Name: custom engineered tissue culture molds from laser-etched masters
Uploaded: 2018-05-21T13:00:00
Description: Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

作者承认来自 NIH R00 HL115123 和布朗大学工程学院的资助。他们也感谢布朗设计车间和克里斯公牛为训练和支持与激光切割机。

1. 创建矢量格式主模具设计
<ol><li>使用矢量图形程序将所需的模具几何组合成矢量格式 (请参阅材料、设备和软件表)。选择文件 |新建并创建具有 RGB 颜色格式的适当维度画布。使用左侧面板中的形状工具创建所需的几何图形: 在窗口顶部输入所需尺寸 (单击顶部的变换按钮 (如果不是最初可见), 精确定义形状大小。 注: 模具几何应允许至少6毫米的边界之间的最外层的?...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+antifibrinolytic+agents+on+the+life+span+of+fibrin+sealant.">Effects of antifibrinolytic agents on the life span of fibrin sealant.</a> J. Surg. Res. 53, 402-407 (1992).</li><li>Roberts, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Cells+in+Dense+Collagen+Promote+Cardiomyocyte+and+Microvascular+Patterning+in+Engineered+Human+Heart+Tissue.">Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue.</a> Tissue Eng. Part A. 22, 633-644 (2016).</li><li>Ye, K. Y., Sullivan, K. E., Black, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+Cardiomyocytes+in+a+Fibrin+Hydrogel+for+Cardiac+Tissue+Engineering.">Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering.</a> JoVE. , (2011).</li><li>Zimmermann, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+of+a+Differentiated+Cardiac+Muscle+Construct.">Tissue Engineering of a Differentiated Cardiac Muscle Construct.</a> Circ. Res. 90, 223-230 (2002).</li><li>McCain, M. L., Agarwal, A., Nesmith, H. W., Nesmith, A. P., Parker, K. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-Etched+Designs+for+Molding+Hydrogel-Based+Engineered+Tissues.">Laser-Etched Designs for Molding Hydrogel-Based Engineered Tissues.</a> Tissue Eng. Part C Methods. 23, 311-321 (2017).</li><li>Zhang, H., Chiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fouling+Coatings+of+Poly(dimethylsiloxane)+Devices+for+Biological+and+Biomedical+Applications.">Anti-fouling Coatings of Poly(dimethylsiloxane) Devices for Biological and Biomedical Applications.</a> J. Med. Biol. Eng. 35, 143-155 (2015).</li></ol>

定制的与组织培养相适应的注塑模几何图形在调整重要的工程组织性能方面有很大用处, 如细胞对准、扩散速率和有效刚度。此外, 这些模具对于为需要几何的分析应用程序准备组织非常有用, 如机械测试16,17。从激光切割负主模具中制备这些器件提供了一种快速、简便、低成本的利用这些工具的方法, 特别是与传统微细加工的时间和成本相比较。激光切?...

在过去的两年中, 软光刻被广泛地用作一种制造技术来支持科学研究, 特别是在微流体、材料研究和组织工程领域, 1, 2, 3。复制成型, 其中一个对象具有一个理想的形状是由负主模创建, 提供了一个方便和低成本的方法, 生产积极的, 可用于铸造成型水凝胶。但是, 所需的负主模具通常是使用昂贵、耗时、尺寸有限的微细加工技术生产的, 需要干净的房间空间和精良的设备。虽然3D 打印提供了一种潜在的替代方案, 但由于低成本打印机的分辨率限制以及常见的3D 打印机聚合物和可抑制固化的聚硅烷之间的化学作用, 它的实用性受到了一定的限制。
激光切割和蚀刻材料, 如塑料, 木材, 玻璃和金属的切割系统最近变得大大降低成本, 因此更容易为制造研究工具。商用级激光切割机能够制造厘米刻度上的物体, 其最小特性小于25µm, 而且还需要最少的训练、专门知识和使用时间。在微流体设备的制造过程中, 对其进行了激光消融, 但据我们所知, 没有手稿描述了从激光切割负主模具中可以制造毫米和厘米级模的工艺 4.
我们使用这一技术主要是为了改善营养扩散, 细胞对齐, 和机械性能5,6,7, 以控制工程组织的形状。然而, 这种技术的通用性允许在任何领域使用模压水凝胶感兴趣, 如药物输送和材料科学研究8。利用激光切割机, 可对几乎任何几何无悬垂 (这将抑制去除没有一个多部分的模具, 这是超出本手稿的范围), 并适合在激光床的尺寸。

<table border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Item</td>
			<td style="width:167px;"></td>
			<td style="width:169px;"></td>
			<td style="width:141px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630-5G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine thrombin</td>
			<td>Sigma</td>
			<td>T6634-250UN</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine aprotinin</td>
			<td>Sigma</td>
			<td>10820-25MG</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I, 4 mg/mL</td>
			<td>Advanced Biomatrix</td>
			<td>5153-100MG</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodim chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicone elastomer</td>
			<td>Corning</td>
			<td>4019862</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tape</td>
			<td>Fisher</td>
			<td>15-901-5R</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylic, 1/4&#34; thick</td>
			<td>McMaster-Carr</td>
			<td>8560K356</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffer, 1 M</td>
			<td>Sigma</td>
			<td>H3537-100ML</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 medium, powder</td>
			<td>Fisher</td>
			<td>31800-089</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>Fisher</td>
			<td>AC423520250</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate</td>
			<td>Fisher</td>
			<td>M33 500</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate dibasic heptahydrate</td>
			<td>Sigma</td>
			<td>S9390-500G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G5767-25G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Frozen block molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin</td>
			<td>Fisher</td>
			<td>3530 1</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Y</td>
			<td>Fisher</td>
			<td>AC152880250</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast green FCF</td>
			<td>Fisher</td>
			<td>AC410530250</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Illustrator</td>
			<td>Adobe Systems</td>
			<td></td>
			<td>Vector Graphics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inkscape</td>
			<td>(Open Source)</td>
			<td></td>
			<td>Vector Graphics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UCP (Universal Control Panel)</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>Laser Cutter Interface</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PLS6.75 Laser Cutter</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>Laser Cutter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromechanical Analyzer</td>
			<td>Aurora Scientific</td>
			<td>1530A with 5 mN load cell</td>
			<td>Mechanical Analysis</td>
		</tr>
	</tbody>
</table>

custom engineered tissue culture molds from laser-etched masters

随着组织工程领域的不断成熟, 对各种组织参数 (包括组织形态) 的兴趣也越来越大。在千分尺上操作组织形状可以直接对细胞进行排列, 改变有效的机械性能, 并解决与养分扩散有关的限制。此外, 制备组织的容器可以对组织施加机械约束, 导致应力场, 从而进一步影响细胞和基质结构。具有高度可重现性的形状组织还具有用于体外检测的实用程序, 其中样本维度至关重要, 例如整个组织机械分析。
本手稿描述了利用激光蚀刻丙烯酸制备的负主模的替代制造方法: 这些模具与烷的性能良好, 允许设计厘米刻度和特征的尺寸尺寸小于25µm, 可快速设计和制造的低成本和最低的专业知识。最低的时间和成本要求, 使激光蚀刻模具被迅速地重复, 直到一个优化的设计是确定的, 并易于适应, 以适应任何兴趣的检测, 包括那些超出组织工程领域。

激光切割机的光学将导致蚀刻区域有非常轻微减少的尺寸, 因为蚀刻深度增加, 并导致模具墙与一个非常微妙的斜角, 由于激光束的渐狭。这将有助于去除铸造的注塑模, 但应仔细考虑, 如果非常深刻蚀刻负主模具 (> 6 毫米) 是必需的 (图 1)。
随着时间的推移, cellularized 结构将会因基体重塑而紧凑, 尽管这种?...

Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

Custom Engineered Tissue Culture Molds from Laser-etched Masters

在此, 我们提出了一个快速, 简便, 低成本的方法, 以制造定制的烷模具, 可用于生产水凝胶为基础的工程组织复杂的几何。我们还描述了机械和组织学评估的结果, 对使用这种技术生产的工程心脏组织进行了研究。

由激光蚀刻大师定制的组织培养模具

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...