这项研究主要由美国心脏协会科学家发展补助金 (12SDG12050083)、国家卫生研究院 (R21HL102773、R01HL118245) 和国家科学基金会 (CBET-1263455 和CBET-1350240 到动力局)。

1. 水凝胶合成材料的制备 <ol> <li> 在无菌条件下准备 PEGDA、锂 phenyl-2,4,6-trimethylbenzoylphosphinate (膝) 和纤维连接蛋白的库存解决方案, 并基于计算 (表 1A). 
	<ol> <li> 在磷酸盐缓冲盐 (PBS) 中称量和溶解化合物。通常, 保持 PEGDA 工作溶液浓度在50和200毫克/毫升 (5-20% 重量/体积)。通过 0.22 #181; m 注射器过滤器进行消毒, 以 PEGDA 溶液. 
		注: 和 #160; 保持 PEGDA 解决方案覆盖的铝箔包装, 以保护他们免受光。为每个实验准备一个新的 PEGDA (推荐) 解决方案. </li>
		<li> 根据制造商和 #39 的建议重组纤维连接蛋白粉末。维护纤维连接蛋白库存解决方案在1毫克/毫升, 分到60至100和 #181; 等分, 并存储在-20 和 #176; C 直到使用。在4和 #176 解冻等分; C 在使用前几个小时或隔夜。如果没有在无菌条件下提供蛋白质库存, 使用0.22 和 #181; m 注射器过滤器进行灭菌. 
		注意: 纤维连接蛋白用于细胞附件。其他 ECM 蛋白可以替代. </li>
		<li> 为库存溶液称量圈并将其溶解在 PBS 中; 典型的股票浓度是25毫克/毫升。几种如果有任何溶解的麻烦。通过 0.22 #181; m 过滤器进行灭菌。用铝箔包装纸盖上一圈, 并将其保持在4和 #176; C 长达几个月. 
		注: 圈是用于光化学的光. </li>
	</ol> </li> <li> 为水凝胶形成准备无菌玻璃滑动模具。
	<ol> <li> 高压釜聚酯板材; 每个凝胶模具都需要一个。浸泡两个玻璃片和三塑料垫片 (0.5 毫米厚) 为每一个凝胶模具在70% 乙醇至少2小时, 但一般允许他们浸泡过夜。在使用前10分钟, 在70% 乙醇中浸泡每一个凝胶模具五活页夹. </li>
		<li> 将玻璃滑块、垫片和活页夹放到细胞培养罩中的一个小的蒸压板上, 使其风干几个小时. </li>
		<li> 准备水凝胶模具, 在玻璃滑梯的边缘放置塑料垫片。将第二个玻璃滑梯放在上面。用活页夹紧紧紧挨着的间隔, 两个长边和一个在上面. 
		注: 如果没有正确组装, 凝胶溶液将会泄漏. </li>
		<li> 在使用前30分钟用细胞培养罩 UV 光对模具进行表面消毒。在中途, 翻转模具, 露出两个表面. </li>
	</ol> </li> </ol> 2。用游离的巯基修饰蛋白质 <ol> <li> 使用电子表格中的计算方法准备库存解决方案, 将胺转化为巯基蛋白 (表 1A). 
	<ol> <li> 进行反应缓冲, 将 PBS 调整为 pH 值 8, 并添加5毫米 EDTA。吸管反应缓冲器为0.22 和 #181; m 注射器过滤器为绝育. 
		注: EDTA 是重要的, 因为它保护硫不形成二硫键。硫醇基团必须保持在其还原形式的反应发生. </li>
		<li> 称量 2-iminothiolane (Traut 和 #39; s 试剂), 并将其溶解在反应缓冲液中, 以储存溶液浓度2毫克/毫升 (14 mM)。通过 0.22 #181; m 注射器过滤器进行灭菌。将库存解决方案存储在4和 #176; C 最多几个月. 
		注: 2-iminothiolane 是分子, 反应与溶剂暴露的游离胺的蛋白质和转换成自由硫. </li>
		<li> 在0.1 和1毫克/毫升浓度之间的反应缓冲中重组蛋白质。除非以前不育, 吸管这个解决方案通过一个0.22 和 #181; m 注射器过滤器灭菌. 
		注: 这种蛋白质是将被图案化到水凝胶表面的生化信号。此外, 虽然蛋白质浓度可以变化, 较高的浓度是理想的更强的蛋白质模式. </li>
	</ol> </li> <li> 将两个股票解决方案混合在一起, 以 2-iminothiolane 反应蛋白质; 使用 表 1B 计算正确的音量比率。通常, 使用 8:1 2-iminothiolane 的摩尔比率与蛋白质. 
	注: 对于较大的蛋白质或更稀的浓度, 使用更高的摩尔比 2-iminothiolane。请参考制造商和 #39 的协议 15 . </li>
	<li> 室温下孵育1小时的反应。在制造商和 #39 的协议 16 之后, 使用脱盐自旋微柱从剩余的反应物中去除巯蛋白产品. 
	注: 此树脂的排除限值为 5 KDa. </li>
	<li> 使用埃尔曼和 #39 的测定来定量测量每种蛋白质的自由硫数。按照检测 17 的制造商和 #39 的协议进行. </li>
</ol> 3。水凝胶形成 <ol> <li> 根据从 表 1C 计算的值创建凝胶前驱体解决方案。混合 PEGDA, 纤维连接蛋白和膝部卷。强力吸管解决方案, 以确保一个均匀的解决方案, 但避免产生气泡。保持解决方案免受光照的保护. </li>
	<li> 在凝胶模具的两个玻片之间小心地移出凝胶前驱体溶液。通常, 在800和1000之间添加一个卷, #181; L 到模具。将模具中的凝胶溶液暴露于 UV 光 (波长: 365 nm, 功率: 3-4 兆瓦/cm 2 ), 以形成水凝胶的1-2 分钟. 
	注意: 不要将水凝胶暴露在长时间的紫外线下, 因为它会限制表面蛋白的图案功能. </li>
	<li> 取下活页夹夹, 并通过向两个侧面垫片施加相反的压力, 轻轻地移除顶部的玻璃幻灯片. </li>
	<li> 使用适当大小的活检穿孔来切割水凝胶样品。从凝胶矩形中冲出多个水凝胶作为复制和控制样品. </li>
</ol> 4。水凝胶刚度测量 <ol> <li> 用 8 mm 直径的活检穿孔来冲出一个 8 mm 平行平板流变仪。将一个水凝胶样品装入流变仪 (请参见 材料表 ). </li>
	<li> 降低直径为8毫米的平行板几何, 直到与凝胶表面接触。保持0.5 毫米的间隙距离为0.5 毫米厚的水凝胶样品. </li>
	<li> 执行时间扫描5分钟, 在0.1% 应变, 0.1 赫兹频率, 37 和 #176; c. 平均 G 和 #39; 每次扫描值。运行独立的水凝胶, 复制每个组成. 
	注: G 和 #39; 值应在时间点上稳定;如果不是, 则应执行百分比应变和频率扫描来选择适当的值 14 . </li>
</ol> 5。蛋白质图案 <ol> <li> 使用计算机辅助设计 (CAD) 程序为掩准备所需的模式。使用高分辨率打印机在透明纸张上打印掩。在使用前10分钟将掩浸泡在70% 乙醇中。在使用前, 允许掩在细胞培养罩中风干. </li>
	<li> 将步骤2中准备的巯蛋白溶液添加到表面图案的水凝胶表面。吸管1-2 和 #181; L/厘米 2 巯蛋白溶液到每一个切口凝胶的表面. 
	注意: 重要的是要尽量减少蛋白质体积;只有添加足够的均匀覆盖整个表面的水凝胶样品. </li>
	<li> 小心地将掩放在水凝胶表面。不要允许掩和水凝胶表面之间有气泡。轻轻按下面罩, 以删除任何形成的气泡. </li>
	<li> 将水凝胶暴露于第二轮 UV 光 (波长:365 nm, 功率: 3-4 兆瓦/cm 2 ) 为 30-60 s. 
	注意: 重要的是要曝光的模式, 以足够的紫外线, 创造一个强大的模式, 而不会造成蛋白质功能的损失. </li>
	<li> 用 PBS 清洗水凝胶去除未的种类, 并将每个凝胶放入井板中。将凝胶放入井中时要小心;确保图案曲面朝上。在 PBS 4 和 #176 清洗凝胶;凝胶在4和 #176 至少两周稳定; C. </li>
</ol> 6。细胞播种用水凝胶的制备 <ol> <li> 在37° C 的基础介质中孵育5-10 分钟的凝胶, 然后在播种前进行 #176;对于人脐静脉内皮细胞 (内皮), 使用无生长因子的 EGM-2 培养基。尽量减少介质的体积, 以防止水凝胶漂浮。对于 48-井板, 使用250和 #181; L 体积的中型. </li>
	<li> 将板在 300 x g 下旋转3分钟, 以确保水凝胶位于井的底部并且不漂浮。在单元播种前立即执行此项. </li>
</ol> 7。水凝胶上的细胞播种 <ol> <li> 使用标准的无菌哺乳动物细胞培养程序进行细胞播种和实验程序。使用标准细胞剥离协议从组织培养瓶中去除内皮。用无菌 PBS 冲洗组织培养皿, 用胰蛋白酶孵育3-5 分钟, 在 37 #176; C. </li>
	<li> 用过量的细胞培养基或胰蛋白酶中和剂溶液淬火 trypsinized 细胞悬浮液. </li>
	<li> 将离心机上的悬浮液在 300 x g 下旋转5分钟, 小心地去除上清, 保持细胞颗粒. </li>
	<li> 重基底 EGM-2 中的细胞团, 无生长因子。使用例进行单元计数. </li>
	<li> 添加7.5万单元格/cm 2 每个水凝胶表面的移慢慢地进入每个井的中心, 以免扰乱凝胶。将井板放入37和 #176 的细胞培养箱中;数天, 定期删除该盘进行观察. </li>
</ol> 8。生物活性的评价 <ol> <li> 区域性 大肠杆菌 一夜之间悬浮在 LB 肉汤的轨道悬浮在37和 #176; C 和 200 rpm。将 大肠杆菌 的培养, 醒酒, 并在最小的 PBS 体积内重组细胞颗粒。称量溶菌酶, 并重建在1毫克/毫升的股票溶液. </li>
	<li> 将溶菌酶溶液中的小容积 (25 和 #181; L) 移入离心管。添加不同级别的膝光 (0-12 mM), 并将样品暴露于1分钟的 UV 光中。在一个单独的组中, 添加相同量的膝部 (2 毫米) 到溶菌酶溶液, 并将样品暴露在不同的紫外光时间 (0-4 分钟)。包括每个处理的复制. </li>
	<li> 将相同体积的浓缩 大肠杆菌 溶液添加到每个溶菌酶样品中, 最终溶菌酶蛋白浓度为0.5 毫克/毫升。在室温下孵育4小时的混合溶液. 
	注意: 这使得功能性溶菌酶能够溶解细菌细胞壁并释放蛋白质到溶液中. </li>
	<li> 使用未经治疗的溶菌酶孵育与 大肠杆菌 为阳性对照; 考虑这100% 生物活性测量。使用 PBS 单独孵育溶菌酶溶液作为阴性对照. </li>
	<li> 向下旋转样品以除去细胞碎片并保持上清液。运行布拉德福德化验, 以量化在上清液中蛋白质的总浓度, 以测量细菌裂解液的数量. </li>
	<li> 根据制造商和 #39 的协议 18 运行布拉德福德检测。计算蛋白质浓度与阴性对照的褶皱变化. </li>
</ol>

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作者没有什么可透露的。

该协议提供了一种生物活性蛋白模式的生物学应用的方法。对于不同的实验, 可以进行一些修改以适应此协议。首先, 对于不同的单元格类型, 单元格附件要求会有所不同。如果最初观察到凝胶的不良细胞附着, 则建议在前体溶液中增加 ECM 蛋白的浓度。其他 ECM 蛋白可以用来代替纤维连接蛋白, 包括不同类型的胶原蛋白, 层粘连蛋白, 或其组合。对于每个新的细胞类型, 细胞附件应优化之前, 水凝胶模式。该协议还允许用户设计的光。基于所需的应用, 光可以产生各种功能的大小, 形状和整体模式。在无掩的情况下可以实现均匀固定。
该协议有一定的局限性。正如代表性结果中突出显示的, 此方法对每个步骤中的 UV 光量都很敏感。暴露在水凝胶形成步骤限制了可用的丙烯酸基团后表面 bioconjugation。因此, 该协议中的一个关键步骤是管理好每个步骤的 UV 光曝光时间。此外, 这个协议需要一个高浓度的蛋白质储存成功的模式。低浓度的蛋白质会导致不良的表面形态。此外, 光也限制了它们只能产生离散模式。使用类似的方法可以实现更复杂的模式, 但需要更高级的方法。
该协议对于现有的方法具有重要意义, 因为它提供了一种调整基板刚度和蛋白质图案的简单方法。使用丙烯酸酯化学的水凝胶的形成允许在生理范围内的基板刚度范围。简单地调节前体溶液中 PEGDA 的浓度, 就能控制水凝胶的刚度。此外, 使用单击化学蛋白质模式允许快速结合水凝胶基质和巯修饰蛋白。这是一个关键的设计功能, 因为它允许本协议适用于任何蛋白质或多肽的利益。
PEG 水凝胶是一种有前途的生物材料, 可以用来探索新的平台, 显示生物系统的生物化学线索。无论是均匀的表面固定或空间特定的模式, 这些技术提供了新的方法来控制细胞的行为。前进, 生物材料技术的进步将提供新的洞察到细胞行为和进一步我们的能力重述在体内信号主题在体外系统。这对干细胞分化和在受控实验系统内的发育信号的建模都是有益的。

有许多刺激会影响细胞的行为。通常, 典型的细胞培养技术依赖于可溶性因素来诱发细胞反应;但是, 这种方法存在局限性。这些方法无法准确地显示在体内常见的所有信号图案。这种信号机制包括隔离生长因子, 细胞信号, 和空间特定的生化线索。此外, 基板刚度在细胞行为和干细胞分化中起着重要的作用, 使用普通细胞培养方法也不容易操作1,2。生物材料的方法提供了一个新的平台, 开始探索这些信号机制。特别是, 水凝胶是优化的候选基板刚度3,4, 固定蛋白和肽5,6, 并创建空间特定模式7,8。
水凝胶通常用作组织工程的支架, 因为它们具有生物物理和生物化学的共性, 细胞外基质 (ECM)9,10。天然高分子材料是支架的常见选择, 因为它们具有生物相容性, 并且在人体的许多组织中都能找到。使用天然高分子材料作为基质的局限性在于, 它们缺乏易于操作的化学基 bioconjugation。另一方面, 合成水凝胶, 如 PEG, 是优秀的平台为靶向化学11,12。此外, PEG 水凝胶不会引起细胞反应, 因此被用作建立生物活性支架的惰性骨干。
为建立生物活性水凝胶, 聚合和硫醇-烯的点击化学反应被使用。这些 photoreactions 需要一个光和一个紫外线光源。当引发被引入紫外光时, 键会断裂形成自由基。这些自由基是启动反应所必需的, 但会对蛋白质的生物活性产生负面影响12,13。因此, 最重要的是优化光和紫外线暴露时间, 以保持蛋白质的生物活性。
该方法通过丙烯酸酯-丙烯酸酯链生长聚合合成水凝胶。PEG-丙烯酸酯 (PEGDA) 单体相互反应形成分支聚合物网络, 负责水凝胶的结构。在凝胶前驱体溶液中 PEGDA 单体的浓度将控制基体的刚度。由于水凝胶的小孔径大小, ECM 蛋白, 如纤维连接蛋白, 可以很容易地纳入水凝胶的目的是细胞附件。最后, 这些水凝胶可以是表面图案的生物活性肽或蛋白质通过硫醇-烯的点击化学反应。在这里, 未免费的丙烯酸酯在水凝胶系统将反应与自由硫位于蛋白质或肽时暴露在紫外线。在水凝胶表面固定蛋白质或多肽后, 水凝胶可在4° c 贮存数周而不会失去生物活性。这为实验室提供了方便、灵活的实验计划和协作的可能性。总的来说, 这个平台允许生物力学和空间生化控制, 相互独立, 从而有机会影响细胞的行为。

<table width="1237">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">PEG-diacrylate (PEGDA)</td>
			<td style="width:112px;">Laysan Bio</td>
			<td style="width:223px;">ACRL-PEG-ACRL-3400</td>
			<td style="width:665px;">Can also be synthesized or purchased through other venders. Different molecular weights can be used.</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:237px;height:60px;">Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
			<td style="width:112px;"></td>
			<td style="width:223px;"></td>
			<td style="width:665px;">Synthesized in lab</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Fibronectin</td>
			<td style="width:112px;">Corning</td>
			<td style="width:223px;">356008</td>
			<td style="width:665px;">Other cell attachment proteins can be used, such as laminin, matrigel</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:223px;">D8537-500ML</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Photomask</td>
			<td style="width:112px;">FineLine Imaging</td>
			<td style="width:223px;">n/a</td>
			<td style="width:665px;">Custom prints on transparent sheets with high resolution DPI.</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Binder Clips</td>
			<td style="width:112px;">Various Vendors</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="width:237px;height:52px;">Compact UV Light Source (365nm)</td>
			<td style="width:112px;">UVP</td>
			<td style="width:223px;">UVP-21</td>
			<td style="width:665px;">Other UV light sources can be used, calibration of power is required.</td>
		</tr>
		<tr height="57">
			<td height="57" style="width:237px;height:57px;">2-iminothiolane (Pierce Traut&#8217;s Reagent)</td>
			<td style="width:112px;">Thermo Sci.</td>
			<td style="width:223px;">26101</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="82">
			<td height="82" style="width:237px;height:82px;">Ellman&#8217;s Reagent: DTNB; 5,5-dithio-bis(2-nitrobenzoic acid)</td>
			<td style="width:112px;">Thermo Sci.</td>
			<td style="width:223px;">22582</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="width:237px;height:52px;">human umbilical vein endothelial cells (HUVECs)</td>
			<td style="width:112px;">Lonza</td>
			<td style="width:223px;"></td>
			<td style="width:665px;">passage number between 6- 10</td>
		</tr>
		<tr height="52">
			<td height="52" style="width:237px;height:52px;">EGM-2 Media</td>
			<td style="width:112px;">Lonza</td>
			<td style="width:223px;">CC31-56, CC-3162</td>
			<td style="width:665px;">EGM-2 without growth factors was used in experiments. Full EGM-2 media was used for cell maintainance</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">0.25% Trypsin EDTA</td>
			<td style="width:112px;">Life Tech</td>
			<td style="width:223px;">25200-056</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Trypsin Neutralizer</td>
			<td style="width:112px;">Life Tech</td>
			<td style="width:223px;">R-002-100</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Centrifuge</td>
			<td style="width:112px;">Various Venders</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Hemocytometer</td>
			<td style="width:112px;">Hausser Sci. Bright-line</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="width:237px;height:51px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:223px;">E6758</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">0.22&#181;m filter</td>
			<td style="width:112px;">Cell Treat</td>
			<td style="width:223px;">229743</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">1mL Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Glass Microscope Slides</td>
			<td style="width:112px;">Fisher Sci.</td>
			<td style="width:223px;">12-550C</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Plastic spacers</td>
			<td style="width:112px;">Various Venders</td>
			<td style="width:223px;"></td>
			<td style="width:665px;">0.5mm thickness</td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">70% Ethanol</td>
			<td style="width:112px;">BICCA</td>
			<td style="width:223px;">2546.70-1</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Bio-shield</td>
			<td style="width:112px;">Bio-shield</td>
			<td style="width:223px;">19-150-0010</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Bradford Reagent&#160;</td>
			<td style="width:112px;">BIO-RAD</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Desalting Resin - Sephadex G-25</td>
			<td style="width:112px;">GE Healthcare</td>
			<td style="width:223px;">95016-754</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">Microspin Columns</td>
			<td style="width:112px;">Thermo Sci.</td>
			<td style="width:223px;">PI69725</td>
			<td style="width:665px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="width:237px;height:35px;">AR-G2 rehometer</td>
			<td style="width:112px;">TA Instruments</td>
			<td style="width:223px;"></td>
			<td style="width:665px;"></td>
		</tr>
	</tbody>
</table>

patterning bioactive proteins or peptides on hydrogel using photochemistry for biological applications

有许多生物刺激可以影响细胞行为和干细胞分化。一般细胞培养方法依赖于培养基中的可溶性因子来控制细胞的行为。然而, 可溶性添加物不能模仿某些信号的图案, 如基质结合生长因子, 细胞信号, 和空间生化线索, 这是常见的影响细胞。此外, 基体的生物物理特性, 如基底刚度, 在细胞的命运中发挥重要作用, 这是不容易使用传统的细胞培养实践操作。在这种方法中, 我们描述了一个简单的协议, 提供图案生物活性蛋白的合成聚乙二醇 (PEG) 水凝胶使用光化学。该平台可以独立控制基板的刚度和空间生化信号。这些水凝胶可以达到很大范围的生理相关的刚度值。此外, 这些水凝胶的表面可以 photopatterned 与生物活性肽或蛋白质通过硫醇-烯的点击化学反应。这些方法已经过优化, 以保留蛋白质功能后, 表面固定。这是一个多才多艺的协议, 可以适用于任何蛋白质或多肽的利益, 以创造各种模式。最后, 在这些生物活性水凝胶表面上播种的细胞可以随着时间的推移对空间特定的信号进行监测。

在 PEG 水凝胶表面建立生物活性模式的协议在图 1中进行了说明。开发了一个电子表格来计算每个股票解决方案的体积和浓度 (表 1A)。蛋白质被固定在水凝胶的表面被修改用 2-iminothiolane (图 1B)。此反应是使用表 1B中的卷执行的。前体水凝胶溶液用10% 重量/体积的 PEGDA 与膝部 (图 1A)。各种前驱 PEGDA 浓度可用于产生所需的基底刚度 (图 2A)。纤维连接蛋白是包括在这个前体溶液的细胞附着的目的。经过彻底混合后, 此解决方案被 pipetted 到准备好的模具中, 并暴露在 UV 光中 (图 1C)。应尽量减少紫外线照射;曝光量应该刚好足以产生水凝胶。水凝胶样品被冲压到适当的直径为期望的井板 (图 1C)。对于表面图案, 改性蛋白溶液 pipetted 到水凝胶表面, 均匀分布。应使用最小体积;蛋白质体积应该刚好足以覆盖整个表面的水凝胶。预先设计的掩直接放在水凝胶表面上;应该避免面罩和水凝胶之间的气泡。第二轮 uv 光被用来将紫外线暴露的蛋白质与水凝胶结合。水凝胶样品被漂洗去除未蛋白质并且显露固定化蛋白质样式 (图 1D)。
重要的是要尽量减少光浓度和紫外线暴露时间时, 蛋白质的存在。以溶菌酶生物活性为指标, 我们发现膝光浓度应小于2毫米 (图 2B), 而 UV 曝光时间应总少于2分钟 (图 2C) 以保留蛋白质生物活性大于80%。
在水凝胶形成和蛋白质模式的紫外线曝光时间是开发一个成功的协议的重要参数 (图 3)。首先, 在水凝胶形成过程中最小化紫外线暴露对于维持游离的丙烯酸酯功能基团对随后的蛋白质固定反应至关重要 (图 3A)。暴露在紫外线下超过2分钟的水凝胶无法形成固定化的蛋白质模式。此外, 随着紫外线照射到蛋白质模式的增加, 更多的蛋白质对表面的反应 (图 3B)。
最后, 细胞可以被培养到这些图案化的水凝胶基质上, 以操纵细胞行为。为了显示固定化模式对水凝胶的潜在作用, 我们用基底 EGM-2 培养基 (图 4) 对内皮细胞和培养的内皮进行了生长因子。内皮被均匀地播种在血管内皮生长因子型 PEG 水凝胶 (图 4A) 的表面上。播种后两天, 内皮被观察到迁移到含有固定化血管内皮生长因子的水凝胶的空间区域 (图 4B, C)。这是一个例子的生物活性蛋白模式的 PEG 水凝胶被用来影响细胞的行为。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55873/55873fig1.jpg"> 
图 1:水凝胶形成和蛋白质模式示意图(A) 准备前体水凝胶溶液, PEGDA 单体、光和胞外蛋白进行细胞附着。(B) 通过与 2-iminothiolane 反应对游离巯基的蛋白质进行修饰。(C) 将前体溶液移入所准备的模具中, 并将其暴露在 UV 光中以形成水凝胶。冲出所需尺寸的水凝胶样品。(D) 将经过修饰的蛋白质溶液移到水凝胶表面, 在表面放置一个掩, 并将其暴露在第二轮 UV 光中。在成像和细胞播种之前冲洗凝胶以去除未物种。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55873/55873fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55873/55873fig2.jpg"> 
图 2: 调节刚度和蛋白质生物活性.(A) 前体凝胶溶液中 PEGDA 单体浓度的变化改变了水凝胶的硬度。(B) 在1分钟的紫外线照射后, 增加膝光的浓度可降低蛋白质功能。(C) 增加紫外线照射时间可降低 2 mM 膝部的蛋白质功能。所有误差线表示复制的标准偏差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55873/55873fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55873/55873fig3.jpg"> 
图 3:模式和水凝胶紫外线照射时间优化的蛋白质模式.(A) 最小化水凝胶形成的紫外线照射时间, 允许表面图案。(B) 增加表面图案的紫外线曝光时间可增加图案强度。缩放栏 = 200 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55873/55873fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55873/55873fig4.jpg"> 
图 4:内皮细胞响应 VEGF 的模式.(A) 在水凝胶上播种的均匀内皮。(B和C) 内皮感觉 vegf 的形态并向固定化 vegf 迁移。图像在播种后两天后 (B) 4X 和 (C) 10X 放大。缩放条形图 = 500 µm 内皮-rfp (红色) 和 VEGF-488 模式 (绿色) 分别在528/553 和465/495 被激发和发射过滤器捕获。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_u href=""></a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_u>pload/55873/55873fig4large. jpg "目标 =" _blank ">> 请点击这里查看这个数字更大的版本。
<img alt="Table 1" src="/files/ftp_upload/55873/55873table1.jpg"> 
表 1: 库存和凝胶前驱体解决方案的计算.红色框表示用户定义的值, 如分子量, 期望的股票浓度, 和 weighed-out 的质量。蓝色方框表示已根据用户定义的值计算的值。

Watch this Scientific Journal Video about Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications at JoVE.com

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

在这个方法中, 我们使用聚合和点击化学技术在聚乙二醇 (PEG) 水凝胶表面创造蛋白质或肽模式, 提供固定化的生物活性信号来研究细胞反应体外.

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 ...

patterning-bioactive-proteins-or-peptides-on-hydrogel-using

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.1K 次观看.  Rensselaer Polytechnic Institute. 在这个方法中, 我们使用聚合和点击化学技术在聚乙二醇 (PEG) 水凝胶表面创造蛋白质或肽模式, 提供固定化的生物活性信号来研究细胞反应体外.

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

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

科研

生物工程

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案:二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

就地 液体生物组件的 TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

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 ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

快速, 可伸缩的组装和负载的生物活性蛋白和免疫增强剂到不同的合成 Nanocarriers 通过闪光 Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

采用两种 Electrohydrodynamic 射流的高分辨率模式: 需求下降和近场静电纺丝

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...