这项工作得到了由教育部、科学技术部 (MEST) [NRF-2015R1D1A1A01060397] 资助的韩国国家研究基金会 (NRF) 和生物 & 医疗技术发展的基础科学研究项目的支持。由科学、信息和通信技术 & 未来规划部资助的 NRF 计划 [NRF-2017M3A9C6029563]。

这项研究是由韩国大学 Anam 医院的机构审查委员会批准的 (IRB 号。ED170495)。所有程序都是按照《赫尔辛基宣言》及其后来的修正案进行的。
1. 用抛光制备铝 (铝) 基板
警告:抛光溶液具有腐蚀性和毒性。佩戴个人防护用品, 包括丁腈橡胶手套、护目镜和实验室大衣。在通风罩中执行此步骤。
<ol><li>在 60% L 双夹克烧杯 (c1h2oh: HClO5 = 4) 中混合乙醇 (c2H5OH, 99.9%) 和高氯酸 (HClO4, 4:1) 制备抛光溶液。</li>
	<li>将双夹克烧杯连接到一个循环器, 并将温度设置为摄氏7摄氏度。把双夹克烧杯放在磁力搅拌器上。把溶液搅拌至少30分钟, 直到温度降到7摄氏度。</li>
	<li>将超纯铝板 (99.999%, 20 x 50 x 1 毫米3) 和碳计数器电极浸入抛光溶液中, 使用鳄鱼夹子和铜线。调整铝板和碳计数器电极的位置以面对面。 注:必须仔细安装剪辑, 以免碰到解决方案。当与溶液接触时, 从夹子流出的杂质会污染铝板。</li>
	<li>将铝板连接至正极端子, 并将碳计数器电极与直流 (直流) 电源的负极端子相连。</li>
	<li>关闭磁力搅拌器并应用电压 20 v 保持此状态为4分钟。</li>
	<li>打开磁力搅拌器, 让电流再流6分钟。 注:静态条件去除了铝板中的大部分杂质和粗糙度, 动态条件提高了抛光的最终质量。</li>
	<li>关闭电源, 并手动将铝板与剪辑分开。用去离子水严格清洗铝板, 除去所有剩余的溶液。</li>
	<li>用氮气干燥铝板, 在惰性气体下贮存。在步骤1和2的所有实验结束时进行此干燥过程。 注:在注入压缩空气时, 使空气从抛光部分流向另一侧。在相反的情况下, 杂质可以从不抛光的部分逃脱, 污染铝板。</li>
</ol>2. 磷酸电解质梯度阳极氧化铝模具的制备
警告:甲醇和它的烟是眼部毒性。连续接触铬会导致严重的铬中毒。在通风罩中执行此步骤。
<ol><li>原阳极氧化<ol>
<li>制备 1 L 磷酸电解质, 将590毫升的去离子水装入玻璃瓶中。</li>
		<li>将400毫升甲醇 (CH3OH, 99%) 和10毫升磷酸 (H3PO4, 85%) 放入玻璃瓶中。通过手动摇动或用磁力搅拌混合解决方案。</li>
		<li>将500毫升的电解质倒入1升双夹克烧杯中。用鳄鱼夹子和铜线将抛光的铝板和碳计数器电极浸入溶液中。</li>
		<li>倒入额外的电解质, 以定位在溶液表面上方的抛光2-3 毫米的边界。 注:如果用抛光接触溶液的边界进行阳极氧化, 铝板在阳极氧化过程中往往会燃烧。</li>
		<li>在 U 形底座上安装竖轴。附加一个架空搅拌器, 和叶轮使用金属夹具。将叶轮的螺旋桨定位在两个电极的下端附近。设置在200和 300 rpm 之间的架空搅拌器的转速。</li>
		<li>将双夹克烧杯连接到一个循环器, 并将温度设置为摄氏-10 摄氏度。使系统至少1小时的搅拌, 直到温度降到-10 摄氏度。 注:不要用水作为冷却剂。因为水在低温下结冰, 所以循环不会正常工作。建议使用混合物的水和乙醇的比例为1:1 的冷却剂。</li>
		<li>施加195伏的电压, 搅拌16小时。 注:如果电流在施加电压后在2或3小时内上升超过50毫安, 那么燃烧的概率就会很高。立即停止的过程, 并更换铝板与一个新的。如果重复出现此问题, 请检查循环器的温度控制没有异常。如果没有异常, 改变电解质。</li>
		<li>停止电源, 并手动将铝板与剪辑分开。用去离子水清洗阳极氧化铝板, 除去所有剩余的溶液。</li>
	</ol>
</li>
	<li>氧化铝蚀刻<ol>
<li>在4毫升的去离子水中溶解9.0 克氧化铬 (3) 和20.3 毫升磷酸 (H3PO85%, 500), 制备铬酸蚀刻液。</li>
		<li>将蚀刻液倒入1升双套管烧杯中。将温度设置为摄氏65摄氏度。</li>
		<li>将阳极氧化铝板浸入蚀刻液中至少10小时搅拌。用铝箔封住烧杯的顶部。</li>
		<li>用去离子水冲洗蚀刻过的铝板几次。</li>
	</ol>
</li>
	<li>二次阳极氧化<ol>
<li>设置与步骤2.1.1 到2.1.7 相同的实验条件。</li>
		<li>将蚀刻铝板加载到系统的阳极上, 并对 6 h 施加195伏的电压。然后, 关闭电源并手动将铝板与剪辑分开。立即用去离子水清洗阳极氧化铝板。 注:当洗涤时间延迟时, 由于残余磷酸的存在, 氧化铝的孔隙大小不自觉地增加。</li>
	</ol>
</li>
	<li>梯度孔隙加宽<ol>
<li>在500毫升的去离子水中溶解5.765 克磷酸, 制备孔隙加宽溶液。把溶液倒入1升双夹克烧杯中。</li>
		<li>将双套管烧杯连接到循环器, 并将温度设置为摄氏30摄氏度。在烧杯旁垂直放置一个线性移动的舞台。将支架连接到线性舞台的移动部分。</li>
		<li>将线性舞台的移动部分设置为 home 位置。把夹子贴到支架上, 然后装上阳极氧化铝板。</li>
		<li>手动操纵铝板的位置, 这样铝板就会出现在溶液表面的上方。 注:在这一步, 铝板不应该接触的解决方案。当铝板到达溶液时, 孔的加宽过程就开始了。</li>
		<li>将铝板逐渐浸入溶液中, 速度为4.86 µm 120 分钟, 使35毫米氧化铝模具的尺寸梯度从 120 nm 到 200 nm (GP 120/200)。在铝板触及溶液表面的那一刻开始秒表。</li>
		<li>为了使 gp 200/280 和 gp 280/360 模具, 分别浸入2或 4 h 的孔加宽溶液中的 gp 120/200 模具的整个区域。</li>
		<li>多次用去离子水冲洗梯度铝板。</li>
	</ol>
</li>
</ol>3. 自组装单层梯度氧化铝结晶器防粘层的沉积
注:执行步骤 3.2.1 3.3.3 在一个手套箱。将真空泵和干氮气注射器连接到手套箱。在除湿过程之前, 将所有样品、试剂和器具放在手套箱中。重复疏散和氮气注入循环三次以上, 以充分去除从手套箱的水分。让干燥的氮气流过实验。
<ol><li>食人鱼处理对氧化铝结晶器羟基的改性研究<ol>
<li>在聚四氟乙烯 (PTFE) 烧杯中倒入140毫升硫酸 (H2,4, 95%)。添加60毫升过氧化氢滴状 (H2O2, 30%)。保持30分钟, 直到解决方案冷却下来。 警告:做食人鱼的解决方案时要格外小心。从加入过氧化氢的那一刻起, 溶液就会剧烈沸腾, 产生高温。在通风罩上进行实验。</li>
		<li>使用非金属镊子浸泡在溶液中的氧化铝模具三十年代。</li>
		<li>多次用去离子水冲洗阳极氧化铝模具。将模具浸泡在去离子水中30分钟, 再将其放入甲醇中再加30分钟。 警告:当丢弃使用的食人鱼溶液时, 用大量的自来水稀释溶液。</li>
		<li>在真空下完全干燥氧化铝模具3小时。</li>
	</ol>
</li>
	<li>20× HDFS 库存液的制备 注:HDFS 是 (heptadecafluoro-1,1,2,2,-tetrahydrodecyl) dimethylchloro 硅烷的缩写<ol>
<li>用分子筛填充1/3 的1升正己烷瓶。用石蜡膜封住瓶子, 将其贮存在干氮气下24小时。</li>
		<li>用玻璃注射器和0.2 µm 聚四氟乙烯注射器过滤200毫升正己烷。</li>
		<li>将80毫升过滤过的正己烷倒入100毫升玻璃瓶中。加入2毫升的 HDFS。</li>
	</ol>
</li>
	<li>自组装单层沉积<ol>
<li>在100毫升烧杯中负载57毫升的过滤正己烷。将阳极氧化铝模具浸入溶剂中。</li>
		<li>在正己烷中加入3毫升的 HDFS 溶液, 使2.9 毫米 HDFS 溶液。等待10分钟的反应进行。</li>
		<li>将阳极氧化铝模具转移到新的正己烷。关闭并密封所有试剂瓶。关闭氮气阀, 然后从手套箱取出样品。 注:HDFS 对湿气非常敏感。将包含 HDFS 的所有试剂存储在干燥中。</li>
		<li>浸泡在60毫升的 methoxynonafluorobutane (C10小时6F18O2, 99%) 的氧化铝模具。超声波清洗在烧杯中的氧化铝模具为十年代每一次, 以消除物理吸附 HDFS 分子。重复清洗程序10次。 注:在不间断的情况下, 长时间暴露阳极氧化铝模具, 会损害氧化物层。</li>
		<li>在真空下完全干燥氧化铝模具24小时。 注:一个成功地存放防粘层是超级防水和稳定的几个月, 当存储在一个干燥。协议可以在这里暂停。</li>
	</ol>
</li>
</ol>4. 热印迹制备梯度 Nanopattern 板
注:在洁净室执行步骤4.2 至4.7。
<ol><li>用印刷电路板 (PCB) 切割机将1.1 毫米厚的聚苯乙烯片切成2.9 毫米宽3.7 毫米长的尺寸。</li>
	<li>用夹钳将氧化铝模具从底部切开35毫米。在背面标明样品的名称和制造日期。</li>
	<li>用小剂量的乙醇清洁 8.0 "晶片。将聚苯乙烯片放在晶片上, 然后装上阳极氧化铝模具。</li>
	<li>把底膜放在热印刷机的抽屉里。将顶部薄膜连接到垫片上。</li>
	<li>把晶片放在底膜上。把垫片放在晶片上。确保晶片上没有灰尘。</li>
	<li>关闭热印刷机的抽屉。应用165摄氏度的热量和620.52 帕的压力100s。</li>
	<li>把样品冷却到室温。轻轻拧下聚苯乙烯板, 释放阳极氧化铝模具。</li>
</ol>5. 梯度 Nanopattern 板的杀菌和亲水性改性
<ol><li>把 nanopattern 盘子放在方形盘子上。倒入100毫升70% 乙醇并暴露在紫外线照射下30分钟。</li>
	<li>用氮气干燥 nanopattern 板。将 nanopattern 板转移到氧气等离子发生器上。</li>
	<li>疏散房间直到它到达1.33 宾夕法尼亚州然后, 注入氧气的速率为30厘米3/分钟. 应用射频功率 60 w 维持氧气等离子九十年代。 注:建议在14天内使用等离子处理的 nanopattern 板。</li>
	<li>用一滴甲苯将 nanopattern 板贴在细胞培养皿的底部。</li>
	<li>将样品密封在袋内, 用低温等离子灭菌器消毒。</li>
</ol>6. hECFCs 的种植
注:执行所有离心程序在4°c, 除非另行注明。
<ol><li>在隔离外周血单个核细胞 (PBMCs) 之前, 在37摄氏度的1小时内孵化出胶原涂层的12井培养皿。</li>
	<li>用1×无菌磷酸盐缓冲盐水 (PBS) 清洗12井培养皿。</li>
	<li>用肝素抑制剂管收集50毫升人体血液。</li>
	<li>将6毫升亲水性多糖溶液放入15毫升管中, 并在亲水性多糖溶液中加入8毫升血液。 注:慢慢将血液注入亲水性多糖溶液中。</li>
	<li>离心管在 1020 x g 20 分钟, 以颗粒细胞。</li>
	<li>收获不透明的细胞层, 并转移到一个新的15毫升管。</li>
	<li>添加10% 胎牛血清 (血清) 含 PBS 和离心管在 1020 x g 10 分钟, 以颗粒细胞。</li>
	<li>取出上清液并添加红细胞溶解缓冲剂。在冰上保持5分钟。</li>
	<li>添加10% 的含血清 PBS 和离心管在 1020 x g 5 分钟, 以颗粒细胞。</li>
	<li>取出上清液并添加10% 个含血清的 PBS。离心管在 1020 x g 5 分钟, 以颗粒细胞。</li>
	<li>去除上清液, 添加1毫升内皮细胞扩张培养基, 补充10% 的血清。种子 8.0 x 106细胞每井每一个胶原涂层的菜肴。</li>
	<li>每天改变培养基1周。然后, 每2天更改一次培养基。 注: hECFCs 可以在10-14 文化日后找到。hECFCs 显示了典型的鹅卵石状形态, 形成了一种能在相衬显微镜中观察到的菌落。对血管内皮钙黏蛋白 (CD144) 和 vWF 的免疫荧光染色也可用于 hECFCs 细胞扩张后的特征。</li>
	<li>培养 hECFCs, 使用内皮细胞膨胀培养基补充5% 血清和青霉素/链亲和素在37°c 的湿润气氛中含有 5% CO2。每天更换一次培养基。</li>
	<li>hECFC 诱导后, 将细胞生长到7通道进行进一步实验。</li>
</ol>7. 梯度 Nanopattern 板上的细胞播种和培养
注:步骤7描述了 hECFCs 在梯度 nanopattern 板上的培养, 但也可以使用其他细胞源。
<ol><li>在室温下, 在 PBS 中涂上1毫升0.1% 蛋白涂层溶液的梯度 nanopattern 板以10分钟。 注:0.1% 蛋白涂层不包括 nanopillar 特征。</li>
	<li>用胰蛋白酶用标准细胞分裂法使 hECFCs 悬浮在培养皿中。</li>
	<li>稀释细胞悬浮液以达到理想的汇流。种子 hECFCs 到梯度 nanopattern 板和扁平控制 (例如, 1.0 x 104细胞/cm2)。 注:当 hECFCs 被播种在 1.0 x 104细胞/cm2在梯度 nanopattern 板材, 细胞融合通常到达 70-80% 在2天以后。</li>
	<li>培养细胞在平的或梯度 nanopattern 板2天在孵化器。</li>
</ol>8. 观察和分析
<ol><li>扫描电镜 (SEM) 成像<ol>
<li>固定 hECFCs 培养在扁平或梯度 nanopattern 板上, 2.5% 戊二醛在4°c 过夜。</li>
		<li>室温下治疗1% 锇毒气1小时。用 PBS 冲洗样品。</li>
		<li>从低到高浓度 (50%, 70%, 80%, 90% 和 100%) 的乙醇脱水样品每步10分钟。</li>
		<li>室温下治疗 hexamethyldisilazane (HMDS) 15 分钟。之后, 用新鲜的 HMDS 清洗样品, 在通风罩下留下样品至少1天, 直到剩余的 HMDS 完全蒸发。</li>
		<li>用铂溅射涂布机将样品涂上5分钟, 用 SEM 检查样品。</li>
	</ol>
</li>
	<li>透射电镜 (TEM) 成像<ol>
<li>固定 hECFCs 培养在平或梯度 nanopattern 板与2% 多聚甲醛和2.5% 戊二醛混合物在 PBS 在4°c 过夜。</li>
		<li>在8.1.3 中使用1% 锇毒气和脱水样品进行后固定。</li>
		<li>在环氧树脂中嵌入样品。使 60 nm 厚的部分从块, 并放置一个部分在 TEM 网格。</li>
		<li>用100毫升甲醇和0.1 毫克柠檬酸铅在100毫升蒸馏水中混合10毫克的醋酸铀来染色切片。用 TEM 获取图像。</li>
	</ol>
</li>
	<li>荧光染色<ol>
<li>固定 hECFCs 培养在平的或梯度 nanopattern 板与4% 多聚甲醛在室温下15分钟。</li>
		<li>Permeabilize 和块样本, 5% 山羊血清和0.1% 辛基醚在 PBS (PBST) 室温下30分钟。</li>
		<li>用抗人纽蛋白原抗体 (1:500 PBST) 在室温下孵育2小时, 用 PBST 冲洗样品三次。</li>
		<li>用荧光共轭罗丹明 (1:1, 000 PBST) 孵化样品, 在室温下用荧光共轭二次抗体 (1:1, 000 在 PBST), 用 PBST 冲洗样品2次。</li>
		<li>在室温下孵化 4 '、6-diamidino-2-苯基吲哚 (DAPI、1:1、000 PBST) 的样品, 5 分钟。</li>
		<li>在梯度 nanopattern 表面放置10µL 的安装介质。在安装介质上放置一个盖玻片。</li>
		<li>将样品倒在样品台上, 用共焦荧光显微镜获取图像。</li>
	</ol>
</li>
	<li>图像分析<ol>
<li>将 hECFCs 捕获的图像传输到图像分析系统。</li>
		<li>选择罗丹明染色图像的随机场。调整阈值并创建一个二进制图像, 其中单元格与背景不同。</li>
		<li>在单元格周围绘制轮廓以测量单元格区域和周长, 并手动计算每个单元格的丝状伪足数。</li>
		<li>选择纽蛋白染色图像的随机场。调整阈值并创建焦点粘附区域的二进制图像。 注:适当调整图像阈值, 以避免局部粘附区的人为过度填充或不足。</li>
		<li>计算每个单元格的焦点粘附数。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

阳极氧化铝的制备常常受到裂纹、孔隙形状不规则和燃烧等缺陷的影响。这些缺陷的主要原因称为电解击穿, 这是严重影响了金属基板的本质是电化铝和电阻率的电解质21。由于电解质的电阻率随温度的不同而变化, 从电极上连续消除热量是保持电解质在这种高压阳极氧化条件下的位置温度稳定的关键点。在本协议中, 我们通过修改 Masuda 的两步阳极氧化22,</su...

近年来, 在细胞工程1、2、3、4等领域, 对表面形貌的物理刺激对细胞的反应关注。因此, 更多的注意力集中在三维纳米结构在细胞附着表面5。据报道, 整合素是细胞的表面识别装置, 通过机械转导6传递了 ECM 微纳米结构驱动的物理刺激。这种机械刺激调节细胞行为通过接触指导7和诱导骨架重组改变形状, 除了病灶粘连和僵硬的细胞8。
人体内皮祖细胞 (hEPCs) 在体内与周围 ECM9的微环境紧密地相互作用。这表明 ECM 的物理状态作为特定细胞基质粘附复合物形成的一个重要参数, 与血流产生的剪应力10有关。据报道, 表面 nanotopography 增强了 hEPCs11毛细管网络的体外形成, ECM/生物可溶性因子联合系统使 hEPCs 能够识别功能失调的基底, 并促进伤口愈合12,13。然而, ECM 和 hEPCs 之间的关系还没有得到明确的理解。
虽然许多研究人员试图澄清细胞反应和物理线索之间的关系, 从不同的基质14,15,16, 这些研究只使用固定尺寸的纳米结构或nanopatterns 与不规则的安排有限制, 以阐明纳米结构和细胞行为的大小之间的关系。这里的问题是缺乏合适的工具来筛选细胞的反应, 可以取代现有的繁琐和迭代的方法, 以找到最佳尺寸的纳米结构。因此, 需要一个简单的技术来筛选细胞反应的物理刺激不重复。
在这里, 我们描述了一个方法, 在我们以前的报告17,18,19产生一个梯度 nanopattern, 其中排列的 nanopillars 直径逐渐增加。此外, 我们还描述了如何培养和分析 hECFCs 在梯度 nanopattern 板上的行为, 以确定物理刺激对细胞的影响。采用轻度阳极氧化、渐进蚀刻和防粘层涂敷的方法, 制备了梯度氧化铝模具。采用热印迹光刻技术, 以成本效益高、简便的方式生产出相同的聚苯乙烯梯度 nanopatterns。利用梯度 nanopatterns, 确定纳米结构的大小对一组实验中的细胞行为有很大的影响是可行的。我们期望这种梯度 nanopattern 有助于理解血液衍生 hECFC 或其他细胞与不同尺寸的纳米结构之间的相互作用机制。

<table><tbody><tr><td>Perchloric acid 60%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>6512-4100</td><td></td></tr><tr><td>Ethyl alcohol, absolute 99.9%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>4118-4100</td><td></td></tr><tr><td>Phosphoric acid 85%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>6532-4400</td><td></td></tr><tr><td>Methyl alcohol 99.5%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>5558-4400</td><td></td></tr><tr><td>Chromium(VI) oxide</td><td>Daejung Chemicals &amp;amp; Metals</td><td>2558-4400</td><td></td></tr><tr><td>Sulfuric acid 95%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>7781-4100</td><td></td></tr><tr><td>Hydrogen peroxide 30%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>4104-4400</td><td></td></tr><tr><td>n-hexane 95%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>4081-4400</td><td></td></tr><tr><td>Toluene 99.5%</td><td>Daejung Chemicals &amp;amp; Metals</td><td>8541-4400</td><td></td></tr><tr><td>(heptadecafluoro-1,1,2,2,-tetrahydrodecyl)dimethylchlorosilane</td><td>Gelest</td><td>SIH5840.4</td><td>Moisture sensitive</td></tr><tr><td>Methoxynonafluorobutane 99%</td><td>Sigma aldrich</td><td>464309</td><td></td></tr><tr><td>Collagen solution</td><td>Stemcell</td><td>#4902</td><td></td></tr><tr><td>Gelatin</td><td>Sigma aldrich</td><td>G1890</td><td>Protein coating solution</td></tr><tr><td>Ficoll-Paque</td><td>GE Heathcare</td><td>17-1440-03</td><td>Hydrophilic polysaccharide solution</td></tr><tr><td>EGM-2MV</td><td>Lonza</td><td>CC-3202</td><td>Endothelial cell expansion medium</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010031</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>12483-020</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma aldrich</td><td>P6148</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma aldrich</td><td>G5882-100ML</td><td></td></tr><tr><td>Osmium tetroxide</td><td>Sigma aldrich</td><td>201030-1G</td><td></td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma aldrich</td><td>440191</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma aldrich</td><td>X100-100ML</td><td>Octylphenol ethoxylate&amp;nbsp;</td></tr><tr><td>Goat serum</td><td>Gibco</td><td>26050-088</td><td></td></tr><tr><td>anti-human vinculin primary antibody&amp;nbsp;</td><td>Sigma aldrich</td><td>V9131</td><td></td></tr><tr><td>F-actin probe</td><td>Molecular Probes</td><td>A12379</td><td>Fluorescence-conjugated phalloidin</td></tr><tr><td>Alexa Fluor 488-conjugated anti-mouse&amp;nbsp;IgG antibody</td><td>Molecular Probes</td><td>A11001</td><td>Fluorescence-conjugated secondary antibody&amp;nbsp;</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole&amp;nbsp;</td><td>Sigma aldrich</td><td>D9542</td><td></td></tr><tr><td>Mounting medium</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>Anti-human vWF primary antibody&amp;nbsp;</td><td>DAKO</td><td>A0082</td><td></td></tr><tr><td>Anti-human&amp;nbsp;CD144 primary antibody&amp;nbsp;</td><td>BD Biosciences</td><td>#555661</td><td></td></tr><tr><td>Eponate 12&amp;trade; Embedding Kit, with BDMA</td><td>Ted Pella</td><td>18012</td><td>Epoxy resin</td></tr><tr><td>Uranyl Acetate, 25g</td><td>Ted Pella</td><td>19481</td><td></td></tr><tr><td>Lead Citrate, Trihydrate, 10g</td><td>Ted Pella</td><td>19312</td><td></td></tr><tr><td>Ultra pure aluminum plate</td><td>Goodfellow</td><td>26050-088</td><td></td></tr><tr><td>Polystyrene sheet</td><td>Goodfellow</td><td>ST313120</td><td></td></tr><tr><td>8.0&quot; silicon wafer</td><td>Siltron</td><td>29-01024-03</td><td>Single side polished, 725 &amp;micro;m thick</td></tr><tr><td>Vacuum desiccator, 4.4 L</td><td>Kartell</td><td>KA.230</td><td></td></tr><tr><td>Vacuum pump</td><td>Vacuumer</td><td>V3.VOP100</td><td></td></tr><tr><td>Power supply</td><td>Unicorntech</td><td>UDP-3003</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Daihan scientific</td><td>SL.SMS03022</td><td></td></tr><tr><td>Overhead stirrer</td><td>Daihan scientific</td><td>HT120DX</td><td></td></tr><tr><td>Circulator</td><td>Daihan scientific</td><td>WCR-P12</td><td></td></tr><tr><td>Linear moving stage</td><td>Zaber</td><td>A-LSQ300A-E01-KT07</td><td></td></tr><tr><td>Angle bracket, 90 degrees</td><td>Zaber</td><td>AB90M</td><td>Accessory of the linear moving stage</td></tr><tr><td>PMP forcep, 145 mm</td><td>Vitlab</td><td>67995</td><td>Nonmetallic tweezer</td></tr><tr><td>PTFE beaker, 250 mL</td><td>Cowie</td><td>CW007.25</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Branson</td><td>B2510MTH</td><td></td></tr><tr><td>PCB cutter</td><td>Hozan Tool Industrial</td><td>K-110</td><td></td></tr><tr><td>Nanoimprint device</td><td>Nanonex</td><td>NX-2000</td><td></td></tr><tr><td>Oxygen plasma generator</td><td>Femto Science</td><td>CUTE</td><td></td></tr><tr><td>Low temperature sterilizer</td><td>Lowtem</td><td>Crystal 50</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Panasonic</td><td>MCO-18AC</td><td></td></tr><tr><td>Confoal laser scanning microscope</td><td>Carl Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Scanning electron microscope</td><td>JEOL</td><td>JSM6701</td><td></td></tr><tr><td>Transmission electron microscope</td><td>Hitachi</td><td>H-7500</td><td></td></tr></tbody></table>

fabrication of gradient nanopattern by thermal nanoimprinting technique and screening of the response of human endothelial colony-forming cells

Nanotopography 可以在身体周围的各种细胞外基质 (ECMs) 中找到, 并且已知对细胞反应有重要的调控作用。然而, 由于缺乏适当的筛查工具, 很难确定纳米结构的尺寸与细胞的反应之间的关系。在这里, 我们展示了可重现的和经济高效的梯度 nanopattern 板的开发, 用于操作细胞反应。以阳极氧化铝 (氧化铝) 为主要模具, 采用热印迹技术制备了 nanopillars 直径范围增大的梯度 nanopattern 板 [120-200 nm (gp 120/200)、200-280 nm (gp 200/280) 和 280-360 nm (gp 280/360)]。这些梯度 nanopattern 板的设计, 以模仿不同大小的 nanotopography 在 ECM 和被用来筛选的反应, 人类内皮细胞集落形成单元 (hECFCs)。在本协议中, 我们描述了在细胞工程中制作梯度 nanopattern 板的分步过程, 从人外周血中培养 hECFCs 的技术, 以及在 nanopattern 板上培养 hECFCs。

图 1显示了根据其类型和位置, 制备的梯度氧化铝模具的 SEM 图像。图 2显示了具有正圆 nanopillars 的梯度 nanopattern 板的 SEM 图像,图 3是 nanopillar 直径的量化数据。表 1列出了预制 nanopillars 的特点。
图 4A显示了血液衍生 hECFCs ...

Watch this Scientific Journal Video about Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells at JoVE.com

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

在这里, 我们提出了一个通过热 nanoimprinting 制备梯度 nanopattern 板的协议, 以及筛选人类内皮祖细胞对纳米结构的反应的方法。通过使用所描述的技术, 有可能产生一个脚手架, 可以操纵细胞行为的物理刺激。

热 Nanoimprinting 技术制备梯度 Nanopattern 及其对人血管内皮细胞菌落形成的响应筛选

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

fabrication-gradient-nanopattern-thermal-nanoimprinting-technique

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.9K 次观看.  Korea University. 在这里, 我们提出了一个通过热 nanoimprinting 制备梯度 nanopattern 板的协议, 以及筛选人类内皮祖细胞对纳米结构的反应的方法。通过使用所描述的技术, 有可能产生一个脚手架, 可以操纵细胞行为的物理刺激。

视频: Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

科研

生物学

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

使用 PRIMO 微模式系统对蛋白质的光诱导分子吸附，研究细胞对细胞外基质蛋白的反应

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...