Name: cardiac muscle cell-based actuator and self-stabilizing biorobot - part 2
Uploaded: 2017-05-09T15:00:00
Description: Watch this Scientific Journal Video about Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2 at JoVE.com

MT Holley由路易斯安那州理事会研究生研究员计划支持，C. Danielson由霍华德·休斯医学院教授课程支持。本研究得到NSF批准号：1530884的支持。

这里描述的所有程序都是使用经批准的方案并按照圣母大学机构动物护理和使用委员会的规定进行的。 细胞播种与培养<ol><li>在开始之前，准备所需的物品：一个小漏斗，移液管和补充有10％胎牛血清（FBS）和1％青霉素抗生素（DMEM完成）的Dulbecco改良Eagle培养基（DMEM）。 </li><li>将T-25烧瓶与其内的功能化装置（生物执行器或biorobot）一起使用。有关细胞播种前的器械制备，功...

<ol>
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这里概述的步骤描述了一种成功的基于PDMS的致动器和双子座的种子方法，其有助于心肌细胞的附着。此外，描述了表征单元的行为和设备的性能的图像采集和后续分析的过程。 24小时后观察到悬臂的细胞自发收缩;收缩强度随着时间的推移不断增加，在第6天达到最大值，其后强度缓慢下降。尽管生物致动器的悬臂只有4毫米长，但观察到高达2.5毫米的大偏转，特别是在培养6天...

双子座是基于活细胞的装置，其结合在通常由软弹性材料如PDMS或水凝胶1组成的机械骨架内。细胞会自发地或响应于刺激而进行节律性收缩，从而起到致动器的作用。从细胞收缩产生的力量驱动各种biorobots。由于其收缩性质，哺乳动物心脏细胞（心肌细胞）和骨骼肌细胞通常用于生物手术致动。除了心肌细胞和骨骼肌细胞外，还使用了其他细胞类型，例如昆虫肌肉组织2和外植体肌肉组织3 。昆虫肌肉组织使得能够在室温下操作生物致动器。 biorobot的功能和性能主要取决于生物致动器的强度和一致性（ 即，。肌肉细胞），而机械骨架结构主要决定运动，稳定性和功率的机制。由于这些装置仅由电池产生的力驱动，所以没有化学污染物或操作噪声。因此，它们与其他常规机器人形成了节能替代品。各种文献来源讨论了将活细胞和组织整合到biorobots 1,4,5中的不同方法。微型加工和组织工程技术的进步使得能够开发可以步行，抓地力，游泳或泵送的5号飞机的6,6。通常，将细胞作为汇合的细胞片材直接培养到机械（聚合物）主链上，或者它们被模制成支架例如环和条带中的3维致动结构。最常见的是biorobots使用心肌细胞片6,7制造 ，因为这些细胞具有在没有外部刺激的情况下表现出自发收缩的先天能力。另一方面，关于骨骼肌细胞片的报道是有限的，因为它们需要刺激以在体外引发收缩以引发膜去极化8 。 该协议首先描述了如何将心肌细胞种植在由薄PDMS悬臂制成的功能化生物致动器上。然后详细描述游泳剖面的种子和分析。悬臂用细胞粘附蛋白如纤连蛋白进行功能化，并与心肌细胞融合。随着细胞在装置上的接合，它们使悬臂弯曲，从而作为致动器。随着时间的推移，随着细胞的成熟，我们通过分析视频来跟踪设备表面应力的变化悬臂弯曲。这里开发的生物致动器可用于确定任何细胞类型的收缩性质，例如成纤维细胞或诱导的多能干细胞，因为它们经历分化。 早期的Biorobots研究一直集中在开发生物执行器上，而Biorobot架构和功能能力的优化在很大程度上被忽视。最近有一些研究表明，在自然界受到启发的Biorobots中实施游泳模式。例如，已经设计了具有基于鞭毛的运动6 ，水母推进9和生物混合射线4的游泳芭比娃娃。与文学中的其他作品不同，这里我们着重于改变机械骨架的性质，以创建一个自稳定结构。在这项研究中开发的biorobot能够保持恒定的音调，滚动和静音游泳深度，游泳。可以通过改变每个基础复合材料的厚度来修改这些参数。在开发PDMS执行器，潜水手电筒和设备功能化过程中涉及的制造步骤在本文的两部分文章的第1部分以及我们最近的工作中进行了详细描述7。这里开发的技术可以铺设开发用于各种应用的新颖，高效的Biorobots的方式，例如货物交付。 本研究中遵循的隔离过程类似于早期工作10中描述的过程，以及最近发表的工作。用于制造PDMS执行器和双子座装置的微加工方法在这两部分手稿的第1部分中有详细描述。该手稿的协议部分描述了将心肌细胞接种到制造的PDMS上的步骤a在使用细胞粘附蛋白进行功能化之后，它们可以用于制造和制造。

<table>
	<tbody>
		<tr>
			<td>Chemicals and reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiomyocytes (primary cardiac cells)</td>
			<td>Charles River</td>
			<td>NA</td>
			<td>Isolated from 2-day old neonatal Sprague Dawley rats</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle&#8217;s media (DMEM)</td>
			<td>Hyclone Laboratories</td>
			<td>16750-074</td>
			<td>with 4500 mg/L glucose, 4.0 mM L-glutamine, and 110 mg/L sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetalclone III serum</td>
			<td>Hyclone industries, GE</td>
			<td>16777-240</td>
			<td>Fetal bovin serum (FBS)</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffer (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole dihydrocholride powder (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Solution (1 mg/ml)</td>
		</tr>
		<tr>
			<td>Calcein-AM and ethidium homodimer-1 kit (Live/Dead Assay)</td>
			<td>Molecular Probes</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Fluo-4, AM</td>
			<td>Molecular Probes</td>
			<td>F14217</td>
			<td>calcium indicator dye</td>
		</tr>
		<tr>
			<td>Tyrodes salt solution</td>
			<td>Sigma-Aldrich</td>
			<td>T2397</td>
			<td>buffer solution</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Molecular Probes</td>
			<td>P3000MP</td>
			<td>nonionic surfactant-20 % solution in Dimethylsiloxane (DMSO)</td>
		</tr>
		<tr>
			<td>16% Parafomaldehyde</td>
			<td>Electron microscopy</td>
			<td>15710</td>
			<td>Caution: Irritant and combustible</td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100 100 mL</td>
			<td>cell lyses detergent, (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether)</td>
		</tr>
		<tr>
			<td>ProLong gold antifade reagent</td>
			<td>Molecular Probes</td>
			<td>P10144</td>
			<td>Mounting agent</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Phalloidin</td>
			<td>Molecular Probes</td>
			<td>A12381</td>
			<td>Actin filament marker</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 594 conjugate</td>
			<td>Molecular Probes</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>pha</td>
			<td>Molecular Probes</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-connexin 43 antibody</td>
			<td>Abcam</td>
			<td>ab11370</td>
			<td>Gap junction marker</td>
		</tr>
		<tr>
			<td>Anti-cardiac troponin I antibody</td>
			<td>Abcam</td>
			<td>ab10231</td>
			<td>Contractile protein</td>
		</tr>
		<tr>
			<td>16% EM grade paraformaldehyde solution</td>
			<td>Electron microscopy</td>
			<td>100503-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Elsevier</td>
			<td></td>
			<td>Sylgard 184</td>
		</tr>
		<tr>
			<td>Materials and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Thor Labs</td>
			<td>DCC1545M</td>
			<td></td>
		</tr>
		<tr>
			<td>LED light strip</td>
			<td>NA</td>
			<td>NA</td>
			<td>Any white LED without spectrum emission</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikkon C2</td>
			<td>NA</td>
			<td>Confocal microscope with three filter set.</td>
		</tr>
		<tr>
			<td>Zooming lens</td>
			<td>Infinity</td>
			<td>Model# 252120</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>NA</td>
			<td>Used in Section 4) for biological actuator analysis.</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>National Institute of Health</td>
			<td>NA</td>
			<td>Java-based image processing software. Used in Section 5) for biorobot analysis. 
			Free Image Processing and Analysis software in java. (https://imagej.nih.gov/ij/)</td>
		</tr>
		<tr>
			<td>Thor Cam</td>
			<td>Thor Labs</td>
			<td>NA</td>
			<td>Camera operating software</td>
		</tr>
	</tbody>
</table>

cardiac muscle cell-based actuator and self-stabilizing biorobot - part 2

近年来，已经开发了由与合成机械骨架集成的活细胞或组织组分组成的混合装置。被称为双子座的这些装置仅由活体部件的收缩活动所产生的力量驱动，并且由于其许多固有的优点，可以替代传统的全人造机器人。在这里，我们将描述在这篇两部分文章的第一部分中设计，制造和功能化的生物致动器和biorobot的种子和特征的方法。由聚二甲基硅氧烷（PDMS）和薄膜悬臂组成的制造的生物致动器和双穴装置功能化用于纤连蛋白的细胞附着。在功能化之后，将新生大鼠心肌细胞以高密度接种到PDMS悬臂上，产生汇合的细胞片。这些装置每天都被成像，并且运动着分析杠杆臂。在播种后第二天，我们观察到由于在自发收缩期间由细胞施加的力，悬臂的弯曲。通过对悬臂弯曲的定量分析，观察到细胞随着时间的推移逐渐增加，细胞表面应力逐渐增长。同样，我们观察到由于作为翅片的PDMS悬臂的致动而引起的Biorobot的运动。在对装置的游泳轮廓进行定量时，观察到各种推进模式，其受到翅片的静止角度的影响。运动方向和跳动频率也由翅片的静止角确定，观察到最大游泳速度为142μm/ s。在这份手稿中，我们描述了使用心肌细胞填充制造的装置的过程，以及生物致动器和biorobot活动的评估。

由薄PDMS悬臂（25μm厚）和心肌细胞构成的生物致动器构成游泳制冷机的核心， 如图1所示装置的示意图和截图所示 。细胞在培养24小时后开始出现收缩，第2天观察到悬臂的弯曲。每天记录装置的侧面轮廓，并且使用以下方法从悬臂的弯曲量化表面应力定制图像分析脚本7 。静态和动态应力每天从表面应力中提取（ <str...

Watch this Scientific Journal Video about Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2 at JoVE.com

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

在这项研究中，生物致动器和具有功能化的弹性悬臂的自稳定游泳混合动物用心肌细胞接种，培养并表征其随时间的生物化学和生物力学性质。

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生物工程

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

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

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 ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

心肌肌细胞基致动器和自稳定Biorobot  - 第1部分

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

为高浓度单分子显微镜制造零模式波导

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

评估人体骨骼肌微组织骨骼肌健康的功能指标

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...