Name: bioprinting cellularized constructs using a tissue-specific hydrogel bioink
Uploaded: 2016-04-21T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink at JoVE.com

作者非常感谢由空间和海战系统中心太平洋（SSC PACIFIC）的合同号N6601-13-C-2027下的国防威胁降低局（DTRA）的资金。这种材料的出版并不构成此结果或结论的政府批准。

1.水凝胶生物油墨配方及制备方法<ol><li>为了提供组织特异性生化型材，制备如前对肝所述组织特异性的ECM消化的解决方案。20 注意:一般来说，此ECM摘要将包括被使用的最终水凝胶生物油墨体积的40％。的ECM的消化溶液数百毫升可以制备，等分，并在-80℃下以供将来使用冷冻。 </li><li>到水凝胶制剂，溶解光引发剂，2-羟基-4&#39;前 - 在0.1％的（2-羟基乙氧基）-2-甲基苯丙酮，水重量/体积。 注意:卷在50-100毫升范围可提前制备，并在4℃避光保存屏蔽几个月。 </li><li>为了形成水凝胶bioinks，首先溶解在水中，光引发剂溶液的透明质酸（HA）的水凝胶的试剂盒的基材组件。 <ol><li>分别溶解巯基HA和硫醇盐明胶在水净化hotoinitiator溶液（步骤1.2），以使2％w / v的溶液。 </li><li>溶解聚乙二醇二丙烯酸酯（PEGDA），在水凝胶的试剂盒的交联剂，在水 - 光引发剂溶液（步骤1.2），以使8％w / v的溶液。 </li><li>在水 - 光引发剂溶液（步骤1.2）溶解聚乙二醇（PEG）8-臂炔（10 kDa的MW），以使8％w / v的溶液。 </li></ol></li><li>在一般情况下，形成使用下面的方案水凝胶，虽然附加的定制是可能的。 <ol><li>结合4份2％的硫醇化的HA，4份2％的硫醇化明胶，1份交联剂1，1份交联剂2与8份组织的ECM溶液和2份肝细胞培养基（HCM）（或10份水作为一种通用的非组织特异性水凝胶）。 注:其他未修饰的HA或明胶可以被添加以使生物油墨挤出更加顺畅。这在下面描述。 </li></ol></li><li> VORTEX高所得的混合物（速度10满分10分），持续10秒前混合使用。&lt;/ LI&gt; <li>水凝胶生物油墨使用<ol><li>用于挤出或生物印刷测试，将混合物转移到一个注射器或打印盒，并允许它在37℃下自发交联30分钟（第一阶段交联）。 </li><li>为流变学测量的混合物转移到35毫米的培养皿中，并允许其交联30分钟。 注:混合物立即开始通过硫醇 - 丙烯酸酯键形成交联，并将开始粘度提高。该混合物应转移到注射器，墨盒或目标位置在10分钟内，以避免传输期间吸液管或注射器的堵塞。 </li><li>当二次交联（阶段2）是理想的，照射第1阶段的交联凝胶用紫外光（365纳米，18瓦/厘米2）以引发硫醇炔聚合反应。 注意:照射时间是依赖于材料的表面积。在一般情况下，材料的一个平方厘米只要求1-2秒在这个UV功率的紫外线照射。 </li></ol></li></ol> 2.打印机兼容性测试<ol><li>之前与生物印刷设备，测试挤出特性使用标准的注射器和小规格针头（20-30表压）简单挤压试验的实验台上集成测试。 <ol><li>通过标准注射器推生物油墨实现与很少或没有颠簸水凝胶的顺利挤出长丝。线条或图案简单的挤压足以确定成功。 </li></ol></li><li>对于生物印刷器集成，通过吹打他们入打印机的墨盒装载生物油墨制剂，并允许在生物油墨30分钟到暗盒内自发阶段1的交联。 注:生物油墨的体积取决于具体的应用和应该由用户来确定。打印机墨盒可能类似于或者是注射器是与生物印刷设备兼容。 </li><li>评估挤压兼容性通过印刷使用生物油墨简单的生物印刷图案。例如，打印由平行线7×7毫米形状。施加压力（ 例如，20千帕的气压），而在约300mm /分钟的速度在XY平面中的打印头移动。 注意:各种尺寸的打印头喷嘴的直径都可以使用，但具有400-500微米直径的开口的锥形喷嘴是最佳的在250-350微米范围内打印的球体。 <ol><li>如果被挤出的材料是块状或不规则，见步骤2.4，或者降低PEGDA的量以软化阶段1交联材料。准备妥当生物油墨配方中挤出顺利，允许期望的图案或结构的精确沉积。 注:生物印刷步骤中所述使用在内部设计的专门用于组织构造打印的定制的3-D生物印刷装置32这样，特定的打印参数而变化显着地为每种类型的设备和需要TESTIN。由用户克</li></ol></li><li>以提高挤出性能，补充未修饰的HA和明胶的bioinks（1.5毫克/毫升和30毫克/毫升，分别）。 </li></ol> 3.验证生物印刷由原发性肝癌构造<ol><li>通过悬滴法作为细胞成分33 ​​准备的3-D原代细胞肝癌球体 注:生物印刷可以在不球体悬浮在水凝胶bioinks以及单个细胞中进行，而是。球状​​体在这里用来加速细胞 - 细胞相互作用和构造的功能。采用球状体或细胞的数量取决于特定的应用和应该由用户来确定。这些步骤应在无菌条件下使用无菌用品中进行。 <ol><li>通过将HCM补充组分试剂盒的解冻内容到肝细胞基础培养基（HBM）和无菌过滤制备的HCM。 <ol><li>解冻的补充成分未直到液体。 </li><li>添加补充部件（抗坏血酸，0.5毫升，牛血清白蛋白[无脂肪酸]，将10毫升;硫酸庆大霉素/两性霉素B，0.5毫升，氢化可的松21-半琥珀酸酯，0.5毫升，胰岛素，0.5毫升，人重组表皮生长因子，0.5毫升，转移，0.5毫升）以500毫升的HBM。 </li><li>通过使用的瓶顶过滤器单元或一个注射器尖端过滤0.45微米或0.22微米的过滤器无菌过滤。 </li></ol></li><li>通过后的各细胞类型已根据制造商的说明被解冻上血球计数确定原代人肝细胞，库普弗细胞，星形细胞的细胞密度。 </li><li>结合原代人肝细胞，库普弗细胞，星形细胞在HCM中媒体的80:10:10的比例由细胞数目已经加热到37℃的锥形管中。 注意:要使用的介质的体积取决于整个细胞数量特定于应用程序，并应通过第被确定E用户。 </li><li>离心细胞悬浮液5分钟，在520 xg离心20℃。 </li><li>吸出上清液，留下细胞团块的后面。 </li><li>重悬在HCM媒体细胞沉淀，得到含有每40微升媒体1000细胞的细胞悬浮液。对正在生产的球状体的数量总成交量依赖。 </li><li>细胞悬液转移到96孔格式悬滴板。总共约1,000个细胞添加到每个孔中HCM和在37℃下保持，在5％CO 2 3天期间，多细胞球状体形成。 </li><li>收集使用移液器从悬滴板肝球体。转移到无菌的15毫升锥形管中。 </li></ol></li><li>在肝特异性的水凝胶生物油墨的BioPrint肝球状体<ol><li>如步骤1中描述的，采用8％PEGDA和8％8臂PEG炔作为交联剂制备肝脏含有ECM的水凝胶生物油墨的制剂。使用该组合其在地区环境部门一道能力lting密切剪切弹性模量，以原生肝组织水凝胶。 </li><li>让球体沉降到它们被放置在步骤3.1.7的锥形管的底部。这种变化的基础上球体的大小和密度，但一般1-2分钟内发生。通过仔细地吸或用移液器取出所有介质。 </li><li>转移新鲜制备的水凝胶生物油墨溶液的所需体积含有该球状体的锥形管中。通常，一个合适的体积为10％，比3-D构建体的体积要打印更大-25％。仔细吸取上下悬浮球体在水凝胶生物油墨溶液。传送到使用移液器或血清吸管一个生物印刷盒。 </li><li>的生物印刷器盒内部，使溶液经受第一次交联阶段（硫醇 - 丙烯酸酯反应）30分钟。 注意:根据球体大小，盒可能需要缓慢地旋转或内容可能需要与混合无菌刮刀，以保持整个生物油墨分布在球体的第一阶段交联时。这是少悬浮细胞，而不是球体准备bioinks的必需品。 注:按照第1阶段的交联，用户有几个小时的操作窗口。然而，建议快速执行生物印刷过程中提高细胞生存力。 </li><li>继第一阶段的交联，使用设备生物印刷来创建一个包含原发性肝癌球体（或其他细胞）所需的水凝胶结构。 注意:此技术提供了一种系统，用于biofabricating各种各样的结构。参数，如总体积，细胞或球状体，印刷结构几何的数量，以及基板，其上印刷的结构是高度依赖于用户的目标。 </li><li>沉积成所需配置后，辖紫外光2-4秒以引发二次交联机理，稳定的共nstructs和增加刚度至期望的水平。 注:PEG-炔的浓度，并且因此整体最终的交联密度，主要控制最终的构建刚度。 </li><li>重复步骤3.2.4和3.2.5以便创建多层构建体。 </li></ol></li></ol>

作者什么都没有透露。

有几个部件是当试图biofabricate三维组织构建，以便最终使用在人类或用于体外筛选应用考虑的关键。使用适当的细胞成分决定了端电位的功能，而生物制造装置本身确定的通用方法用于到达末端构建体。第三成分，该生物材料，是同样重要的，因为它提供双重角色。具体地，生物材料组件必须与两个生物制造硬件（ 即 ，bioprinters），并且还支持潜在脆弱生物细胞组分相容。优化这两...

近年来，各种各样的技术已经变得可用，通过谋求制造，或biofabricate，他们解决了对功能的器官和组织的替代来源的需求。生物印刷已成为最有希望的，这些技术中的一个。生物印刷可以被认为是生物份的机器人添加剂制造的一种形式，可以使用3维建立或图案上可行的器官样或组织样结构。1在大多数情况下，生物印刷采用3维（3 -D），其由计算机涉及细胞和生物材料存入的精确位置，从而扼要打印装置解剖学-模仿生理结构。2这些设备打印"生物油墨"，它可以采取细胞聚集体的形式，包封在水凝胶的细胞或粘性流体或细胞接种微载体，以及无细胞聚合物，其提供机械结构或作为无细胞PLAceholders 3,4继生物印刷过程中，所得到的结构可以成熟为功能性的组织或器官的结构，并用于其预期的最终应用。5,6-迄今为止，一个完整的全功能人大小器官尚未印制，但它仍然是生物印刷的研究和开发的主要长期目标。2然而，小规模的，目前正在在许多应用，包括病理学建模，药物开发和毒理学筛选实施"类器官"组织构建。 一位研究人员在生物印刷应用技术已经遇到的主要障碍是，很少有材料已用于生物印刷的明确目的开发的。要有效地生物印刷成功，生物材料必须符合4个基本要求。生物材料需要有1）的适当的机械性能，以允许沉积（无论是通过一个喷嘴作为凝胶或一个i-挤出nkjet作为液滴），2），以保持它的形状作为淀积后的3-D结构的一个组成部分的能力，3）的能力为的2现有特征的用户控制，以及4）一个细胞友好和支持的环境在所有生物印刷的过程的阶段。7历史上，工作生物印刷已经常常试图在雇用生物印刷设备现有的传统生物材料而不考虑其兼容性，而不是设计的生物材料有必要与生物印刷后续印刷后的应用程序的性能。 各种bioinks的最近研制与沉积和制造硬件更好的界面。标准水凝胶系统造成显著的问题，因为他们一般既可以是前体的力学性能不足，或者如果可以打印喷嘴堵塞或变成打散在挤压过程中聚合水凝胶流体解决方案。我们的团队，以及行吟诗人RS，已经探索各种水凝胶制剂，以解决这些生物印刷问题，包括细胞球体印刷成水凝胶基质，从微毛细管5,8-细胞和凝胶长丝挤出，9-11挤出透明质酸（HA）-gold纳米颗粒的水凝胶用动态交联的属性使用光聚合的水凝胶刚度的12时间控制甲基丙烯酸HA和明胶，13为基础的纤维蛋白原凝血酶交联，14,15离子交换海藻胶原凝胶，16日和最近的快速聚合紫外线（UV） -引发交联，17 这些例子表明，可以通过有效地bioprinted生成材料的可行性。然而，除了与硬件集成，以成功地产生可行的和功能性的3-D组织构建，生物材料必须包含生物化学和机械线索在维持细胞援助生存力和功能。这些额外的因素，生物化学和机械型材，可以对bioprinted组织构建成功函数显著影响。 两个小区和本机外基质（ECM）是负责呈现宽范围的信号分子如生长因子和其它细胞因子的其他细胞。这些信号的组合从组织到组织而变化，但也可以是在调节细胞和组织的行为非常有效和有影响。18从不同器官用人组织特异性细胞外基质成分和实施为水凝胶或水凝胶的一部分已探索与成功。19-21这种方法，它包括脱细胞的给定组织，粉碎它，并且将其溶解的，可用于从任何组织产生组织特异性生化信号，并且可以在三维水凝胶构建体并入。22 另外，它被广泛记载，在身体组织占据了广泛刚度。23这样，能够调整生物材料，如弹性模量E&#39;或剪切弹性模量G&#39;的机械性能，是在组织工程的有用工具。如上所述，在生物油墨机械性能控制允许使用一个软的凝胶，然后可以通过二次交联在后一点上，可以实现的弹性模量的水平在哪些进一步操作基于挤出的生物制造匹配的靶器官类型的那个。例如，生物材料可定制以满足5-10千帕的刚度本地人一样肝脏，23或匹配10-15千帕的刚度像原生心脏组织，理论上24,25增加这些组织体发挥作用的能力以类似的方式，以它们的天然组织对应物。环境刚度对细胞表型的影响已经EXPlored在近年来，特别是相对于干细胞。恩氏等人证实在驾驶间充质干细胞（MSC）朝向与组织弹性匹配的衬底的谱系计算机辅助该基板的弹性。25这一概念已进一步探索分化成肌肉，心脏功能，肝表型，造血干细胞的增殖和维护的干细胞的治疗潜力的。24,26-29如果能够调谐的水凝胶，以不同的弹性模量是将用于biofabricate组织构建的生物材料的一个重要特征。30 在这里，我们描述了代表在我们的实验室用于配制可挤出bioprinted水凝胶系统中的通用方法，和自定义为1）包含特定的组织类型的生物化学轮廓和2）模仿组织类型的弹性模量的协议。通过解决这些需求，我们的目标是为provide可以概括在体内的生理化学和生物特性的材料的组织。31本文中所描述的模块化的水凝胶的复合系统利用多交联的方法，以得到可挤出bioinks，并允许二次交联，以稳定和增加的刚度高端产品来匹配一系列的组织类型。生化定制是通过组织特异性细胞外基质成分满足。作为示范，我们聘请了肝脏特异性品种这水凝胶系统来的BioPrint功能性肝类器官结构。描述的协议，使用自定义的3-D设备生物印刷。在一般情况下，该协议可以适应大多数基于挤出的打印机，具体印刷参数显着变化对于每种类型的设备，并且需要由用户测试。

<table><tbody><tr><td>Hyaluronic acid</td><td>Sigma</td><td>53747</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G6144</td><td></td></tr><tr><td>2-Hydroxy-4&amp;prime;-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>Sigma</td><td>410896</td><td></td></tr><tr><td>Hyaluronic acid and gelatin hydrogel kit (HyStem-HP)</td><td>ESI-BIO</td><td>GS315</td><td>Kit contains the components Heprasil (thiolated and heparinized hyaluronic acid), Gelin-S (thiolated gelatin), and Extralink (PEGDA)</td></tr><tr><td>PEG 8-Arm Alkyne, 10&amp;nbsp;kDa</td><td>Creative PEGWorks</td><td>PSB-887</td><td></td></tr><tr><td>Primary human hepatocytes</td><td>Triangle Research Labs</td><td>HUCPM6</td><td></td></tr><tr><td>Primary human liver stellate cells</td><td>ScienCell</td><td>5300</td><td></td></tr><tr><td>Primary human Kupffer cells</td><td>Life Technologies</td><td>HUKCCS</td><td></td></tr><tr><td>Hepatocyte Basal Media (HBM)</td><td>Lonza</td><td>CC-3199</td><td></td></tr><tr><td>Hepatocyte Media Supplement Kit</td><td>Lonza</td><td>CC-3198</td><td>HCM SingleQuot Kits (contains ascorbic acid, 0.5&amp;nbsp;ml; bovine serum albumin [fatty acid free], 10&amp;nbsp;ml; gentamicin sulfate/amphotericin B, 0.5&amp;nbsp;ml; hydrocortisone 21-hemisuccinate, 0.5&amp;nbsp;ml; insulin, 0.5&amp;nbsp;ml; human recombinant epidermal growth factor, 0.5&amp;nbsp;ml; transferring, 0.5&amp;nbsp;ml)</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td>Other manufacturers are ok.</td></tr><tr><td>Ammonium hydroxide</td><td>Fischer Scientific</td><td>A669</td><td>Other manufacturers are ok.</td></tr><tr><td>Fresh porcine cadaver tissue</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Lyophilizer</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Freezer mill</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Bioprinter</td><td>n/a</td><td>n/a</td><td>The bioprinter described herein was custom built in-house. In general, other devices are adequate provided they support computer controlled extrusion-based printing of hydrogel materials.</td></tr><tr><td>Hanging drop cell culture plate</td><td>InSphero</td><td>CS-06-001</td><td>InSphero GravityPlus 3D Culture Platform</td></tr></tbody></table>

bioprinting cellularized constructs using a tissue-specific hydrogel bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

当上述过程被正确执行，水凝胶应包含特异于靶组织类型的生物化学信息，20允许对生物印刷和最终弹性模量，34的高度控制和支持存活功能性细胞中的组织构建。 水凝胶定制 到最 ​​好的模拟天然肝，水凝胶生物油墨是由肝脏ECM解决方案和生长因子阵列补充 20 ECM解决?...

Watch this Scientific Journal Video about Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink at JoVE.com

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

我们描述一组一起提供组织模仿水凝胶生物油墨与官能的和可行的3-D组织构建可bioprinted在体外筛选应用中使用的协议。

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

bioprinting-cellularized-constructs-using-tissue-specific-hydrogel

Research

JoVE Journal

Bioengineering

16.7K 次观看.  Wake Forest Univeristy Health Sciences. 我们描述一组一起提供组织模仿水凝胶生物油墨与官能的和可行的3-D组织构建可bioprinted在体外筛选应用中使用的协议。 

视频: Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

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

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

科研

免疫与感染

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.

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 ...

生物工程

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

在三维生物打印中使用多层水凝胶生物油墨，实现均匀细胞分布

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

用于骨髓源性肥大细胞培养的结晶纳米纤维素包埋琼脂糖生物材料墨水的制备

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

用于生成骨类似物的细胞悬浮液中的陶瓷全向生物打印

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...