Name: three-dimensional acoustic assembly device for mass manufacturing of cell spheroids
Uploaded: 2023-10-13T12:30:00.000+00:00
Duration: 5 min 17 s
Description: Watch this Scientific Journal Video about Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture at JoVE.com

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><tbody><tr><td>0.22-&amp;mu;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&amp;nbsp;</td><td>Gibco</td><td>15250061</td><td>Cell counting</td></tr><tr><td>Trypsin-EDTA&amp;nbsp;</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

1.0K 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 

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

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 ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

生物化学

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 ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

用于基于 FLIM 的代谢和缺氧分析的多细胞球状体形成

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

作者聚焦:使用荧光寿命成像显微镜标准化用于代谢和氧合分析的球状体形成方法

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

用于细胞球体批量生产的三维声学装配装置

副本

摘要

探索更多视频

此视频中的章节

一个简单的悬滴细胞生成三维球体文化协议

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

平面和立体印刷的导电油墨

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

用于生成多细胞三维球体的 CD 实验室平台

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

用于细胞球体批量生产的三维声学装配装置

副本

摘要

探索更多视频

此视频中的章节

一个简单的悬滴细胞生成三维球体文化协议

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

平面和立体印刷的导电油墨

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

用于生成多细胞三维球体的 CD 实验室平台

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）