Name: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture (Video) | JoVE
Uploaded: 2023-10-13T12:30:00
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

Tis work was supported by the National Key Research and Development Program of China (2022YFA1104600), and the Zhejiang Provincial Natural Science Foundation of China (LQ23H160011).

1. Fabrication of the 3D acoustic assembly device
<ol>
	<li>Begin by preparing four 1 mm thick PMMA sheets through laser cutting30, and then proceed to glue them together to form a square chamber with an inner width of 21 mm and a height of 10 mm.</li>
	<li>Next, attach another 1 mm thick PMMA sheet to the bottom of the chamber to serve as a holder for the bioink.</li>
	<li>Carefully affix three lead zirconate titanate (PZT) transducers (each measuring 20 mm in length, 10 mm in ...

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

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G., Di Marzio, N., Eglin, D., Alini, M., Serra, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+waves+that+make+the+pattern:+a+review+on+acoustic+manipulation+in+biomedical+research.">The waves that make the pattern: a review on acoustic manipulation in biomedical research.</a> Materials Today Bio. 10, 100110 (2021).</li><li>Harley, W. 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Efficient and stable fabrication of cell spheroids with high throughput using technologies like the 3D acoustic assembly device holds great promise for advancing biomedical engineering and drug screening1,2,3. This approach simplifies the mass production of cell spheroids through straightforward procedures.
However, there are critical factors to consider when using this acoustic device. The creation o...

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.

<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

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.

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

This study designed a 3D acoustic assembly device for mass manufacturing of cell spheroids. The acoustic device comprised a square chamber with two PZT transducers attached to the X-plane and Y-plane on the outer surface of the chamber and one PZT transducer on the chamber&#39;s bottom (Figure 1A,B). Three output channels from two function generators were connected to three power amplifiers to generate three independently sinusoidal signals to actuate the PZT transducers (<s...

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

Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture (Video) | JoVE

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.

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