我们感谢 h. Tsutsui 和 d. 栗原为我们提供了转基因植物, 包括T fournieriRPS5Ap::H2B-tdTomato线和南芥 UBQ10pro::H2B-mClover线分别。这项工作得到了名古屋大学变革性生物分子研究所和日本先进植物科学网的支持。这项工作的财政支助是由日本科技署 (ERATO 项目赠款) 提供的。JPMJER1004 为金斗勋), 为创新领域的科学研究提供资助 (编号JP16H06465 和 JP16H06464 为金斗勋), 和日本促进科学协会 (jsp) 资助为挑战性的探索性研究 (批准26600061为纽约和授予 nos 25650075 和15K14542 为林宏)。

1. 制备 Microdevice 研究生长的花粉管和苔藓 Protonemata
注: 我们使用了无掩模光刻仪器在硅片上制备了硅烷模具。本手稿中省略了有关系统操作的详细信息。使用光掩模的标准光刻技术9 也可用于创建本手稿中所描述的注塑模具。
<ol><li>将11克的预聚合物塑料混合物 (弹性体底座: 固化剂的比例为 10:1) 倒入每4英寸的模具中。</li>
	<li>将步骤1.1 中准备的模具放在真空室中20分钟。</li>
	<li>在非对流式烤箱中固化65摄氏度90分钟后, 将其从模具上剥离, 然后使用活检拳将进入孔的穿孔进入射流通道。 注: 对于花粉管装置, 必须调整孔的大小以反映雌蕊的直径。因此, 这一价值将因物种而异。在这个实验中, 我们打了一个1毫米孔的雌蕊入口和1.5 毫米孔的液体介质油藏。对于青苔 protonemata 装置, 一个4毫米孔被用于样品水库。</li>
	<li>五十年代, 将该层和玻璃底盘 (直径 3.5-5 厘米) 暴露在空气等离子体中。</li>
	<li>在非对流式烤箱中按下玻纤层进入玻璃底盘, 加热65摄氏度30分钟, 完全封闭微流控网络。</li>
</ol>2. Microdevice 根毛的制备
<ol><li>重复步骤 1.1-1.3 使用模具, 为根和根头发 microdevices 准备两个注塑层。 注: 我们在根 microdevice 中使用了一个2毫米的液体介质储层孔。</li>
	<li>在五十年代将两个聚硅烷层暴露在空气等离子体中。</li>
	<li>使用定制的桌面光刻, 在显微镜下组装两层。 注意: 这些微的层间必须面对面。在组装之前, 请确保两个图层上的对齐标记匹配。</li>
	<li>在非对流式烤箱中加热65摄氏度30分钟, 完全封闭微流控网络。</li>
	<li>从所构造的 microdevice 上取下盖板, 将设备放在直径为5厘米的玻璃底部盘上。</li>
</ol>3. 花粉管的体外细胞培养培养基的制备 (蓝猪耳耳)
<ol><li>准备修改后的 Nitsch 介质, 如前所述15和蒸压釜的介质在121°c 为20分钟。 注意: 修饰 Nitsch 的介质的组成是 NH4没有3 (80 毫克/升), 是一个3 (125 毫克/升), Ca (无3)2‧ 4H2O (500 毫克/升), MgSO4‧ 7H2O (125 毫克/升), KH2PO4 (125 毫克/升), MnSO4‧ 4H2O (3 毫克/升), ZnSO4‧ 7H2O (0.5 毫克/升), H3BO3 (10 毫克/升), 丘索4‧ 5H2o (0.025 毫克/升), Na2MoO4‧ 2H2O (0.025 毫克/升),蔗糖 (5万毫克/升), 酪蛋白 (500 毫克/升)。所制备的培养基至少可贮存在4°c 4 月。</li>
	<li>用蒸压去离子水制备 26% (w/v) 聚乙二醇, 用0.3 µm 孔过滤器过滤溶液。 注: 所制备的培养基可储存在4摄氏度, 1 月。</li>
	<li>将步骤3.1 和3.2 中准备的试剂混合在 1:1 (v/v) 的比率中。 注: 此介质应为每次使用而准备新鲜。</li>
</ol>4. 制备根毛的体外细胞培养培养基 (拟南芥)
<ol><li>准备一个解决方案, 0.215% (w/v) Murashige & Skoog 培养基, 0.05% (瓦特/v) MES, 1% (瓦特/v) 蔗糖和 1% (w/v) 琼脂在去离子水。蒸压釜溶液在121°c 为20分钟。</li>
</ol>5. 制备苔藓 Protonemata 的体外细胞培养培养基 (Physcomitrella 专利)
<ol><li>在培养皿中, 在 BCDAT 中等的16中预孵化苔藓 protonemata。 注: BCDAT 介质的组成为 1 mM MgSO4, 10 mM 熟悉3, 45 µM FeSO4, 1.8 毫米的.2PO4 (pH 值调整为6.5 与 KOH), 微量元素解决方案 (0.22 µM 丘索4, 0.19 µM ZnSO4, 10 µM H3BO3、0.1 µM Na2MoO4、2µM MnCl2、0.23 µM CoCl2、0.17 µM 基)、1 mm CaCl2和5毫米二铵 (+) 酒石酸。</li>
	<li>在 microdevice 的 BCDATG 培养基中培养苔藓。 注: BCDATG 培养基为 BCDAT 培养基, 含 0.5% (瓦特/v) 葡萄糖。所制备的培养基可贮存在4°c 1 月。</li>
</ol>6.体外培养 Microdevice 中的T. 耳花粉管
<ol><li>将花粉管 microdevice 在真空室中, 再加20分钟。</li>
	<li>将 microdevice 从真空室中取出, 并使用一个非常精细的微将生长培养基引入雌蕊入口。用同样的介质把剩余的水井填满。等待几分钟, 直到所有的微都是由微内产生的真空填充介质。</li>
	<li>在玻璃底部的盘子里放一些湿纸巾, 以保持盘子里的湿度。</li>
	<li>用解剖针将花粉粒从野生型耳' 蓝色和白色 ' 的花朵转移到其柱头上。 注意: 我们还进行了这项实验的转基因T. 耳' 皇冠紫罗兰 ' 花 ( RPS5Ap::H2B-tdTomato线17), 与花粉管含有荧光标记精子细胞和营养细胞核。</li>
	<li>使用刀片切割授粉方式 (1 厘米长)。</li>
	<li>将剪切样式插入设备的入口, 如图 2所示。</li>
	<li>把盘子盖上, 用胶带封住。</li>
	<li>在黑暗中将设备放置在28摄氏度的孵化器中, 5-6 小时。</li>
</ol>7.体外培养 Microdevice 中的南芥根毛
注: 步骤 7.1-7.9 (除7.3 和 7.5) 应在层流罩中进行。
<ol><li>通过浸泡在无菌液体中 (5% (v/v) 家用漂白剂和 0.02% (v/v) 海卫 X-100), 将种子从南芥哥伦比亚 (Col-0) 和转基因线UBQ10pro::H2B-mClover (含有荧光核标记) 中消毒5分钟。</li>
	<li>用蒸压水彻底冲洗灭菌过的种子。</li>
	<li>把漂洗过的种子在水中存放2天, 在4摄氏度的黑暗中。</li>
	<li>在紫外线照射下 microdevice 消毒。</li>
	<li>将 microdevice 放在真空室中, 再加20分钟。</li>
	<li>使用微将生长介质引入设备的井中。等待几分钟, 直到真空填满所有的微与介质。</li>
	<li>将蒸压湿纸巾放在玻璃底部的盘子里, 以保持盘子里的湿度。</li>
	<li>将被灭菌的种子转移到设备的入口。</li>
	<li>把盘子盖上, 用胶带封住。</li>
	<li>在连续白光下, 将设备垂直放置在22摄氏度的孵化器中。</li>
	<li>检查根头发生长后 4-10 天的孵化。使用倒置显微镜获得明亮的场和荧光图像。</li>
</ol>8.体外在微流控装置中培养P. 专利(苔藓) Protonemata
注: 步骤 8.2-8.6 (除 8.3) 应在层流罩中执行。
<ol><li>Preculture P. 专利应变 Gransden 200418在 BCDAT 培养基上, 用玻璃纸在培养皿中覆盖一周, 连续白光在25摄氏度。</li>
	<li>在 microdevice 的紫外线照射下, 在层流罩中消毒。</li>
	<li>将 microdevice 放在真空室中, 再加20分钟。</li>
	<li>用微将生长介质引入井中。等待几分钟, 直到真空填满所有的微与介质。</li>
	<li>将蒸压水添加到 microdevice 周围的盘子中, 以保持盘子里的湿度。</li>
	<li>将一小块苔藓 protonemata 组织移植到 microdevice 的入口, 并在连续白光下25摄氏度的培养皿中组织。</li>
	<li>检查 2-3 周的孵化后苔藓 protonemata 生长。使用显微镜获得明亮的场图像。</li>
</ol>9.耳花粉管生长的时间推移成像
<ol><li>将 microdevice 放在装有图像采集硬件 (e. g) 和软件的倒置荧光显微镜上。找到 microgap 的位置。 注: 对于图像采集软件, 我们使用了商业上可用的产品 (材料表)。开放源码显微镜软件, 如µManager 也可以在线 (https://micro-manager.org/wiki/Micro-Manager)。我们建议在显微镜上安装显微镜冷凝器, 以获得更高分辨率的图像。</li>
	<li>每十年代使用显微镜图像采集软件捕捉明亮的现场图像。</li>
	<li>观察RPS5Ap::H2B-tdTomato线花粉管中的荧光标记的精子细胞和营养细胞核, 用 561 nm 激光照射试样, 并使用带通光过滤器 (578/105 nm)。 注: 虽然花粉管生长的时间推移图像经常被捕获, 但成像并没有影响生长。但是, 总是建议尽量减少激光强度、曝光时间和时间间隔, 以尽量减少荧光的毒性和漂白。</li>
	<li>调整图像的亮度和对比度, 并使用 ImageJ 软件 (https://imagej.nih.gov/ij/) 准备视频文件。</li>
</ol>

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作者声明他们没有竞争的财政利益。

为了获得上述结果, 必须遵循议定书中的几个关键步骤。首先, 在粘合之前, 必须在足够的时间内用等离子处理该层和玻璃底碟表面。否则, 当尖端生长的细胞穿过 microgaps 时, 该层可能会在本地与玻璃表面分离。根头发和苔藓 protonemata 协议的另一个关键步骤是 microdevice 的灭菌。通常, 根毛和苔藓 protonemata 细胞需要培养在 microdevice 几个星期。在不杀菌设备的情况下, 微生物可能会蓬勃发展, 这可能会...

花粉粒在柱头上发芽后, 每粒谷物都会产生一个花粉管, 将精子细胞带到卵细胞和胚珠中的中心细胞进行双重受精。花粉管拉长的风格, 并最终达到胚珠通过感知多个指导线索沿其方式1。在伸长过程中, 花粉管遇到一系列物理障碍;传送的轨道是充满细胞, 和花粉管必须进入分钟珠孔端打开胚珠达到他们的目标 (图1A) 2.因此, 花粉管必须具有穿透物理障碍物的能力, 同时能耐受周围的压力。根毛发是另一种尖端生长的植物细胞, 它必须经受住环境中的物理障碍, 以填充土壤颗粒 (图 1B) 的形式。
研究了花粉管的各种力学特性, 包括细胞顶端区域的膨压力和刚度, 可以用初始质壁分离方法3、4和细胞力显微镜来测量.CFM)5,6分别。然而, 这些方法本身并没有揭示花粉管是否能够通过其生长路径上的物理屏障拉长。一种替代技术, 允许花粉管伸长率被监测体内是两个光子显微镜7。然而, 用这种方法很难观察到胚珠组织内的单个花粉管的形态学变化。此外, 土壤中的根毛发生长可以用 X 射线计算机断层扫描 (CT) 和磁共振成像 (MRI)8来可视化, 尽管分辨率较低。在这里, 我们提出了一个方法, 可用于获取高分辨率图像的细胞的变形过程, 在常规显微镜。
这里描述的方法的总体目标是可视化尖端生长的植物细胞的伸长能力, 包括花粉管, 根毛和苔藓 protonemata, 在极小的空间。本文介绍的聚甲基 (microdevices) 在光学上具有透明和透气的特点, 可以在显微镜下培养活细胞, 观察其生长行为。通过使用模具的软光刻技术9也可以创建微 ~ 纳米尺度空间。这些特性使我们能够研究在物理密闭环境中尖端生长的植物细胞的伸长能力。
在这项工作中, 我们在微流控装置上构造了1µm 宽间隙 (4 µm), 并研究了花粉管穿透这些人工障碍物的能力, 这些障碍远小于圆柱形花粉管直径 (约8µm)。这个实验平台使我们能够可视化花粉管对 microgaps 的反应, 捕捉响应的时间推移图像, 跟踪细胞的变形过程。我们还开发了 microdevices, 可用于研究根毛和苔藓 protonemata 的穿透能力。已报告了若干 microdevices, 使植物根10、11、12、13和 moss protonemata14在高分辨率下实现可视化。在我们的设备, 一系列根毛发生长通道垂直连接到一个根生长室, 和个别根毛 (约7µm 直径) 被引导到射流通道与1µm 宽的差距。我们还在含有 microgaps 的 microdevice 中培养苔藓 protonemata (直径约20µm), 以检查它们对这些物理屏障的反应。本文提出的微流控方法使我们能够探索各种尖端生长的植物细胞在极小的空间中拉长的能力, 目前尚无任何其他方法可供研究。

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
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			<td height="18" style="height:19px;width:216px;">PDMS</td>
			<td style="width:165px;">Dow Corning Co.</td>
			<td style="width:119px;">Sylgard184</td>
			<td style="width:427px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Murashige &#38; Skoog Medium</td>
			<td style="width:165px;">Wako Pure Chemical</td>
			<td style="width:119px;">392-00591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MES</td>
			<td style="width:165px;">Dojindo</td>
			<td style="width:119px;">345-01625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:165px;">Wako Pure Chemical</td>
			<td style="width:119px;">196-00015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mm glass-bottom dish</td>
			<td style="width:165px;">Matsunami Glass</td>
			<td style="width:119px;">D210402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom dish</td>
			<td style="width:165px;">Iwaki&#160;</td>
			<td style="width:119px;">3971-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:165px;">Feather</td>
			<td style="width:119px;">No.11</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">biopsy punches</td>
			<td style="width:165px;">Harris</td>
			<td style="width:119px;"></td>
			<td>Uni-Core</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading tips</td>
			<td style="width:165px;">Bio-Bik</td>
			<td style="width:119px;">124-R-204</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:119px;">IX83</td>
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		<tr height="21">
			<td height="21" style="height:22px;width:216px;">CSU-W1</td>
			<td style="width:165px;">Yokogawa Electric</td>
			<td style="width:119px;"></td>
			<td>No Catalog number is avairable for this customized microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MetaMorph imaging software</td>
			<td style="width:165px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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development of microfluidic devices to study the elongation capability of tip-growing plant cells in extremely small spaces

在体内, 尖端生长的植物细胞需要克服一系列物理障碍;然而, 研究人员缺乏在这种限制性条件下可视化细胞行为的方法。为了解决这一问题, 我们开发了生长室, 用于尖端生长的植物细胞, 其中含有一系列狭窄的, 微型制造的缺口 (1 µm) 在一个多甲基 (基板) 基质。这种透明材料允许用户在 microgap 穿透过程中通过时间推移成像监测单个细胞中的尖端伸长率。利用这个实验平台, 我们观察了花粉管的形态学变化, 因为它们穿透了 microgap。在这个过程中, 我们捕获了一个荧光标记的营养细胞核和精子细胞的形状的动态变化。此外, 我们展示了根毛和苔藓 protonemata 的能力, 以穿透1µm 的差距。此体外平台可用于研究单个单元格对物理受限空间的响应方式, 并可提供对尖端生长机制的洞察力。

如图 1所示, 尖端生长的植物细胞在其生长路径体内中遇到一系列物理屏障。本研究提出的微流控体外细胞培养平台使三种植物细胞 (花粉管、根毛和苔藓 protonemata) 的尖端生长过程能够通过1µm 人工间隙进行检查 (图 3,图 4,图 5)。对于花粉管的研究, 用活细胞成像监测花?...

Watch this Scientific Journal Video about Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces at JoVE.com

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

我们描述了一种方法来调查尖端生长的植物细胞, 包括花粉管, 根毛和苔藓 protonemata, 以拉长通过极窄的差距 (约1µm) 在微流控装置。

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

development-microfluidic-devices-to-study-elongation-capability-tip

Research

JoVE Journal

Bioengineering

7.3K 次观看.  Nagoya University. 我们描述了一种方法来调查尖端生长的植物细胞, 包括花粉管, 根毛和苔藓 protonemata, 以拉长通过极窄的差距 (约1µm) 在微流控装置。

视频: Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...