Name: a paired bead and magnet array for molding microwells with variable concave geometries
Uploaded: 2018-01-28T13:00:00
Description: Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

这项研究由科学、ICT 和未来规划部 (NRF-2014R1A1A2057527 和 NRF-2016R1D1A1B03934418) 资助的韩国国家研究基金会 (NRF) 支持。

1. 通过孔阵列铝板和磁铁阵列的制备
<ol><li>准备两个50毫米 x 50 毫米 (或更大) 的铝板。每个板材的厚度为300µm, 是珠粒直径的一半。</li>
	<li>在其中一个铝板上形成一个 30 x 30 通孔阵列, 使用带有Φ550µm 微钻头的 CNC 旋转雕刻机, 其转速为30毫米/秒, 主轴速度为 8000 RPM。每个孔之间的距离 (中心到中心) 是 1 mm (图 1a和图 2a, i)。</li>
	<li>使用与 ...

<ol>
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这种制作方法面临的主要挑战是在铝板的通孔阵列中的珠子的安全固定。为了解决这一难题, 以 30 x 30 磁铁阵列的形式使用磁力来稳固地固定珠子, 如图 6和7所示。磁体阵列的磁通密度与极性相反, 在每个磁体表面的中心最强。因为磁力的强度取决于磁通密度, 所以珠子被引导到每个磁铁的顶面中心, 在那里他们被放置在位置。如果使用一个大尺寸的磁铁 (5 厘米 x 5 cm x 1 厘?...

生长成球状形态的细胞比二维平面区域性1更类似于实体组织。考虑到这一优势, 采用椭球的方法来改善细胞与细胞相互作用的研究2,3, 免疫激活4, 药物筛选5, 和差异性6。此外, 椭球结合多种细胞类型最近已应用于 organoids (近生理三维 (3D) 组织), 这是非常有用的研究人类发展和疾病7。几种方法已被用于生产椭球。最简单的方法是利用非粘合表面, 使细胞聚集在一起, 形成椭球。培养皿可以用牛血清白蛋白、pluronic F-127 或疏水性聚合物 (如聚 2-乙甲基丙烯酸酯) 来处理, 使其表面不粘附8, 9。微调烧瓶方法是另一种众所周知的生产大量椭球10,11的方式。在这种方法中, 细胞通过搅拌来保持悬浮, 以防止它们附着在基体上。相反, 浮动单元格聚合以形成椭球。无粘结面法和微调瓶法均能产生大量的椭球。但是, 它们受到限制, 包括控制球体尺寸的困难, 以及每个球体的跟踪和监测。作为对此类问题的一种补救方法, 另一种球形生产方式, 即悬挂跌落法可以采用12。这包括将细胞悬浮液沉淀在培养皿盖子的下侧。这些下落通常是15到30µL 在大小并且包含大约300到3000个细胞13。当盖子是倒置的, 下落由表面张力举行。每个下落的微重力环境集中细胞, 然后形成唯一椭球在自由的液体空气接口。悬挂下降法的优点是它提供了一个受控的大小分布, 而它很容易跟踪和监测每个球体, 相对于非胶粘剂表面和微调烧瓶的方法。然而, 这种方法在椭球的大量生产和生产过程本身是过度劳动密集型的情况下产生了一个不利因素。
微阵列是一个平板, 有许多微尺寸的井, 每一个直径不等, 从100到1000µm。在使用 microwells 时, 球体的生产原理与非粘性表面方法相似。好处包括 microwells 提供空间之间的 microwells 分离的细胞或椭球, 这样, 它是易于控制的球体大小, 同时也使它易于监测每个单一的球体。随着大量的 microwells, 高吞吐量球体的生产也成为可能。microwells 的另一个优势是根据用户独特的实验目的, 选择不同形状的水井 (六面体、圆柱形、三角形棱柱)。然而, 一般来说, 一个三维 (3D) 凹 (或半球) 形状被认为是最适合生产统一尺寸的单椭球。因此, 凹 microwells 的用处已经报告了许多细胞生物学研究, 如那些检测胚胎干细胞分化的心肌细胞14, 胰岛细胞簇的胰岛素分泌,15,肝细胞酶活性为16, 肿瘤耐药性椭球17。
不幸的是, 制作 microwells 往往需要专门的微设施;传统的光刻方法需要曝光和开发设备, 而反应离子蚀刻的方法需要等离子和离子束设备。这类设备费用高昂, 加上复杂的制作过程, 给无法进入微的生物学家带来了高门槛。为了克服这些问题, 提出了其他成本效益高的方法, 如冰光刻18 (使用冷冻水滴) 和柔性膜法14 (使用膜、通孔衬底和真空)。然而, 这些方法也有严重的缺点, 如难以控制的模式大小, 实现高宽比, 和生产大面积 microwells。
为了克服上述问题, 我们提出了一种新的凹微制造方法, 利用孔基板、钢珠和磁铁阵列。利用这种方法, 利用磁力辅助的自锁金属珠 (图 1) 的机理, 可以制作数以百计的凹球形 microwells。制作过程涉及使用极少数昂贵和复杂的设施, 不需要许多先进的技能。因此, 即使是不熟练的人也很容易承担这种制作方法。为了证明所提出的方法, 在凹 microwells 培养人脂肪干细胞, 产生椭球。

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CNC rotary engraver</td>
			<td style="width:111px;">Roland DGA</td>
			<td style="width:119px;">EGX-350</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro drill bit</td>
			<td style="width:111px;">HAM Pr&#228;zision</td>
			<td style="width:119px;">30-1301 TA</td>
			<td>&#934; 0.55 and 0.75 mm</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sulfuric acid 98%</td>
			<td style="width:111px;">Daejung</td>
			<td style="width:119px;">7683-4100</td>
			<td style="width:167px;">For cleaning aluminum plate. 
			Dilute with distilled water with 15% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neodymium magnet</td>
			<td style="width:111px;">Supermagnete</td>
			<td>W-01-N</td>
			<td>1 x 1 x 1 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bearing ball</td>
			<td style="width:111px;">Agami Modeling</td>
			<td style="width:119px;">SUJ2</td>
			<td>&#934; 600 &#956;m steel bead</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:111px;">Dowcorning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluronic F-127</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">p2443</td>
			<td>Dilute with phosphate buffered saline to 4% (w/v) solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s modified eaggle&#39;s medium (DMEM)</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s phosphate buffered saline (D-PBS)</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adipose-derived mesenchymal stem cells</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">ATCC PCS-500-011</td>
			<td></td>
		</tr>
	</tbody>
</table>

a paired bead and magnet array for molding microwells with variable concave geometries

椭球区域性是理解细胞行为的有用工具, 因为它提供了一个在体内类似的三维环境。各种球体的生产方法, 如无粘结表面, 微调烧瓶, 悬挂滴, 和 microwells 已被用于研究细胞间的相互作用, 免疫活化, 药物筛选, 干细胞分化, 和化生成。在这些方法中, microwells 与三维凹几何已经得到了科学家和工程师的注意, 由于他们的优势, 统一大小的球体生成和轻松的反应, 个别椭球可以监测.尽管提出了使用柔性膜和冰光刻等成本效益高的方法, 但这些技术存在着严重的缺点, 如难以控制模式尺寸、实现较高的长宽比和生产大面积的 microwells。针对这些问题, 提出了一种在不需要复杂的高成本设备的情况下制作凹 microwells 的鲁棒方法。该方法利用了 30 x 30 通孔阵列、几百微米级的钢珠和磁力, 在 3 cm x 3 cm 烷 (microwells) 基底上制造了900个。为了证明我们的方法对细胞生物学应用的适用性, 我们培养脂肪干细胞3天, 成功地生产椭球使用我们的微平台。此外, 我们进行了一个静磁场模拟研究的机制, 即磁力被用来捕获钢珠在通孔。我们认为, 所提出的微制备方法可以应用于许多基于球体的细胞研究, 如药物筛选、组织再生、干细胞分化和肿瘤转移。

通过以下步骤2.1 到 3.7, 成功地制作了凸模和微模式。(图 4)。商业钢珠被困在 30 x 30 通孔阵列。珠子被紧紧地抱着, 珠子和相应的通孔之间没有缝隙 (图 4a)。预制凹微的形状为凹半球形, 直径为600µm, 与钢珠相同 (图 4b)。凹微的横截面 (图 4c) 显示相邻微的距离为1毫米 (中心至中心), 与?...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

本文介绍了一种在不需要复杂的高成本设备的情况下, 制作凹 microwells 的鲁棒方法。采用磁力、钢珠和通孔阵列, 在 3 cm x 3 cm 烷 (microwells) 衬底上形成几百个。

变凹形形状 Microwells 的配对磁珠和磁体阵列

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

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Education

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Physics

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Unit 1

Magnetic Forces and Fields

SCIENTISTS IN ACTION

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

科研

生物工程

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

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 ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...