The authors thank Dr. Jeff Caplan and Mr. Michael Moore at the Delaware Biotechnology Institute Bio-Imaging Center for their support with confocal microscopy. Access to microscopy equipment was supported by the National Institutes of Health (NIH) shared instrumentation grants (S10 RR0272773 and S10 OD016361) and the State of Delaware Federal Research and Development Grant Program (16A00471). This research was supported by grants from the Institutional Development Award (IDeA) from the NIH National Institute of General Medical Sciences (P20GM103446), the American Cancer Society (14-251-07-IRG), the University of Delaware Research Foundation (14A00778), the Cancer Prevention and Research Institute of Texas (CPRIT) (RR140013), and the NIH National Library of Medicine (4 R00 LM011390-02).

<ol>
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The authors have nothing to disclose.

Critical in overriding the standard functions of the microscope software to enable the use of virtual masks for image-guided, laser-based degradation, the microscope macro must be setup and manipulated carefully. How the microscope software interfaces with the microscope macro can sometimes be counterintuitive, and much effort was invested in determining the optimal settings in both programs to develop this method. A general understanding of basic laser-scanning confocal microscope use is recommended, especially with the &#34;Stage&#34; and &#34;Focus&#34; windows for finding and correctly positioning the hydrogel in the x, y, and z dimensions, before attempting laser-based degradation. Additionally, the fabrication of an inlet channel is critical to the protocol; suspended fluorescent species must be able to enter the microfluidic systems for successful imaging and network characterization. It is important to note that this protocol applies to a specific laser-scanning confocal microscope configured with a specific femtosecond pulsed laser, running specific versions of the microscope software and the microscope macro (see details in the List of Specific Materials/Equipment). We have discovered that newer versions of the microscope macro lack the necessary control and functionality needed for image-guided laser control. While other microscope platforms and pulsed laser sources can be used, this protocol applies specifically to this system and will need modification according to the platform, laser source, and software implemented. The underlying principles throughout this protocol still apply, however.
A potential modification to this protocol involves changing the hydrogel composition. Here, we utilized 5 wt%, 3.4 kDa PEGDA hydrogels, but laser-based degradation (for purposes aside from microfluidic network generation) has been implemented to degrade both synthetic and natural hydrogels, including PEGylated fibrinogen21,22, silk protein hydrogels23, and collagen24. Adjustment of the laser power, scan speed, spacing between Z-slices, and number of repetitions will aid in determining the optimal parameters to fabricate microfluidic networks in other hydrogel formulations.
One current limitation of the technique is the overall volume of hydrogel that can be degraded in a feasible amount of time. To create open voids or microfluidic features within the hydrogel, the laser dwell time must be at or above 8.96 &#181;s/pixel (or a laser scan speed of 0.021 &#181;m/&#181;s) when using a laser fluence of 37.7 nJ/&#181;m2. With these settings, it takes 1.4 h to degrade vessels within a 0.014 mm3 volume (as seen in Figure 1). Using a laser dwell time of 4.48 &#181;s/pixel or below, the energy delivered is not enough for full degradation of the hydrogel formulation used here. Implementing a different hydrogel composition could overcome this limitation. The use of photolabile gels that contain light-sensitive components34-36 or hydrogels that have large multiphoton cross-sections21-24 are good options that would enable the use of less energy and would result in faster degradation. With respect to dimensional limitations, features have been fabricated up to 1.5 mm deep in the hydrogel7. The depth achievable is a function of the working distance of the objective and can be increased using ultra-long working distance objectives optimized for in vivo imaging. The smallest measured channel7 created using a 20X (NA1.0) water immersion objective had a width of 3.3 &#181;m and depth of 8.9 &#181;m, which is on par with the size of the smallest channels generated in Figure 1. While other labs have used lower NA objectives21,23,24, we anticipate that microfluidic networks with smaller features can be generated using higher NA objectives at the expense of a reduced working distance. Ultimately, however, the resolution of the technique is a function of the point spread function of the focused laser beam, the laser scanning properties, the amount of energy delivered by the laser, and the laser absorption properties of the material being degraded.
Furthermore, laser properties (speed, fluence, spacing between virtual masks, and number of repetitions) must be optimized based on hydrogel properties (crosslink density, macromer/monomer molecular weight, weight percent, and type of hydrogel: protein-based vs. synthetic), as these will inherently alter interactions with the laser and thus the ultimate resolution of the process. As the energy delivered is also influenced by the objective used, additional optimization is required when switching between objectives. With respect to the costs involved, access to a microscope with a pulsed laser is required, and while many different objectives can be used, those with a high NA and long working distance (for in vivo imaging) can be expensive.
Above and beyond the protocol detailed here, microfluidic networks generated using this technique can also be functionalized with cell-adhesive peptides post-channel fabrication to induce endothelial cell adhesion and lumen formation7. Additionally, the incorporation of cell-degradable peptide sequences in the bulk material9 prior to channel degradation could allow for the study of cell migration into and through the hydrogel. For continuous fluidization of the microfluidic network within the hydrogel, hydrogels can be photopolymerized in larger fluidic devices or housings, as demonstrated in Figures 2 and 37.
The generation of vascular networks via vasculogenic or angiogenic self-assembly8-12 provides a straightforward approach to induce vascularization throughout a relatively large hydrogel volume. While this approach results in perfusable fluidic networks, it is difficult to directly control the size, tortuosity, density, and overall network architecture. Due to this limitation, the flow profile and shear rates in the vasculature may differ between experiments. Alternatively, microfabrication techniques13-16 allow for direct control over network architecture but are often limited by their inability to generate small, capillary-sized channels or the fabrication of dense networks that mimic in vivo vascular architecture. While the laser-based hydrogel degradation technique outlined here has been newly repurposed for microfluidic generation, it simultaneously overcomes both the architectural control and size-based limitations of existing microfabrication methods by enabling the creation of 3D biomimetic microfluidic networks that recapitulate the density, tortuosity, size range, and overall architecture of in vivo vasculature. Furthermore, multiple fluidic networks can be generated in a single hydrogel, allowing the study of inter-network transport7. For tissue engineering applications that strive to more closely replicate in vivo transport processes in vitro, the highly resolved hydrogel-embedded microfluidic networks outlined here are well suited. We anticipate that this protocol will be useful in developing tissue constructs that more accurately mimic in vivo transport for use as drug screening devices and in vitro disease models.

血管系统，包括两个淋巴和cardiovasculature的，形式高度致密的网络，是用于营养和氧气的传输和用于去除代谢废物是必不可少的。因此，居住在血管化组织细胞从来没有超过50-100微米远离容器1。重现体内血管结构体外的能力，关键是要精确地模拟使用工程结构在体内的运输过程。随着最近的驱动开发用于高通量药物器官上的单芯片器件2筛选3,4和疾病建模5,6，方法创建一个概括的合成或天然水凝胶在体内般的交通正在绘制微流控网络显著的兴趣。以提高在这些设备中使用microtissues的仿生学，我们开发了利用三dimensiona一个图像引导，基于激光的水凝胶降解法作为模板天然脉管l的（3D）图像栈以生成嵌入PEGDA水凝胶7血管源性的微流体网络中。这个协议概括了使用配备有飞秒脉冲激光经由图像引导，基于激光的降解来制造在PEGDA凝胶血管源性，仿生微流体网络的市售激光扫描共焦显微镜的。 来流化水凝胶当前的方法包括的血管8-10或血管生成10-12内皮细胞自组装和微制造技术中的诱导对内皮13-16创建预定义的信道。而自组装网络概括密度和微血管的复杂结构，它们往往比体内网络11,17,18，这可能是在造型运输用于药物筛选应用问题更加可渗透的。自组装网络由physiolog的ically相关，毛细管尺寸的容器，但它们可能难以与散装流体流动整合由于在产生较大的动脉大小船只限制。因为是在这些网络的装配没有直接控制，最终的结构可以从样本变化来样，使得难以重复地制备具有相同的流体流动和输运性质网络。 为了产生3D，水凝胶的嵌有重复的几何形状和良好定义的体系结构的微流体网络中，若干微细加工技术得到了发展，其中包括模块式组件13，牺牲材料16，直接写入组件14，和全向印刷15的三维印刷。应用这些方法，微流体结构，因此，流体流动和输运性质，可以在许多结构被重复制造。这些方法的主要限制，然而，这是无法创造微法luidic网络与毛细管大小的功能，4〜10微米19。多数的微细加工技术往往局限于特征在直径13,16从150至650微米。一些现有技术能够在很宽的直径范围内具有通道产生分层网络，10至300微米为直接写入组件14，和18至600微米为全向印刷15，但它们在他们的产生密集网络或能力有限到单个构建体7内产生极为接近的多个微流体网络。 为了克服这些限制，我们开发的图像制导，激光为基础的水凝胶降解技术，使仿生的重复制造，即概括体内微血管的结构层次微流体网络。要做到这一点，一个790纳米，140飞秒脉冲激光在80 MHz工作是光栅扫描我Ñ所需的水凝胶内的三维位置， 通过体内脉管系统的图像作为限定。我们推测，降解过程通过水的激光诱导光学击穿，所得等离子体形成时，水的随后的快速热弹扩张，和水凝胶的局部降解操作作为水膨胀20。这种机制从基于蛋白的水凝胶21-24的基于激光的降解略有不同。不像PEGDA，其具有低多光子截面，蛋白质常常具有大的多光子截面，因此通过多光子吸收引起的化学断裂23降解。以生成图像引导的微流体网络中，激光快门被图像衍生虚拟口罩，其由限定微流体结构的兴趣9区域的镶嵌的控制。使用这种方法，我们已经证明，制作三维血管源性仿生微流体的能力集成电路网络，概括在体内脉管系统的密集和曲折结构，以便通过改变能量的降解期间输送的量局部地控制水凝胶的孔隙率。我们也已经能够产生交织在接近（15微米），但从未直接连接7两个独立的微流控网络。我们还证明，通过交降解官能与整结扎肽序列endothelialize激光恶化微通道的能力，精氨酸-甘氨酸-天冬氨酸-丝氨酸（RGDS），以促进内皮细胞的粘附和管腔形成7。 有了这个协议，复杂的微流控网络在PEGDA水凝胶的生成是使用在许多大学校园访问市售显微镜通过影像引导下，基于激光的下降成为可能。作为降解过程是由数字，虚拟掩模导向，这法布里卡化技术是适合于微流体网络的创意设计，允许其在各种应用中使用。我们预计，在这里所描述的方法将是在设计能够复制生物运输过程建模药物转运2重要的仿生器官和人体上的单芯片器件最有利的。这种制造技术也可用于在体外疾病模型，包括癌症转移5,6-和血脑屏障模型25的产生兴趣。如基于激光的水凝胶降解以前已用于创建神经元向外生长21-23的引导轨道，该技术的图像引导的延伸可在高级组织工程策略证明是有用的定位在用户定义三维空间排列的细胞。

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">MATLAB</td>
			<td style="width:199px;">Mathworks</td>
			<td style="width:171px;">R2015a</td>
			<td style="width:191px;">named &#34;programming software&#34; in protocol; refer to source 9 for details on algorithm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">FIJI (Fiji is Just Image J)</td>
			<td style="width:199px;">NIH</td>
			<td style="width:171px;">version 1.51a</td>
			<td>named &#34;image processing software&#34; in protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">LSM 780 Confocal Microscope</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:171px;"></td>
			<td>named &#34;laser-scanning confocal microscope&#34; in protocol; for laser-based hydrogel degradation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Zen 2010B SP1</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:171px;">release version 6.0</td>
			<td>named &#34;microscope software&#34; in protocol; for use on Zeiss LSM-780</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Multitime, 2010</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:171px;">v16.0</td>
			<td>named &#34;microscope macro&#34; in protocol; for use on Zeiss LSM-780 (in conjunction with Zen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Objective W Plan-Apochromat 20x/1.0 DIC D=0.17 M27 75mm</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:171px;">421452-9880-000</td>
			<td>for use on Zeiss LSM-780</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Chameleon Vision II Modelocked Ti:S Laser</td>
			<td style="width:199px;">Coherent</td>
			<td style="width:171px;"></td>
			<td>named &#34;high-powered pulsed laser&#34; in protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Sodium chloride</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:171px;">S5886-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">HEPES</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:171px;">H3375-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Triethanolamine (TEOA)</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:171px;">90279-100ML</td>
			<td>flammable; skin and eye irritant; work with in a fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Sodium hydroxide</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:171px;">S5881-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">150 mL Vacuum Filtration Cups with 0.2 &#181;m PES Membrane</td>
			<td style="width:199px;">VWR</td>
			<td style="width:171px;">10040-460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">PEGDA</td>
			<td style="width:199px;">synthesized in house</td>
			<td style="width:171px;"></td>
			<td>refer to source 33 for synthesis methods; store under argon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Eosin Y disodium salt</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:171px;">E6003-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">1-vinyl-2-pyrrolidinone (NVP)</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:171px;">V3409-5G</td>
			<td>store under argon; carcinogenic; work with in a fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">3M Double Coated Tape, 9500PC, 6.0 mil</td>
			<td style="width:199px;">Thomas Goldkamp</td>
			<td style="width:171px;">37728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Flexmark 90 PFW Liner</td>
			<td style="width:199px;">FLEXcon</td>
			<td style="width:171px;">FLX000620</td>
			<td>backing for handling of double coated tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Model SC Plotter (adhesive cutter)</td>
			<td style="width:199px;">USCutter</td>
			<td style="width:171px;">SC631E</td>
			<td>used to cut adhesive in ring shapes to connect coverslips to petri dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">60 mm Petri Dish with 20 mm Hole</td>
			<td style="width:199px;">MatTek Corporation</td>
			<td style="width:171px;">P60-20-F-NON</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:531px;">High Intensity Illuminator (white light source)</td>
			<td style="width:199px;">Fiber-Lite</td>
			<td style="width:171px;">4715MS-12WB10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Power Meter</td>
			<td style="width:199px;">Newport</td>
			<td style="width:171px;">1916-R</td>
			<td>detect power at 524 nm when using white light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Slim Profile Wand Detector</td>
			<td style="width:199px;">Newport</td>
			<td style="width:171px;">918D-ST</td>
			<td>for use with power meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:199px;">Dow Corning</td>
			<td style="width:171px;">3097358-1004</td>
			<td>used to make PDMS molds; refer to source 7 for methods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">TMPSA-Functionalized #1.5 Coverslips, 40 mm Round</td>
			<td style="width:199px;">synthesized in house</td>
			<td style="width:171px;"></td>
			<td>refer to source 7 for methods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Dextran, Fluorescein, 2,000,000 MW, Anionic, Lysine Fixable</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:171px;">D7137</td>
			<td>can use alternative tagged dextrans; 2000 kDa does not diffuse readily into a 5% 3.4kDa PEGDA hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">1 mL Syringe, Luer-Lok</td>
			<td style="width:199px;">BD</td>
			<td style="width:171px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:531px;">Acrodisc Syring Filter, 0.2&#181;m Supor Membrane, Low Protein Binding</td>
			<td style="width:199px;">Pall</td>
			<td style="width:171px;">PN 4602</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:531px;">sticky-Slide VI0.4&#160;</td>
			<td style="width:199px;">Ibidi</td>
			<td style="width:171px;">80601</td>
			<td>microfluidic devices that can be used to house hydrogels</td>
		</tr>
	</tbody>
</table>

image-guided, laser-based fabrication of vascular-derived microfluidic networks

此详细的协议概述图像制导，激光为基础的水凝胶降解嵌入在PEGDA水凝胶血管源性微流体网络的制造执行。在这里，我们描述的虚拟面具，使图像引导的激光控制的建立;一个微成形PEGDA水凝胶，适用于微流体网络制造和压头驱动流的光聚合;设置和使用飞秒脉冲钛配对市售的激光扫描共聚焦显微镜:秒激光诱导水凝胶退化;并使用制造的微流体网络的成像荧光物质和共焦显微镜。多协议的集中在显微镜软件和显微镜宏的适当的设置和实施方式中，因为这些是在使用市售的显微镜对包含许多复杂的微流体制造目的的关键步骤。此techniqu的图像引导部件e可以为3D图像堆栈或用户生成3D模型的实现，从而使创意微设计和几乎所有的配置复杂的微流体系统的制造。在组织工程中的预期效果，在该协议中概述的方法可以在先进的仿生microtissue构建用于器官和人体上的单芯片器件的制造提供帮助。通过模仿复杂结构，扭曲，尺寸和体内脉管系统的密度，基本生物运输过程可以在这些构造中复制，导致药代动力学和疾病的更准确的体外模型。

To demonstrate the implementation of image-guided, laser-based hydrogel degradation to generate a 3D, vascular-derived microfluidic network, we utilized a 3D image stack of mouse brain vasculature that was acquired via knife-edged scanning microscopy (KESM)26,27. A selected 3D region of the larger microvascular dataset was converted into a series of virtual masks for image-guided microfluidic network formation, as described above. After fabrication, the resulting microfluidic network was perfused with a solution of FITC-labeled, 2,000 kDa dextran and imaged via confocal microscopy. The image stack of the in vivo vasculature that defined the network architecture (Figure 1A and C) and the fabricated microfluidic network (Figure 1B and D) were used to generate Z-projections (Figure 1A and B) and 3D renderings (Figure 1C and D). The comparison of the images demonstrates that image-guided, laser-based hydrogel degradation enables the fabrication of 3D biomimetic microfluidic networks that recapitulate the size, tortuosity, and complex architecture of in vivo vasculature. The technique is amenable to fabrication of networks containing a wide range of diameters; within Figure 1, channels ranging from 3.3 to 28.8 &#181;m in diameter were generated. Note that the larger rectangular structure in the top left of Figure 1B and in the left corner of Figure 1D is the inlet channel to introduce flow into the network.
<img alt="Figure 1" src="/files/ftp_upload/55101/55101fig1.jpg" /> 
Figure 1: Vascular-Derived Microfluidic Network. A series of virtual masks, derived from an image stack of mouse brain vasculature (A and C), were used to fabricate a 3D biomimetic microfluidic network embedded in a PEGDA hydrogel (B and D) via image-guided, laser-based degradation. The microfluidic network was perfused with 2,000 kDa FITC-labeled dextran and imaged via confocal microscopy. Z-projections (A and B) and 3D renderings (C and D) of the two networks demonstrate the ability to generate microfluidic networks that recapitulate the density, organization, and overall architecture of in vivo vasculature. The arrow (D) marks the rectangular inlet created to introduce the dextran. The scale bar represents 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55101/55101fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We have implemented two methods, pressure heads and syringe pumps, to initiate fluid flow in these embedded microfluidic networks. For example, a 30 by 30 &#181;m rectangular channel was fabricated to connect two wells in a micromolded PEGDA hydrogel (Figure 2A), with fluid flow driven via a pressure head7. Generated in the left well, the pressure head induced flow through the microchannel and into the well on the right. In Figure 2A, an initial front of tetramethylrhodamine (TRITC)-labeled, 70 kDa dextran was observed moving through the channel using time-lapse microscopy. Later, in Figure 2B, a 10-&#181;m diameter fluorescent polystyrene sphere flowed from the left well, through the channel, to the well on the right. With the duration of flow controlled by the volume of fluid present in the micromolded well at the inlet, and the flow-rate controlled by the hydrostatic gradient generated between the inlet and outlet well, pressure head-driven flow in micromolded hydrogels provides a simple method for flow induction, without the need for an external housing or a syringe pump.
<img alt="Figure 2" src="/files/ftp_upload/55101/55101fig2.jpg" /> 
Figure 2: Pressure Head-Driven Flow in a Micromolded PEGDA Hydrogel. PEGDA was micromolded to form a hydrogel containing two wells connected by an embedded microchannel. A pressure head was generated in the left well to initiate flow of TRITC-labeled, 70 kDa dextran (A1-A3) and a 10-&#181;m polystyrene sphere (B1-B3) through a 30 by 30 &#181;m rectangular channel from one well to the other. The scale bar represents 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55101/55101fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Alternatively, to generate constant fluid flow within microfluidic networks, syringe pump-driven flow can be initiated by photopolymerizing the hydrogel inside of a secondary microfluidic device with inlet and outlet ports for syringe pump interfacing. In Figure 3A, a commercially available, pre-fabricated microfluidic device is shown with a 1-mm band of hydrogel photopolymerized across the width of the device7. Laser-based degradation was implemented to fabricate microfluidic channels and networks in housed hydrogels using the same protocol described here for freestanding hydrogels. Syringe pump-generated flow can be seen in Figure 3B, where a 20-&#181;m diameter cylindrical channel was fabricated in a hydrogel housed in a microfluidic device. In this 20-&#181;m diameter channel, individual 2-&#181;m fluorescent polystyrene spheres are easily resolved without fluid flow (Figure 3B1), whereas when a 5 &#181;L/s flowrate is applied, the spheres move through the field of view, as evidenced by streaking (Figure 3B2). This same approach was used to induce flow through a 2D, vascular-derived network to monitor flow of TRITC-labeled, 65 kDa dextran through the network and diffusion into the surrounding hydrogel (Figure 3C).
<img alt="Figure 3" src="/files/ftp_upload/55101/55101fig3.jpg" /> 
Figure 3: Syringe Pump-Driven Flow in PEGDA Hydrogels Polymerized in Microfluidic Housings. A commercially available, pre-fabricated microfluidic device (A) with a 17 x 3.8 x 0.53 mm (x,y,z) microchannel houses a photopolymerized 1 x 3.8 x 0.53 mm (x,y,z) PEGDA hydrogel, indicated by the black arrow. A 20-&#181;m diameter cylindrical channel was degraded through the hydrogel, and 2-&#181;m polystyrene spheres were pumped through the device (B). The spheres are clearly resolved without fluid flow (B1), but they appear as streaks at a flowrate of 5 &#181;L/s (B2). In another housed hydrogel, a 2D vascular-derived network was fabricated and TRITC-labeled, 65 kDa dextran was pumped through the network (C). Time-lapse microscopy was used to monitor the fluorescent dextran as it filled the channels and diffused into the surrounding hydrogel. White arrows in (B) and (C) indicate the direction of fluid flow. The calibration bar in (C) indicates the intensity of the fluorescent dextran. The scale bars represent 20 &#181;m in (B1) and (B2) and 50 &#181;m in (C). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55101/55101fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks at JoVE.com

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

image-guided-laser-based-fabrication-vascular-derived-microfluidic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.8K 次观看.  University of Delaware. This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

视频: Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

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 ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...