作者要感谢来自纽约首都地区研究联盟资助的支持。

1，生物印刷平台建立打印机的修改是基于对HP DESKJET 500热喷墨​​打印机和HP 51626A黑色墨盒。 <ol><li>取下打印机顶部塑料盖，小心地从盖取下控制面板。 </li><li>拆下打印机的顶部和底部之间的3线连接。从底座上拆下打印机的顶部。 </li><li>在打印机顶部，取下墨盒下在右边的小塑料及橡胶配件（打印头清洗系统）。 </li><li>取出进纸托盘与弹簧的基础。 </li><li>取下金属板覆盖塑料纸吧。 </li><li>切断在用手锯或其它切割工具的中间进料轮位置的塑料纸吧。 </li><li>除去前面的步骤之后露出的2进纸轮。轮子的塑料是很难和一个电子锯会有所帮助。 </li><li>使用清洁的空气罐头和乙醇擦拭的灰尘和碎屑。 </li><li>将打印机顶部部分到基座上。 </li><li>使用之前UV消毒至少2小时在层流罩的改性打印机。 </li><li>切断用手锯或其他切削工具的HP 51626A墨盒的盖子。 </li><li>清空墨水和撕下软骨底部以及水库的过滤器。 </li><li>彻底用流动的自来水冲洗墨盒。 </li><li> Ultrasonicate盒在去离子（DI）水10分钟以除去残留的墨水。 </li><li>检查墨盒，以确保所有的墨水已被删除。冲洗或喷雾彻底盒用70％乙醇消毒，接着无菌去离子水。 </li><li>设置一个长波长紫外线灯在印刷平台上提供同步光聚合能力。 </li><li>使用UV光测量紫外线强度在打印平台米。调整UV灯和打印机平台之间的距离如此的强度在打印对象是4-8之间毫瓦/厘米2（大约25厘米的灯，以在打印机平台）。 </li></ol> 2，生物油墨的制备<ol><li>单层软骨细胞扩张<ol><li>板500万的人软骨细胞到每个T175组织培养瓶用于细胞扩增中的Dulbeccos改进的Eagles培养基（DMEM），补充有10％小牛血清和1x青霉素 - 链霉素 - 谷氨酰胺（PSG）。培养细胞在37℃下用含5％CO 2的加湿空气。改变培养基每3天，直到烧瓶85％汇合。从同一通道使用的细胞。 </li></ol></li><li>溶解PEGDA在PBS中的10％w / v的终浓度添加光引发剂I-2959的0.05％w / v的终浓度过滤灭菌的溶液中。 </li><li>暂停在制备PEGDA溶液培养的人软骨细胞以5×10 6个细胞/毫升。 </li></ol> 3，软骨组织印刷<ol><li>关闭打印机和笔记本电脑上。 </li><li>创建一个实心圆与直径4毫米使用Microsoft Word或Adobe Photoshop中的印刷图案。 <ol><li>调整图案的位置，并确保它会准确打印到塑料模具。 </li><li>计算到达支架的所需厚度所需的打印数。为4毫米高度，220的打印需要创建所需的脚手架。 </li></ol></li><li>加载到生物油墨墨盒。盖上铝箔盒在打印过程中，以保护从直接紫外线照射。 </li><li>发送打印命令到打印机。拉纸传感器，当打印机开始打印。整个印刷过程只需要不到4分钟用于直径4毫米和4毫米高支架。 </li><li>转印印刷neocartilage到24孔板中并加入1.5毫升培养培养基到每个孔中。 </li></ol><ol><li>孵育印刷neocartilage在LIVE / DEAD活力/细胞毒性工作在室温下在黑暗中15分钟，溶液中。 </li><li>削减了一半的细胞充满水凝胶，并采取切削区的荧光图像。 </li><li>现场计数（绿色）和死（红）细胞由不知情的观察者五个随机拍摄的图像。通过将活细胞的数目由活细胞和死细胞的总数计算细胞存活率。 </li></ol>

<ol>
	<li>Cui, X., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvasculature+fabrication+using+thermal+inkjet+printing+technology.">Human microvasculature fabrication using thermal inkjet printing technology.</a> Biomaterials. 30, 6221-6227 (2009).</li><li>Cui, X., Breitenkamp, K., Finn, M. G., Lotz, M., D'Lima, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+cartilage+repair+using+three-dimensional+bioprinting+technology.">Direct human cartilage repair using three-dimensional bioprinting technology.</a> Tissue Eng Part A. 18, 1304-1312 (2012).</li><li>Cui, X., Breitenkamp, K., Lotz, M., D'Lima, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+action+of+fibroblast+growth+factor-2+and+transforming+growth+factor-beta1+enhances+bioprinted+human+neocartilage+formation.">Synergistic action of fibroblast growth factor-2 and transforming growth factor-beta1 enhances bioprinted human neocartilage formation.</a> Biotechnol. Bioeng. 109, 2357-2368 (2012).</li><li>Cui, X., Breitenkamp, K., Finn, M. G., Lotz, M., Colwell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+cartilage+repair+using+thermal+inkjet+printing+technology.">Direct human cartilage repair using thermal inkjet printing technology.</a> Osteoarthritis and Cartilage. 19, (2011).</li><li>Cui, X., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+deposition+of+human+microvascular+endothelial+cells+and+biomaterials+for+human+microvasculature+fabrication+using+inkjet+printing.">Simultaneous deposition of human microvascular endothelial cells and biomaterials for human microvasculature fabrication using inkjet printing.</a> NIP24/digital Fabrication 2008: 24th International Conference on Digital Printing Technologies, Technical Program and Proceedings. 24, 480-483 (2008).</li><li>Cui, X., Dean, D., Ruggeri, Z. M., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+damage+evaluation+of+thermal+inkjet+printed+Chinese+hamster+ovary+cells.">Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells.</a> Biotechnol. Bioeng. 106, 963-969 (2010).</li><li>Cui, X., Hasegawa, A., Lotz, M., D'Lima, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+meniscus+cells+promotes+meniscal+phenotype+without+hypertrophy.">Structured three-dimensional co-culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy.</a> Biotechnol. 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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=Development+of+A+High-Resolution+Thermal+Inkjet+Printhead.">Development of A High-Resolution Thermal Inkjet Printhead.</a> Hewlett-Packard Journal. 39, 55-61 (1988).</li><li>Harmon, J. P., Widder, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+the+Printhead+Into+the+HP+Deskjet+Printer.">Integrating the Printhead Into the HP Deskjet Printer.</a> Hewlett-Packard Journal. 39, 62-66 (1988).</li><li>Kim, T. K., 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=Experimental+model+for+cartilage+tissue+engineering+to+regenerate+the+zonal+organization+of+articular+cartilage.">Experimental model for cartilage tissue engineering to regenerate the zonal organization of articular cartilage.</a> Osteoarthritis and Cartilage. 11, 653-664 (2003).</li><li>Sharma, B., 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=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Engineering. 13, 405-414 (2007).</li><li>Cohen, D. L., Lipton, J. I., Bonassar, L. 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作者没有任何金融利益在这项研究中。

这种3D生物印刷系统和同步光聚合产能提供比使用注射器挤出蜂窝海藻酸钠水凝胶16骨软骨缺损的原位印刷的最佳先前报道的方法一个显著更高的打印分辨率。较高的打印分辨率是特别关键的软骨组织工程恢复解剖软骨带状组织。层 - 层组装过程中同时进行光聚合反应是至关重要的，以维持细胞和生物材料支架的三维结构的精确沉积。微细加工与各印刷层也导致带状层之间的?...

生物印刷基于热喷墨印刷是最有前途的应用技术在组织工程和再生医学的领域之一。数字控制和高吞吐量的打印头单元，支架和生长因子可以精确地沉积到所需的二维（2D）和三维（3D）位置的迅速发展。采用该技术在组织工程和再生医学1-9许多成功的应用已经实现。在本文中，生物印刷一个平台，建立与修改后的惠普（HP）DESKJET 500热喷墨​​打印机和同步光聚合体系。由聚（乙二醇）（PEG）配制合成的水凝胶已经表明维持软骨细胞的生存力和促进软骨ECM生成10,11的容量。此外，光交联聚乙二醇在水中具有低粘度，这使得它非常适合同时聚合物：水溶性高在生物印刷3D化。在本文中，人类软骨细胞悬浮在聚（乙烯）乙二醇二丙烯酸酯（PEGDA; 3,400兆瓦）的精确打印，构建neocartilage层 - 层与1,400 dpi的三维解析。观察到沉积的细胞在三维支架材料中均匀分布，即生成软骨组织具有优异的机械性能和增强的ECM生成。与此相反，在手动制造的凝胶，而不是其最初沉积仓的底部由于较慢的聚合骨架，而导致不均匀的软骨形成培养后2,3蓄积的细胞。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">HP Deskjet 500 thermal inkjet printer</td>
			<td style="width:221px;">Hewlett-Packard</td>
			<td style="width:195px;">C2106a</td>
			<td style="width:433px;">Discontinued. Purchased refurbished from internet vendor.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">HP black ink cartridge</td>
			<td style="width:221px;">Hewlett-Packard</td>
			<td style="width:195px;">51626a</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Ultraviolet lamp</td>
			<td style="width:221px;">UVP</td>
			<td style="width:195px;">B-100AP</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">UV light meter</td>
			<td style="width:221px;">General Tools</td>
			<td style="width:195px;">UV513AB</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Zeiss LSM 510 laser scanning microscope</td>
			<td style="width:221px;">Carl Zeiss</td>
			<td style="width:195px;">LSM 510</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Dulbeccos Modified Eagles Medium (DMEM)</td>
			<td style="width:221px;">Mediatech</td>
			<td style="width:195px;">10-013</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Penicillin-streptomycin-glutamine (PSG)</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">10378-016</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Accutase cell dissociation reagent</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">A11105-01</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Phosphate buffered saline (PBS)</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">10010-023</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Live/Dead viability/cytotoxicity Kit</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">L-3224</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Poly(ethylene glycol) diacrylate (PEGDA)</td>
			<td style="width:221px;">Glycosan Biosystems</td>
			<td style="width:195px;">GS700</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Irgacure 2959</td>
			<td style="width:221px;">Ciba Specialty Chemicals</td>
			<td style="width:195px;">I-2959</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Human articular chondrocytes</td>
			<td style="width:221px;">Lonza</td>
			<td style="width:195px;">CC-2550</td>
			<td style="width:433px;"></td>
		</tr>
	</tbody>
</table>

human cartilage tissue fabrication using three-dimensional inkjet printing technology

生物印刷，这是基于热喷墨印刷，是最有吸引力的应用技术在组织工程和再生医学的领域之一。与数字控制单元，支架和生长因子可以精确地沉积到所需的二维（2D）和三维（3D）位置迅速。因此，这种技术是制造组织模仿他们的母语解剖结构的理想方法。为了与原生地带性的组织，细胞外基质成分（ECM）和机械性能工程师软骨，我们开发了使用商用喷墨打印机能够进行3D软骨组织工程同时进行光聚合生物印刷一个平台。人软骨细胞悬浮于聚（乙二醇）二丙烯酸酯（PEGDA），被印刷于通过层 - 层组装三维neocartilage施工。印刷细胞固定在原来的位置存放，由surro支持unding脚手架在同步光聚合。印刷组织的机械性能类似于天然软骨。相对于传统的组织的制造，这需要较长的紫外线曝光，在印刷单元和同步光聚合的存活率显著更高。印刷neocartilage展现了出色的糖胺聚糖（GAG）和Ⅱ型胶原的生产，这与基因的表达相一致。因此，这个平台是理想的精确细胞分布和安排解剖组织工程。

修改后的热喷墨打印机是能够用于细胞和支架材料沉积在高吞吐量和出色的细胞活力。与同时进行光致聚合和光敏生物材料相结合，这种技术能够以固定细胞和其他印刷物质的最初沉积的位置。根据修改后的热喷墨打印机的属性，在2D打印分辨率为300 dpi，采用130 PL单个墨滴体积。有50个发射喷嘴每个打印头与3.6 kHz的发射频率12,13。因此，对于直径4毫米和4毫米的高度，体积和各印刷层的层 ...

Watch this Scientific Journal Video about Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology at JoVE.com

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

本文介绍的方法显示了如何将一个商用喷墨打印机与紫外线同时聚合生物印刷。该打印机能够构建三维组织结构，细胞和生物材料。这里展示的研究构建了一个3D neocartilage。

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

human-cartilage-tissue-fabrication-using-three-dimensional-inkjet

Research

JoVE Journal

Bioengineering

15.6K 次观看.  Rensselaer Polytechnic Institute. 本文介绍的方法显示了如何将一个商用喷墨打印机与紫外线同时聚合生物印刷。该打印机能够构建三维组织结构，细胞和生物材料。这里展示的研究构建了一个3D neocartilage。 

视频: Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术：新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

三维热塑性材料的印刷, 以创建自动注射器泵的反馈控制微流控应用

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...