Protein microcentrifuge tubes</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel handles</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2896-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel blades</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2771-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cell culture flasks (75 cm2)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ultrasonic bath tray, solid (stainless steel)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z613983-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acrylamide (powder)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3553</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">146072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N-Hydroxyethylacrylamide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">697931</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3678</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimetoxysilyl)propyle acrylate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1805</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L2020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Endothelial cell growth medium</td>
			<td style="width:71px;">Cells Applications</td>
			<td style="width:119px;">211K-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues

它已经是公认的，很多细胞功能由细胞与它们的细胞外基质（ECM）的环境中的物理化学和机械线索的相互作用调节。真核细胞中不断地感受到通过表面mechanosensors的局部微环境转导细胞外基质的物理变化成生化信号，​​并将这些信号，实现基因表达的具体变化。有趣的是，ECM可以相互配合物理力学参数来调节细胞的命运。因此，关键是了解机械传导是分离的细胞功能，细胞外基质线索的相对贡献。 在这里，我们提出了一个详细的实验方案，以快速，方便地生成生物相关的水凝胶的机械传导线索体外独立调整。我们化学改性的聚丙烯酰胺水凝胶（聚丙烯酰胺），以克服其内在的非adhes香港专业教育学院通过在聚合过程中掺入羟基官能化的丙烯酰胺单体的特性。我们获得了一种新的聚丙烯酰胺水凝胶，称为羟基聚丙烯酰胺，其允许ECM蛋白的任何所希望的性质的固相化。羟基-聚丙烯酰胺的结合水凝胶与微接触印刷允许独立地控制单细胞的形态，基质硬度的性质和ECM蛋白的密度。我们提供了可以设置在每一个生物实验室体外细胞机械传导过程，研究了一个简单，快捷的方法。我们通过开展对血管内皮细胞表现出细胞外基质的硬度和细胞核之间的机械耦合实验验证了该新型二维平台。

图1A中提出了丙烯酰胺的共聚合（AAM）和双丙烯酰胺（BIS-AAM）用N-羟基乙基丙烯酰胺（HEA）含有无规自由基聚合具有嵌入的羟基形成的聚丙烯酰胺的亲水性网络中的伯羟基的单体（羟基的PAAm） 。在这个协议中，体重65毫克HEA必须稀释在1毫升HEPES的音量。明知HEA的密度约等于1，我们假设我们得到1,065微升（HEA + HEPES）的工作容积。如上述表1所示 ，总加入1065微升至400微升的AAM的和50μlbisAAm的导致了1515微升的体积。因此，3485微升的HEPES的体积，需要调整溶液至5毫升的最终体积。正如在步骤2.6中，22毫米的玻璃盖玻片的表面必须在7分钟的UV /臭氧，以便激活以将其取出，而不会造成任何损害的水凝胶的表面上。事实上，在玻璃表面变成UV /臭氧处理后的亲水性更强，并且可以因此容易地浸入整个系统中的水除去。另外，在UV /臭氧处理可以防止22毫米盖玻片，这是在与水凝胶表面直接接触的化学污染。这种技术允许以获得平坦的圆形聚丙烯酰胺表面（22毫米直径）结合到玻璃盖玻片（直径25毫米）。 如示于图1B中 ，蛋白质微图案可以的羟基聚丙烯酰胺水凝胶通过微接触印刷的表面上被创建。在这个实验过程中，紫外线/臭氧暴露允许形成硅烷醇基团在表面，以减少暂时的PDMS印章的内在疏水性。的确，在185纳米线产生臭氧从分子氧，而254纳米线转换臭氧原子氧。该活性物质攻击硅氧烷巴PDMS的ckbone以形成富氧的SiOx硅石样层和Si-OH基团的表面。被氧化的PDMS表面被称为恢复其疏水性的暴露于空气后的几个小时，由于从本体相的表面上的低分子量的非交联聚合物链的迁移。此策略有助于PDMS压模的表面上的蛋白质溶液的扩散。 一个该方法的主要优点是能够独立调节矩阵刚度（ 图2A），微图案的几何形状（ 图2B），细胞-配体密度（ 图2C），和蛋白质的性质（ 图3A和3B）。如图2A所示 ，羟基-聚丙烯酰胺凝胶上显示从几个千帕的交联剂浓度的弹性模量，以几十千帕的线性相关性，允许精细再生细胞微环境的刚性。 图2B </str翁&gt;呈现的层粘连蛋白（LM）落 ​​射荧光图像的矩形微图案沉积在三个不同的刚度（2.5，15和40千帕）羟基-聚丙烯酰胺水凝胶，显示了清楚的微图案的几何形状和基质硬度的独立调谐。 图2C示出该单元-配体密度可通过改变用于孵化PDMS压模的蛋白质溶液的浓度进行调制， 图3给出了通过使用连续微接触印刷跨层粘连蛋白和纤连蛋白线冲压胶原线的荧光图像。 有趣的是，为读者要注意的是各种尺寸和微图案的形状被盖上有效，无论是水凝胶的硬度。我们已经成功地再现与锋利的边缘（ 例如 ，三角形和星）和直的（ 例如 ，线），或弯曲的（ 例如 ，圆形）的形状的蛋白质的微特征，这表明不存在魔女ř不限于图形的复杂性。对于单细胞的实验中，微图案表面积为400和2500微米2，对应于单个细胞的存活率的极限下限值之间。如许多其他缩印为基础的方法，该方法的主要限制是关于它的空间分辨率。在硬凝胶（E&gt; 10千帕）时，空间分辨率约为千分尺和主要由邮票的分辨率决定的，而在"软"水凝胶，分辨率的限制变得部分地通过在可能出现的表面变形的测定蛋白转移。 的羟基聚丙烯酰胺水凝胶的毒性是通过量化初级内皮细胞接种于1，2，和3天的培养上均匀涂覆的生存能力和缩微软羟基聚丙烯酰胺水凝胶（ 图4A）的影响。约80％的内皮细胞接种在维护微图案的软羟基聚丙烯酰胺水凝胶其生存能力三天，展示出羟基聚丙烯酰胺生物相容性水凝胶的细胞培养物。有趣的是，在我们的实验室与原代皮质神经元细胞获得类似的生存能力。此外，在原代内皮细胞增殖更上的"硬"（500千帕）相比，"软"（25千帕）衬底（ 图4B），这表明，该方法提供了一种合适的微环境在很宽范围的刚性的研究细胞增殖。 去耦基板刚度对机械传导效果及扩展区，原代内皮细胞（HUVEC）接种于矩形FN涂覆的微图案沉积在三个不同的刚度（2.5,8.5羟基-聚丙烯酰胺凝胶（1200微米2恒定区）的和25千帕）。然后，血管内皮细胞进行染色的肌动蛋白丝，纽蛋白和DNA通过以下标准的免疫细胞化学的过程，作为用于乳房连细胞镀在玻璃基板上。在最软的基体，血管内皮细胞表现出低的肌动蛋白纤维的密度和圆形核，而在硬的基材，肌动蛋白纤维是直的且较厚，细胞核变形（ 图5）。在恒定的扩展面积的变化基板刚度调节张力和分布的肌动蛋白应力纤维的，这会导致核的重塑。这一观察表明刚度矩阵，细胞骨架和细胞核之间的机械耦合。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>最后AAM / bisAAm比</td><td> AAM 40％（微升） </td><td> HEA重量在1毫升HEPES（毫克）溶解</td><td>双AAM（2％） （微升） </td><td> HEPES（微升） </td><td>的杨氏模量 （10 3帕） </td></tr><tr><td> 0.2 / 50 0.5 / 50 1/50 2/50 3/50 </td><td> 400 400 400 400 400</td><td> 65 65 65 65 65 </td><td> 50 125 250 500 750 </td><td> 3,485 3,410 3,285 3,035 2,785 </td><td> 〜1.4 〜3.6 〜8.7 〜17.2 〜25 </td></tr></tbody></table> 水凝胶具有不同的刚性表1制备羟基聚丙烯酰胺的。工作溶液以制备羟基聚丙烯酰胺水凝胶具有最终硬度范围从1.4至25千帕。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig1highres.jpg" width="500" /> 图1（A）中的丙烯酰胺（AAM，在黑色），N，N&#39;-亚甲基（双- AAM，蓝色）和N-羟基乙基丙烯酰胺（HEA，红色）混合在一起，以形成羟基<html>聚丙烯酰胺水凝胶（B）的不同阶段对羟基聚丙烯酰胺水凝胶的图案化过程的示意图。蛋白质的溶液中1小时上microstamp（步骤＃1）的结构面首先培养。在水凝胶表面被轻轻地用氮气流（步骤＃2）干燥。该microstamp然后被放置在与羟基聚丙烯酰胺表面正式接触期间1小时（步骤＃3）。连续洗涤后，将缩微表面被钝化沉积的O 2 / N的无菌BSA溶液在4℃下（步骤＃5），以阻止非打印区域。最后，缩微羟基聚丙烯酰胺表面洗涤数次用无菌PBS，并准备好细胞接种（步骤6）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <html> 1010 / 51010fig2highres.jpg"WIDTH ="500"/&gt; 图2（A）的羟基的PAAm水凝胶的硬度的演变是线性相关与双- AAM交联剂（红线是与R 2 = 0995的线性回归）的量（B）的荧光图像矩形的层粘连蛋白微特征标记在不同的刚度（E = 2.5，15和40千帕）的羟基聚丙烯酰胺水凝胶。比例尺对应于（A）65微米，（B）80微米，和（C）50微米（C）为15微米宽度的形式在25千帕羟基聚丙烯酰胺水凝胶个LM梯度平行线，由环境演化荧光强度分布。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> N"SRC ="/文件/ ftp_upload / 51010 / 51010fig3highres.jpg"WIDTH ="500"/&gt; 纤连蛋白的图3（A）的免疫荧光图像（红色）和层粘连蛋白（绿色）相交成90°的条纹（B）纤连蛋白（红色），层粘连蛋白（绿色）和胶原（蓝色）的条纹Multilabeling缩微随后在25千帕聚丙烯酰胺水凝胶基质。比例尺对应30微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig4highres.jpg" width="500" /> 图4（A）的细胞生存力的定量显示了优秀的细胞存活率在24，48，和被镀上后72小时均匀涂覆和微图案化羟基聚丙烯酰胺表面。数表示在每个条的底部对应于细胞计数的生存试验的总数。的内皮细胞增殖（B）的定量在25和500千帕均匀地涂覆基材后1（白色柱条）和7（散列棒）培养天。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig5highres.jpg" width="500" /> 免疫染色的原代内皮细胞的图5的荧光图像上的矩形FN涂覆的微图案，其中沉积在2.5千帕，8.5千帕的羟基聚丙烯酰胺水凝胶中培养，25千帕（从左至右）的肌动蛋白应力纤维（在绿色）染色的Alexa Fluor 488鬼笔环肽，粘着斑（红色）染色，用鼠标polyclo纳尔初级抗体和标记有红色荧光IgG第二抗体和DNA染色为蓝色用DAPI。在比例尺对应17微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig5highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a>

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

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

我们提出了一个新的聚丙烯酰胺水凝胶，称为羟基聚丙烯酰胺，它允许直接结合ECM蛋白以最小的成本或专业知识。羟基聚丙烯酰胺的结合水凝胶与微接触印刷有利于学习的蜂窝mechanostransduction独立控制的自然细胞微环境的许多线索。

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 ...

preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.2K 次观看.  Université de Mons. 我们提出了一个新的聚丙烯酰胺水凝胶，称为羟基聚丙烯酰胺，它允许直接结合ECM蛋白以最小的成本或专业知识。羟基聚丙烯酰胺的结合水凝胶与微接触印刷有利于学习的蜂窝mechanostransduction独立控制的自然细胞微环境的许多线索。 

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

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 ...

科研

生物工程

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

轴突伸展生长:神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

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) ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).