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。

3D in vitro culture models, which provide more in vivo-like structural and morphological characteristics compared to conventional 2D culture models, have been recognized as promising systems in various biomedical applications such as tissue engineering, disease modeling, and drug screening1,2,3. As one type of 3D culture model, cell spheroids typically refer to cell aggregation, creating 3D spheroidal structures characterized by enhanced cell-cell and cell-matrix interactions4,5,6. Therefore, fabricating cell spheroids has become a powerful tool for enabling diverse biological studies.
Various techniques, including hanging drop7, non-adhesive plates8, or microwell devices9, have been developed to obtain spheroids. In principle, these methods commonly facilitate cell assembly by utilizing physical forces such as gravitational force while minimizing interactions between cells and the substrate. However, they often involve labor-intensive processes, have low productivity, and pose challenges for controlling spheroid size10,11. Importantly, the production of spheroids with the desired size and uniformity in sufficient quantity is of utmost importance to satisfy specific biological applications. In contrast to the above-mentioned methods, acoustic waves, as one type of external-force-driven technique12,13,14, have shown potential for mass manufacturing of cell spheroids with high quality and throughput, based on the principle of enhancing cell aggregation through external forces15,16,17,18. Unlike electromagnetic or magnetic forces, acoustic-based cell manipulation techniques are non-invasive and label-free, enabling spheroid formation with excellent biocompatibility19,20.
Commonly, standing surface acoustic waves (SAWs) and bulk acoustic waves (BAWs)-based devices have been developed to generate spheroids, utilizing the acoustic nodes (ANs) produced by corresponding standing acoustic fields21,22,23. Particularly, acoustic assembly devices based on BAWs, with the merits of convenient manufacture, easy operation, and excellent scalability, have gained attention for fabricating cell spheroids24,25. We have recently developed a facile BAWs-based acoustic assembly device with the ability to generate spheroids with high throughput26. The proposed device consists of a square polymethyl methacrylate (PMMA) chamber with three lead zirconate titanate (PZT) transducers arranged respectively in the X/Y/Z plane. This arrangement enables the creation of a 3D dot-array pattern of levitated acoustic nodes (LANs) for driving cell assembly. Compared to previously reported BAWs- or SAWs-based devices, which can only create a 1D or 2D array of ANs27,28,29, the present device enables a 3D dot-array of LANs for rapid cell aggregate formation within the gelatin methacryloyl (GelMA) solution. Subsequently, cell aggregates matured into spheroids with high viability within the photocured GelMA scaffolds after three days of cultivation. Finally, a large number of spheroids with uniform size were easily obtained from the GelMA scaffolds for downstream applications.

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 

用于细胞球体批量生产的三维声学装配装置

3D in vitro culture models, like cell spheroids, have been widely used in the fields of tissue engineering, disease modeling, and drug screening. Our research focuses on developing and invest per engineering method to fabricate spheroids or other 3D in vitro culture models with high quality for diverse biological studies. In the current protocol, the spheroids grew within the GelMA scaffold and are retrieved at the final layback, dissolving the scaffold, causing additional procedures.We will try to acoustically assemble cell aggregates in a cultural medium to enable them to grow within spheroids without scaffold. Compared to magnetic assembly techniques, acoustic waves can directly assemble cells in a label-free manner without potential toxic effects on cells. Besides, our protocol enables cell spheroids to be assembled in a three-dimensional array, substantially increasing throughput.

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

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。

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

777 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 

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

科研

生物工程

Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids

To begin, sterilize the chamber of the acoustic device with 75%alcohol for five minutes. Then, clean the device with sterile PBS and irradiate it with UV light for one hour. Mount the sterile acoustic device onto a microscope stage for top-view observation of the chamber's interior.Position a digital microscope on the side of the device that is free from PZT transducers, enabling side-view observation of the chamber's interior. Independently connect the wires from the three PZT transducers in series to three power amplifiers and three output channels of function generators. Program the settings on the function generators for each PCT transducer, specifying parameters such as sinusoidal waveforms, frequency, and amplitude.Culture 3CA cells in DMEM supplemented with 10%FBS and 1%penicillin streptomycin in a T25 cell culture flask. When the cells become 80%confluent, wash the culture twice with PBS. After removing the PBS, add two milliliters of 0.05%trypsin-EDTA to the flask and incubate at 37 degrees Celsius for cell detachment.Add two milliliters of complete cell culture medium into the flask to stop the trypsinization. Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 G for five minutes.

建立声学组装系统和细胞培养收获以生成均匀的细胞球体

Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids

To prepare the bio ink solution mix an appropriate amount of C3A cell suspension with the sterilized gelMA solution. Prestain the C3A cell spheroids with two micromolar cell tracker solution at 37 degrees for 20 minutes. To wash the cells, first, remove the supernatin by centrifuging and then add fresh cell culture medium.Add at least two milliliters of bio ink into the sterilized acoustic device chamber. Turn on the function generator and power amplifier to initiate the actuation of each PZT transducer to generate a 3D array of cell aggregates. Crosslink the bio ink using blue light for 30 seconds to create 3D hydrogel scaffolds, encapsulating the assembled cell aggregates acoustically.Next, carefully transfer the 3D hydrogel scaffold from the chamber into a Petri dish. Cut it into small pieces using a clean razor, and then add cell culture medium into the Petri dish. Incubate the C3A cell spheroids at different culture time points in one milliliter of PBS containing one microliter of calcium am and two microliters of propidium iodide for 15 minutes at 37 degrees Celsius.Rinse the sample embedded in hydrogel scaffolds twice with PBS. For retrieved spheroids, perform centrifugation at 200 G for five minutes and wash twice with PBS. Using a fluorescence microscope, observe the spheroids and capture the images.The cell aggregates were arranged in the regular 3D dot array pattern with a green fluorescent signal. The assembled C3A gelMA aggregates gradually integrated and formed tight spheroids by day three, accompanied by an increase in spheroid diameter. The viability of the cell spheroids remained good before day three, but slightly decreased after a week of culture.The released spheroids maintained a good spherical morphology with a narrow size distribution along with desirable viability and expression of albumin.