打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养
Summary
àbioprinter被用于创建图案的水凝胶的基础上牺牲的模具。泊洛沙姆模具与第二水凝胶回填，然后洗脱，留下空隙充满了三分之一的水凝胶。此方法使用快速的洗脱和良好的印刷泊洛沙姆产生复杂的生物大分子结构。
Abstract
生物印刷是一项新兴技术，在快速原型制造行业有它的起源。不同的印刷工艺可分为接触生物印刷1-4（挤出，蘸笔，软光刻技术），非接触式生物印刷5-7（激光的正向转 移，喷墨沉积）和基于激光的技术，例如双光子光聚合8。它可用于许多应用，如组织工程9-13，14-16生物传感器的微细加工作为一种工具，回答基本的生物学问题，如影响共培养不同的细胞类型17。与常见的光刻或软光刻的方法不同的是，挤出生物印刷具有的优点在于，它不要求一个单独的掩模或印戳。使用CAD软件，可以快速地改变结构的设计和调整，根据运营商的要求。这使得生物印刷更灵活，比基于光刻的做法。
在这里，我们展示了印刷的牺牲模具来产生多材料的三维结构内的水凝胶，例如，使用一个数组支柱。这些支柱可以代表一个血管网或管内神经导向导管的空心结构。所选择的材料的牺牲模具是泊洛沙姆407，温度敏感型聚合物具有优异的印刷性能，这是液体在4℃，固体其胶凝温度以上〜20℃时为24.5％w / v的溶液18。这个属性允许基于泊洛沙姆牺牲模具洗脱需求在缓慢溶解的固体材料，尤其是狭窄的几何形状具有优势。泊洛沙姆印在显微镜载玻片创建牺牲模具。琼脂糖移液到模具中并冷却，直至凝胶化。泊洛沙姆在冰冷的水洗脱后，在琼脂糖模具的空隙被填充与藻酸盐丙烯酸甲酯藻IKED FITC标记的纤维蛋白原。填充空隙，然后交与UV落射荧光显微镜成像和结构。
Introduction
组织工程方法已经在过去几年取得了很大的进步，对于人体组织和器官的再生19,20。然而，到现在为止，已组织工程的焦点往往是有限的组织，具有简单的结构，或小的尺寸，如膀胱21,22或皮肤23-25。人的身体，但是，包含许多复杂的三维的组织，细胞和细胞外基质被布置在空间定义的方式。为了制造这些组织中，这种技术是必需的，可以放置在指定位置上的一个三维的结构内的细胞和细胞外基质的脚手架。生物印刷有潜力成为这样一种技术，制造复杂的三维组织的愿景，可以实现10,11,26-28。
生物印刷被定义为“材料转让过程使用的图案和组装生物相对埃文特材料-分子，细胞，组织，和可生物降解的生物材料-规定的组织来完成一个或多个生物功能“，它包含了几种不同的技术，工作在不同的分辨率和长度尺度，从亚微米分辨率的两个光子聚合29分辨率为150微米至420微米挤压印刷1,12,30。不是单一材料或材料组合将满足要求每个方法31。挤出印刷，关键参数是粘度和凝胶时间32，高粘度和快速凝胶化是可取的。
3D打印是一种技术，它允许创建复杂的几何形状30,33,34牺牲模具很容易的创建。这个过程是基于模具的建设，利用快速原型技术，如挤压bioprinter。使用创建的牺牲模具难以印刷由于其低的粘度和凝胶时间慢的材料，这些材料以形成复杂的结构。这里介绍的方法涉及到创建一个牺牲模具的材料组成的，在低温下迅速溶解，并可以准确地被挤压。的嵌段共聚物聚（乙二醇）99-聚（丙二醇）67-聚（乙二醇）99（也被称为的Pluronic F127或泊洛沙姆407）满足这些要求。它已经被用于在挤压印刷1中的修改后的版本，但据我们所知，从未被使用未修改的版本，由于它的不稳定性，在液体环境中的打印。泊洛沙姆407还示出了逆热响应行为18 即它从凝胶在冷却时的溶胶。最重要的是，它可以打印任意弯曲成复杂的结构具有非常高的保真度。这允许创建一个结构化的水凝胶从低粘度的材料，在这种情况下慢胶凝琼脂糖，该溶液通过移液到印刷的牺牲模具。具有高的保真度和快速洗脱从铸造的结构化的水凝胶，使得它快速和灵活的方法来创建不同的几何形状的模具，无需使用的平版印刷方法，因为它往往是必需的掩模或邮票打印的牺牲模具的组合。铸造后的结构化的水凝胶，可以进一步与另一种材料是不适合用于挤出印刷由于其低粘度填充。这是在我们的情况下，一个低粘度海藻酸钠丙烯酸甲酯的解决方案。在这里，我们提出的方法反向温敏凝胶图案牺牲模具使用支柱数组的例子。
Protocol
1。下游的泊洛沙姆407的解决方案如果可用，泊洛沙姆溶液进行准备在寒冷的房间（4℃）。如果没有可用的，一个玻璃瓶放置在烧杯中，用冰冷的水填充。在较高的温度下，将泊洛沙姆凝胶点以上，因此无法正确地溶解。 加入60毫升冰冷的PBS溶液倒入玻璃瓶中，并用磁力搅拌器剧烈搅拌。 称取24.5克泊洛沙姆，并将其添加少量的冷PBS。等待，直到泊洛沙姆部分溶…
Representative Results
代表性的结果表明的倒模技术（ 图2中所示）将创建结构化的第二材料，可填充有凝胶。在每次印刷过程中的开头的印刷参数首先优化。分步进行调整参数将导致在描绘在图3和图4单行打印时打印的多层构造。如果层厚度（后一个印刷层的针阀升程）过低时，人们会发现，针触摸前述层。如果针太高，出现波纹的表面上印刷的构造，将出现。此可以看出，在<st…
Discussion
这里，我们提出，在第一次，使用牺牲模，可在冷水中迅速溶出，由于约20℃的泊洛沙姆凝胶 – 溶胶转变温敏聚合物快速创建生物大分子结构不能有足够的分辨率打印速度的全过程，使泊洛沙姆有趣。这里描述的技术可以用于图案形成的水凝胶，水凝胶或在另一个先前已报告的其他材料35的微流体通道的创建。泊洛沙姆作为牺牲模具的优点是，它可以被印在任意的几何形状为固体层由层构?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢德博拉·STUDER的帮助与bioprinter。
这项工作是由欧盟第七框架计划（FP7/2007-2013）根据赠款协议Ñ°NMP4-SL-2009-229292。
Materials
| REAGENTS | |||
| Poloxamer (Pluronic F127) | Sigma | P2443 | |
| PBS | Invitrogen | 10010-015 | |
| CAD software | regenHU | BioCAD | |
| Alginate methacrylate | Innovent e.V Technologieentwicklung Jena | Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292 | |
| Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate | Invitrogen | F13191 | |
| Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) | Innovent e.V Technologieentwicklung Jena | Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292 | |
| Agarose | Lonza | 50004 | |
| EQUIPMENT | |||
| Bioprinter | regenHU | Biofactory | |
| Valve | regenHU | 300 μm Nozzel Diameter | |
| Needle | regenHU | 150 μm Inner Diameter | |
| Zeiss Axioobserver with ApoTome | Zeiss | ||
| UV Light Source | UVP | Blak-Ray B-100AP High Intensity UV Lamp | 100 W |
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Citazione di questo articolo
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