Name: viability of bioprinted cellular constructs using a three dispenser cartesian printer
Uploaded: 2015-09-22T16:00:00
Description: Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

这项工作是由下批准号：EPS-0903795荣获由美国国家科学基金会，美国国立卫生研究院NIDCR R01-DE019355（MJY PI），和格兰特8P20 GM103444（YM PI）政府的支持来支持的。

1.制备明胶含有底物的藻酸盐水凝胶的三维生物印刷<ol><li>准备由Pataky 等 11描述，以避免具有高的内容相关联的存活率下降了钙/明胶底物法以下钙/明胶衬底。钙/明胶底物法如下所示。 <ol><li>结合氯化钙脱水物（1.5重量％），氯化钠（0.9重量％），和猪明胶（2重量％）在蒸馏水和煮沸2分钟，以创建一个100mM的明胶溶液。 </li></ol></li><li>倒入5毫升明胶/钙溶液的入100毫米标准培养皿?...

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T., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+&#8216;Multi-arm+Bioprinter&#8217;+for+Hybrid+Fabrication+of+Tissue+Engineering+Constructs.">Development of a &#8216;Multi-arm Bioprinter&#8217; for Hybrid Fabrication of Tissue Engineering Constructs.</a> Robotics and Computer&#8211;Integrated Manufacturing. 30 (3), 295-304 (2014).</li><li>Kolesky, D. B., Truby, R. L., Gladman, A. S., Busbee, T. A., Homan, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Bioprinting+of+Vascularized,+Heterogeneous+Cell&#8211;Laden+Tissue+Constructs.+Adv+Mater.+X.">Three-Dimensional Bioprinting of Vascularized, Heterogeneous Cell&#8211;Laden Tissue Constructs. Adv Mater. X.</a> Adv Mater. X, x-y (2014).</li></ol>

组织工程的主要重点是通过开发能够恢复，维持或提高当地组织函数的生物替代品来弥补器官短缺和移植需求之间的差距。这导致了支架的具有复杂，解剖学上正确的外部几何形状，而在它的内geometr精确控制的直接制造。三维生物印刷是用于使用层 - 层approac从数字模型生成各种尺寸和形状的三维结构的方法。三维仿生构建体的制造起着组织工程的进步至关重要的作用。 有设...

组织工程使用在功能替代品的开发生物和工程的原理来维持，恢复或提高天然组织和。产生按需三维仿生结构将有助于在组织工程中以及在基于细胞的传感器，药物/毒性筛选，组织或肿瘤模型和其他科学和技术进步的能力。三维组织工程构建的组织是制造方法的一个基本组成部分，因为它必须密切模仿细胞外基质的天然组织的高度组织的相互作用。 可生物降解的和成形的三维支架是在产生新的组织构建因为细胞迁移以形成一个二维的细胞层的关键因素，但缺乏在偏爱三维生长的能力。该支架用作小区的临时基础附着和增殖，因此它必须从材料具有可控孔隙率和生物降解性，和足够的机械IntegrIT公司构成。该支架材料不应该是细胞毒性或创建从主机有不良反应。水凝胶已被普遍使用在组织工程技术，并且由于它们的亲水性，水凝胶允许整个所有制结构流体和气体交换。通过结合不同的水凝胶，合成水凝胶的属性均可修改，以满足不同的应用需求。 传统的组织工程方法涉及创建所用细胞接种后fabricatio脱细胞多孔牺牲支架。许多技术已经被采用，例如纤维粘结，溶剂浇铸和熔融模塑，但被证明是用于组织工程应用最低限度地成功。纤维粘合方法允许纤维在特定的形状对齐，但它们仅能够促ducing非常薄的支架。溶剂浇铸方法产生高度多孔结构，然而最大产生膜仅为3毫米THIC。因此，使用这些技术制造的三维结构是不可行的。熔融模制技术生产三维支架被证明是成功的，但需要这样的高温下不能在生产PROCES掺入生物材料。支架接种后加工在其满足组织工程学的要求的能力，产生三维支架与预定义或控制的微观结构和有限。凭借雄厚的脚手架播种技术的另一个主要问题是血管和机械差的不足。 生物印刷已经被通过使用无毒的，可生物降解，​​热可逆的凝胶，以克服常规的缺陷扩展到三维。少数的实体自由成形制造牛逼目前所采用的echniques是激光辅助生物印刷和喷墨印刷。激光辅助生物印刷技术使用一个脉冲激光源，一个目标板，以及一个接收衬底，以产生立体感。然而，这种技术是由于低的吞吐量，低细胞存活率限定，只能生产制造的结构的有限的安排，因为只有光交预聚物可用于形成交联的水凝胶。喷墨印刷是作为其通过沉积皮升墨再现的基板上的数字图像数据的非接触方法。然而，喷墨印刷不产生高分辨率构建体，构建体经历快速蛋白质变性，和许多细胞的沉积期间被裂解。 目前，新的添加剂制造生物印刷方法已经被开发出来。在这些系统中的细胞，蛋白质，生长因子，和仿生水凝胶典型地集成到矩阵脑膜IALS在制造过程中，用计算机控制的执行器来生成三维支架基细胞载货结构紧密模仿原生的微架构同时沉积。细胞载货水凝胶构成了生物油墨，可以是异质的，由多种细胞类型，或同质的。添加剂制造系统存款生物油墨滴逐滴或层 - 层经由一次性注射器和提示到计算机控制的阶段能够移动在x，y和z方向上的。通过计算机软件，印刷支架的结构可以很容易地根据应用的要求进行操作。不像常规技术，三维医疗技术（核磁共振成像，计算机断层扫描）可以被并入到设计中，生成患者特异性构建体。这些方法也允许制备血管替换的可能性，因为构建体产生具有较高升OCAL细胞密度，从而使细胞 - 细胞相互作用和改善植入后surviva的可能性。 帕尔梅托打印机是使用可编程的机器人的制造方法，以生成三维多相组织构建（图1）一个定制三维多分配器系统。它允许使用多种材料中的独特的组合，以产生异质结构。在生物印刷器的初始化是生物印刷中，因为它允许您设置各种参数，以优化bioprinted结构的印刷适性最重要的步骤之一。 的生物印刷器包括间歇式过程由可编程逻辑控制器（PLC），该用户操作通过交互的触摸屏控制面板上（图1中的A）受控启动，运行和关闭序列。为了防止生物污染的生物印刷器被封闭在带正压力有机玻璃逻辑材料（PMMA）室与高效微粒计重（HEPA）-filtered空气循环系统（图1，B，C）。打印机的内部可以使用内置的紫外线光源（图1 中的D）进行消毒。的生物印刷器的主要组成部分是一个完全可编程的定位机器人，可以再现地放置一个分配端具有10微米（图1 的E）的精度。有三个分配器，其能够沉积体积低至使用旋转螺杆230 NL（图1 中的F）。它们是独立可编程使用支配印刷参数为每个分配器（图1，G）的 ​​单独的计算机。旋转螺杆式分配利用一个马达驱动螺杆旋转生物油墨向下注射器和出注射器尖端的移动。这些分配器被安装在一个气动LY控制工具巢（图2A，B）中 ，使机器人以切换分配器安装到程序控制（图1 中的H）下的Z轴机械臂。 XYZ表机器人接收从计算机上运行设计软件打印指示（ 图1，I）。每个程序都包含分配位置，校准程序，和饮水机变化的协议。产生构建体的设计主要是由XYZ坐标，其中每个分配器将沉积材料。的生物印刷器包括两个光学光传感器（图2C），该确定注射器前端的XYZ坐标。这些传感器发送坐标信息的机器人，它利用这些来计算分配端端部的位置。有一个附加的位移激光器（图2D），该凸出点尺寸的633纳米二极管红色激光束30×100微米，测量与ACCURA距离CY 0.1微米。当光束高度集中在机器人确定打印表面的Z方向的距离。该测量中，与前端部Z中的光学光传感器测量，允许精确ż计算坐标用来放置分配器触头相对于印刷表面。分配器尖端横向移动和垂直地通过X轴导向光学光传感器找到Y和Z的中心，并且横向穿过一个Y轴传感器来找到X轴的中心。斧+用+ CZ = d，来确定所述表面是相对于分配头端的位置：印刷面使用以下公式中的xyz空间中的平面映射。打印机阶段（图1，J），持有样品培养皿达80毫米的直径，并且使用循环水浴，以保持设定的温度（图1，K）。阶段温度可以在范围-20内设定并保持在稳定的。有一个USB摄像头安装到机器人的Z-臂，以提供分配尖端的打印过程（图1 中，L）的放大图。有一个第二照相机安装朝向腔室内部提供在印刷过程中（图1，L）上的生物印刷器的完整视图的顶部。 甲计算机辅助设计绘图软件确定沉积图案，并允许用户生成增量间隔开的液滴和复杂的结构（图3）。三维途径可以手动编码到打印机兼容的设计软件或从单独的计算机辅助设计绘图软件（图4， 表1）导入。打印机兼容的软件允许的印刷参数的变化，例如在注射器前端和实名词之间的沉积方法（单个液滴沉积或连续通路沉积），三维的通路的几何结构，沉积速率，距离率打印表面，以沉淀单个下降，并且高度和速度的注射器时间量被抬起的液滴的沉积之间。每个程序都包含XYZ点胶的位置，提示校准程序，和饮水机变化的协议来提供一个无菌的环境，无需操作员干预，在打印过程中。机器人的可编程逻辑控制器（PLC）接收运行设计软件从计算机指令，并控制事件的来自外部控制器的时序（例如，分配器）。为了做到这一点，PLC使用一个循环机制来控制分配器，机器人定位装置，和环境因素。 利用旋转式螺杆，液体配送系统的立体直接写入生物印刷允许更高效，准确，并比以前的方法更简单的细胞沉积的过程。这项研究显示了定制的生物印刷器能够产生行政长官LL-载货水凝胶结构具有较高的细胞活力。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Positioning Robot (JR2000 XYZ)</td>
			<td>Janome&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensers: SDAV Linear Drive SmartDispensers</td>
			<td>Fishman Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Light Sensors:&#160;</td>
			<td>Keyensce</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Displacement Laser: OD Mini</td>
			<td>SICK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recirculating Water Bath: Polystat</td>
			<td>Cole-Parmer</td>
			<td>EW-12122-02</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cameras: Dino-Lite Premier 5MP</td>
			<td>AnMo Electrionics/YSC Technologies</td>
			<td>AD7013MT</td>
			<td></td>
		</tr>
		<tr>
			<td>Printer-Compatible Computer Design Software: JR-C Points</td>
			<td>Janome</td>
			<td></td>
			<td>Comes with purchase of Janome Robot</td>
		</tr>
		<tr>
			<td>Computer-Aided Design Drawing Software: Visual PathBuilder</td>
			<td>RatioServ</td>
			<td></td>
			<td>Can be downloaded at: www.ratioserv.com/index.php/downloads</td>
		</tr>
		<tr>
			<td>Printer 3 cc Syringes:&#160;</td>
			<td>Fishman Corporation</td>
			<td>122051</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Dispenser Tips</td>
			<td>Fishman Corporation</td>
			<td>Z520122&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Dioxide</td>
			<td>Sigma-Aldrich</td>
			<td>13462-67-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Protanal LF 20/40 Alginate (Sodium Alginate)</td>
			<td>FMC BioPolymer</td>
			<td>9005-38-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Mallinckrodt Baker, Inc</td>
			<td>9300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Periodate</td>
			<td>Sigma-Aldrich</td>
			<td>7790-28-5</td>
			<td></td>
		</tr>
		<tr>
			<td>hADSC</td>
			<td>Lonza</td>
			<td>PT-5006</td>
			<td>Store in vials in liquid nitrogen until use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Gibco Life Technologies</td>
			<td>11965-092</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Gibco Life Technologies</td>
			<td>C3100MP</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>Live/Dead Mammalian Viability Assay Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>L-3224</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>MES Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-(dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>E1769&#160;</td>
			<td>10 G</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, +Calcium, +Magnesium</td>
			<td>Life Technologies</td>
			<td>14040133</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, -Calcium, -Magnesium</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>RGD Peptides</td>
			<td>International Peptides</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin Stain</td>
			<td>Invitrogen Life Technologies</td>
			<td>A22283</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
		<tr>
			<td>(4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) Stain</td>
			<td>Life Technologies</td>
			<td>R37606</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
	</tbody>
</table>

viability of bioprinted cellular constructs using a three dispenser cartesian printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

结果证实了生物印刷器能够精确地沉积在特定的三维位置细胞载货水凝胶和始终如一地利用计算机辅助软件。这些软件决定了放置每个液滴并控制许多参数的分配（ 图3,4）。的生物印刷器的重复性适当沉积的生物材料是根本其在组织工程应用的成功。 细胞活力，一个成功的技术，生物印刷的要求之一，分析1小时和8天后打印。高细胞活力是用于制造仿生构建必要...

Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Research

JoVE Journal

Bioengineering

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

分层海藻酸钠构造：一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Imaging Cell Viability on Non-transparent Scaffolds ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â Using the Example of a Novel Knitted Titanium Implant

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

用运动调节血清治疗韧带结构：转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

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 ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

设计开源、低成本生物墨水和食品熔炼挤出3D打印机

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...