本研究得到了国家重点研发计划（2022YFA1104600）和浙江省自然科学基金（LQ23H160011）的支持。

1. 3D声学装配装置的制造
<ol><li>首先通过激光切割准备四个1毫米厚的PMMA片材30，然后继续将它们粘合在一起，形成一个内宽为21毫米，高度为10毫米的方形腔室。</li>
	<li>接下来，将另一张 1 毫米厚的 PMMA 片材贴在腔室底部，作为生物墨水的支架。</li>
	<li>小心地将三个锆钛酸铅 （PZT） 探头（每个长 20 毫米、宽 10 毫米、厚 0.7 毫米，初级谐振频率为 3 MHz，参见 材料表）分别固定在腔室的三个正交壁的外部。确保底部 PZT 换能器位于腔室下方的中心。</li>
	<li>将焊丝焊接到每个 PZT 传感器的两个导电区域。</li>
	<li>最后，将设备固定在空心底座上，以防止底部 PZT 与其他台面接触。</li>
</ol>2. 设置声学装配系统
<ol><li>首先将声学设备安装到显微镜载物台上，以便俯视观察腔室内部。</li>
	<li>将数码显微镜放置在设备没有PZT探头的一侧，以便从侧面观察腔室内部。</li>
	<li>将三个 PZT 传感器串联的导线独立连接到三个功率放大器和函数发生器的三个输出通道（参见 材料表）。</li>
	<li>对每个 PZT 传感器的函数发生器设置进行编程，指定正弦波形、频率和幅度等参数。</li>
	<li>为确保灭菌，用 75% 酒精填充声学设备的腔室 5 分钟，然后用无菌 PBS 溶液彻底清洁。随后，在洁净的工作台中用紫外线照射腔室至少1小时。</li>
</ol>3. 细胞培养和收获程序
<ol><li>首先，使用补充有 10% 胎牛血清和 1% 青霉素-链霉素的 Dulbecco 改良 Eagle 培养基 （DMEM） 在 T25 细胞培养瓶中培养 C3A 细胞（一种人肝细胞癌细胞系）（参见 材料表）。</li>
	<li>当C3A细胞达到约80%汇合时，按如下方式进行细胞传代：首先，用PBS洗涤培养瓶底部两次。然后，向细胞培养瓶中加入2mL 0.05%胰蛋白酶-EDTA，并在37°C下孵育以促进细胞分离。通过加入 2 mL 完全培养基停止胰蛋白酶化过程。</li>
	<li>将细胞溶液转移到 15 mL 管中，并在室温下以 200 × g 离心 5 分钟以获得细胞沉淀。</li>
</ol>4. 生物墨水的制备
<ol><li>通过将 0.3 g GelMA 和 0.025 g LAP（参见 材料表）溶解在 5 mL 磷酸盐缓冲溶液 （PBS） 中，制备含有 0.5% （w/v） 苯基-2,4,6-三甲基苯甲酰基次膦酸锂 （LAP） 的 6% （w/v） GelMA 溶液。让混合物在47°C水浴中静置1小时。</li>
	<li>在与细胞混合之前，将 GelMA 溶液通过 0.22 μm 过滤器（参见 材料表）进行灭菌。</li>
	<li>将上述细胞沉淀重悬于细胞培养基中（步骤3.3）。用0.4%台盼蓝溶液对少量细胞进行染色，然后使用干净的血细胞计数器进行细胞计数。</li>
	<li>将根据上述获得的细胞密度计算的适量C3A细胞与灭菌的GelMA溶液混合，制备细胞密度为2×106 个细胞/mL的生物墨水。</li>
	<li>为了观察组装的细胞球体，通过在37°C下用2μM细胞追踪剂（DiO染料，参见 材料表）孵育20分钟来预染色C3A细胞。随后，在使用前用新鲜细胞培养基洗涤标记的细胞三次。</li>
</ol>5. 使用声学设备组装细胞球体
<ol><li>将超过 1 mL 的生物墨水移液到灭菌室中。确保液体表面与腔室底部之间的面对面距离是声波长一半的整数倍。 注意：可以通过调整添加的生物墨水的体积来控制这个距离。声波长 （λ） 可以使用公式 λ = c/f 来估计，其中 c 表示介质中的声速，f 是 PZT 换能器的频率。</li>
	<li>打开函数发生器和功率放大器以启动每个 PZT 传感器的驱动。 注意： 建议先单独启动每个 PZT 探头，以实现最佳的平行线蜂窝模式。这可以通过对每个PZT换能器在初级谐振频率附近进行步长为0.001 MHz的频率扫描来实现。然后，将这些最佳信号同时应用于所有三个 PZT 传感器，以获得预期的 3D 点细胞模式。在施加每个输入信号之前，通过轻轻搅拌确保细胞均匀分散在生物墨水中。</li>
	<li>使用蓝光（405 nm，60 mW/cm 2,30 s）交联生物墨水，以创建3D水凝胶支架，该支架封装以声学方式组装的细胞聚集体。之后，关闭函数发生器和功率放大器。</li>
	<li>小心地将3D水凝胶支架从腔室转移到培养皿中，并使用干净的剃须刀将其切成小块（例如，2毫米×2毫米×2毫米）。</li>
	<li>加入细胞培养基进行培养。 注意：请记住每天更换培养基。</li>
</ol>6. 检索细胞球体
<ol><li>首先使用倒置显微镜观察支架不同层的球状体形成。</li>
	<li>培养3天后，取出培养基，用PBS彻底清洗水凝胶支架两次。</li>
	<li>在细胞培养箱中，用细胞培养基以 1：200 的稀释度将支架与超过 2 mL 的 GelMA 裂解缓冲液孵育 30 分钟。此步骤旨在溶解水凝胶支架。</li>
	<li>将上一步的溶液转移到试管中，然后在室温下以200 ×g 离心5分钟以获得细胞球状体沉淀。</li>
	<li>弃去上清液，将沉淀重悬于新鲜培养基中，用于下游应用。</li>
</ol>7. 球状体活力分析
<ol><li>使用活/死染色试剂盒评估水凝胶支架内或检索到的球状体中细胞球状体的活力（参见 材料表）。</li>
	<li>在不同的培养时间点将样品与含有1μL钙黄绿素-AM和2μL碘化丙啶（PI）的1mLPBS溶液在37°C下孵育15分钟。</li>
	<li>对于水凝胶支架内的样品，直接用PBS清洗两次。对于回收的球状体，在室温下以200 ×g 离心5分钟以获得球状体沉淀，并在观察前洗涤两次。</li>
	<li>使用荧光显微镜观察样品并捕获荧光图像。</li>
	<li>使用 ImageJ 计算用钙黄绿素-AM 染色的面积与用钙黄绿素-AM 和 PI 染色的总面积的比率，以确定球状体活力。</li>
</ol>

使用 3D 声学组装装置可扩展生成均匀的单元球体

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作者没有什么可透露的。

使用 3D 声学组装设备等技术高效稳定地制造具有高通量的细胞球体，为推进生物医学工程和药物筛选 1,2,3 带来了巨大的前景。这种方法通过简单的程序简化了细胞球体的大规模生产。
但是，使用此声学设备时需要考虑一些关键因素。驻波的产生对于构建3D点阵声场至关重要。在该设备中，每个维度中只有...

与传统的 2D 培养模型相比，3D 体外培养模型提供了更多类似体内的结构和形态学特征，已被公认为各种生物医学应用（如组织工程、疾病建模和药物筛选）中的有前途的系统 1,2,3.作为 3D 培养模型的一种类型，细胞球体通常是指细胞聚集，创建以增强细胞-细胞和细胞-基质相互作用为特征的 3D 球状体结构 4,5,6。因此，制造细胞球体已成为实现各种生物学研究的有力工具。
已经开发了各种技术，包括悬挂液滴7、非粘性板8 或微孔装置9，以获得球状体。原则上，这些方法通常通过利用重力等物理力来促进细胞组装，同时最大限度地减少细胞与基材之间的相互作用。然而，它们通常涉及劳动密集型过程，生产率低，并且对控制球体大小10,11 提出了挑战。重要的是，生产具有所需尺寸和均匀性且数量足够的球状体对于满足特定的生物应用至关重要。与上述方法相比，声波作为一种外力驱动技术12,13,14，基于通过外力增强细胞聚集的原理，显示出大规模制造高质量和高通量细胞球体的潜力15,16,17,18.与电磁力或磁力不同，基于声学的细胞操作技术是非侵入性和无标记的，能够形成具有出色生物相容性的球状体19,20。
通常，已经开发了基于驻置表面声波 （SAW） 和体声波 （BAW） 的器件来利用相应的驻置声场产生的声学节点 （AN）21,22,23 来生成球体。特别是基于BAW的声学组装装置，具有制造方便、操作方便、可扩展性好等优点，在制造细胞球体方面备受关注24,25。我们最近开发了一种基于 BAW 的简单声学组装装置，能够生成具有高通量的球体26。所提出的装置由一个方形聚甲基丙烯酸甲酯 （PMMA） 腔室组成，三个锆酸铅钛酸酯 （PZT） 探头分别布置在 X/Y/Z 平面上。这种布置可以创建悬浮声学节点 （LAN） 的 3D 点阵图案，用于驱动单元组装。与先前报道的基于 BAW 或 SAW 的设备相比，这些设备只能创建 ANs27、28、29 的 1D 或 2D 阵列，本设备能够实现 LAN 的 3D 点阵列，用于在明胶甲基丙烯酰 （GelMA） 溶液中快速形成细胞聚集体。随后，经过三天的培养，细胞聚集体在光固化的 GelMA 支架内成熟为具有高活力的球状体。最后，从GelMA支架中可以很容易地获得大量大小均匀的球体，用于下游应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22-&#956;m filter</td>
			<td>Merck</td>
			<td>SLGSM33SS</td>
			<td>Used for GelMA solution sterilization</td>
		</tr>
		<tr>
			<td>35 mm-cell culture dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>Used for culturing cells</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>A1RHD25</td>
			<td>Fluorescent cell observation</td>
		</tr>
		<tr>
			<td>DiO dye</td>
			<td>Beyotime</td>
			<td>C1038</td>
			<td>Dye used to stain cells</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12430054</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Rigol</td>
			<td>DG5352</td>
			<td>For RF signal generation</td>
		</tr>
		<tr>
			<td>GelMA</td>
			<td>Regenovo</td>
			<td>none</td>
			<td>Used to prepare bioink</td>
		</tr>
		<tr>
			<td>GelMA lysis buffer</td>
			<td>EFL</td>
			<td>EFL-GM-LS-001</td>
			<td>Used to dissolve GelMA scaffolds</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Ti-U</td>
			<td>Cell observation</td>
		</tr>
		<tr>
			<td>LAP</td>
			<td>Sigma-Aldrich</td>
			<td>900889</td>
			<td>Used as photoinitiator</td>
		</tr>
		<tr>
			<td>Live-Dead kit</td>
			<td>Beyotime</td>
			<td>C2015M</td>
			<td>Cell vability analysis</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010002</td>
			<td>Used as buffer</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td>Prevent cell culture contamination</td>
		</tr>
		<tr>
			<td>Power amplifer</td>
			<td>Minicircuit</td>
			<td>LCY-22+</td>
			<td>Increase the voltage amplitude of the RF signal</td>
		</tr>
		<tr>
			<td>PZT transducers</td>
			<td>Yantai Xingzhiwen Trading Co.,Ltd.</td>
			<td>PZT-41</td>
			<td>Functional units for acoustic assembly device</td>
		</tr>
		<tr>
			<td>T25 cell culture flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>Used for culturing cells</td>
		</tr>
		<tr>
			<td>Trypan blue&#160;</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>Cell dissociation enzyme</td>
		</tr>
	</tbody>
</table>

three-dimensional acoustic assembly device for mass manufacturing of cell spheroids

细胞球体是有前途的三维（3D）模型，在许多生物领域获得了广泛的应用。该协议提出了一种通过机动程序使用 3D 声学组装装置制造高质量和高通量细胞球体的方法。声学组件装置由三个锆钛酸铅 （PZT） 换能器组成，每个换能器都布置在方形聚甲基丙烯酸甲酯 （PMMA） 腔室的 X/Y/Z 平面上。当施加三个信号时，这种配置可以生成悬浮声学节点 （LAN） 的 3D 点阵模式。因此，明胶甲基丙烯酰 （GelMA） 溶液中的细胞可以被驱动到 LAN，在三维空间中形成均匀的细胞聚集体。然后对 GelMA 溶液进行紫外光固化和交联，作为支持细胞聚集体生长的支架。最后，通过随后在温和条件下溶解 GelMA 支架来获得和回收大量成熟的球状体。拟议的新型3D声学细胞组装装置将能够放大细胞球体甚至类器官的制造，为生物领域提供巨大的潜力技术。

本研究设计了一种用于细胞球体大规模生产的 3D 声学组装装置。声学装置包括一个方形腔室，两个PZT换能器连接到腔室外表面的X平面和Y平面上，一个PZT换能器连接到腔室底部（图1A，B）。来自两个函数发生器的三个输出通道连接到三个功率放大器，以产生三个独立的正弦信号来驱动PZT传感器（图1C）。
用于驱动连接?...

Watch this Scientific Journal Video about Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture at JoVE.com

作者聚焦：用于高质量 3D 细胞球状体培养的无支架声学组装方法的开发 

细胞球体被认为是生物应用领域的一种潜在模型。本文介绍了使用 3D 声学组装设备可扩展生成细胞球体的协议，它为稳定和快速地制造均匀细胞球体提供了一种有效的方法。

用于细胞球体批量生产的三维声学装配装置

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

3D体外培养模型与细胞球体一样，已广泛应用于组织工程、疾病建模和药物筛选等领域。我们的研究重点是开发和投资每种工程方法，以制造高质量的球状体或其他 3D 体外培养模型，用于各种生物学研究。在目前的协议中，球体在 GelMA 支架内生长，并在最后的放置时被回收，溶解支架，导致额外的程序。我们将尝试在培养基中声学组装细胞聚集体，使它们能够在没有支架的球状体中生长。与磁组装技术相比，声波可以直接以无标记的方式组装细胞，而不会对细胞产生潜在的毒性作用。此外，我们的协议使细胞球体能够组装成三维阵列，从而大大提高了通量。

three-dimensional-acoustic-assembly-device-for-mass-manufacturing

Research

JoVE Journal

Bioengineering

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

977 次观看.  Hangzhou Dianzi University. 3D体外培养模型与细胞球体一样，已广泛应用于组织工程、疾病建模和药物筛选等领域。我们的研究重点是开发和投资每种工程方法，以制造高质量的球状体或其他 3D 体外培养模型，用于各种生物学研究。在目前的协议中，球体在 GelMA 支架内生长，并在最后的放置时被回收，溶解支架，导致额外的程序。我们将尝试在培养基中声学组装细胞聚集体，使它们能够在没有支...

视频: 作者聚焦：用于高质量 3D 细胞球状体培养的无支架声学组装方法的开发 

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

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

用于生成多细胞三维球体的 CD 实验室平台

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

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 ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.