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 width, 0.7 mm in thickness, and with a primary resonant frequency of 3 MHz, see Table of Materials) to the exterior of the three orthogonal walls of the chamber, respectively. Ensure that the bottom PZT transducer is centered beneath the chamber.</li>
	<li>Solder wires to the two conductive areas of each PZT transducer.</li>
	<li>Finally, secure the device onto a hollow base to prevent the bottom PZT from coming into contact with other countertops.</li>
</ol>
2. Setting up the acoustic assembly system
<ol>
	<li>Begin by mounting the acoustic device onto a microscope stage, allowing for top-view observation of the chamber&#39;s interior.</li>
	<li>Position a digital microscope on the side of the device that is free from PZT transducers, enabling side-view observation of the chamber&#39;s interior.</li>
	<li>Independently connect the wires from the three PZT transducers in series to three power amplifiers and three output channels of function generators (see Table of Materials).</li>
	<li>Program the settings on the function generators for each PZT transducer, specifying parameters such as sinusoidal waveform, frequency, and amplitude.</li>
	<li>To ensure sterilization, fill the chamber of the acoustic device with 75% alcohol for 5 min, followed by a thorough cleaning with sterile PBS solution. Subsequently, irradiate the chamber with UV light in a clean bench for a minimum of 1 h.</li>
</ol>
3. Cell culture and harvest procedure
<ol>
	<li>Begin by culturing C3A cells, a human hepatocellular carcinoma cell line, in a T25 cell culture flask using Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (see Table of Materials).</li>
	<li>When the C3A cells reach approximately 80% confluence, perform cell passage as follows: firstly, wash the bottom of the culture flask twice with PBS. Then, add 2 mL of 0.05% trypsin-EDTA to the cell culture flask and incubate at 37 &#176;C to facilitate cell detachment. Stop the trypsinization process by adding 2 mL of complete culture medium.</li>
	<li>Transfer the cell solution to a 15 mL tube, and centrifuge it at 200 &#215; g for 5 min at room temperature to obtain a cell pellet.</li>
</ol>
4. Preparation of the bioink
<ol>
	<li>Prepare a 6% (w/v) GelMA solution containing 0.5% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) by dissolving 0.3 g of GelMA and 0.025 g of LAP (see Table of Materials) in 5 mL of phosphate buffer solution (PBS). Allow the mixture to sit in a 47 &#176;C water bath for 1 h.</li>
	<li>Before mixing with cells, pass the GelMA solution through a 0.22 &#181;m filter (see Table of Materials) for sterilization.</li>
	<li>Resuspend the cell pellet mentioned above (step 3.3) in cell culture medium. Stain a small volume of cells with a 0.4% trypan blue solution and then use a clean hemocytometer for cell counting.</li>
	<li>Mix an appropriate amount of C3A cells, calculated based on the cell density obtained above, with the sterilized GelMA solution to prepare the bioink with a cell density of 2 &#215; 106 cells/mL.</li>
	<li>For visualization of the assembled cell spheroids, pre-stain C3A cells by incubating them with 2 &#181;M cell tracker (DiO dye, see Table of Materials) at 37 &#176;C for 20 min. Subsequently, wash the labeled cells with fresh cell culture medium three times before use.</li>
</ol>
5. Assembling the cell spheroids using the acoustic device
<ol>
	<li>Pipette more than 1 mL of the bioink into the sterilized chamber. Ensure that the face-to-face distance between the liquid surface and the chamber bottom is an integer multiple of half the acoustic wavelength. 
	NOTE: One can control this distance by adjusting the volume of bioink added. The acoustic wavelength (&#955;) can be estimated using the formula &#955; = c/f, where c represents the speed of sound in the medium, and f is the frequency of the PZT transducer.</li>
	<li>Turn on the function generator and power amplifier to initiate the actuation of each PZT transducer. 
	NOTE: It is recommended to individually actuate each PZT transducer first to achieve the optimal parallel-line cellular pattern. This can be achieved by conducting a frequency sweep with a step size of 0.001 MHz near the primary resonant frequency for each PZT transducer. Afterward, simultaneously apply these optimal signals to all three PZT transducers to obtain the expected 3D-dot cellular pattern. Before applying each input signal, ensure that the cells are evenly dispersed in the bioink by gently agitating.</li>
	<li>Crosslink the bioink using blue light (405 nm, 60 mW/cm2, 30 s) to create a 3D hydrogel scaffold that encapsulates the cell aggregates assembled acoustically. Afterward, turn off the function generator and power amplifier.</li>
	<li>Carefully transfer the 3D hydrogel scaffold from the chamber into a Petri dish and cut it into small pieces (e.g., 2 mm &#215; 2 mm &#215; 2 mm) using a clean razor.</li>
	<li>Add cell culture medium for cultivation. 
	&#8203;NOTE: Remember to change the culture medium every day.</li>
</ol>
6. Retrieving cell spheroids
<ol>
	<li>Begin by observing spheroid formation at different layers of the scaffolds using an inverted microscope.</li>
	<li>After 3 days of culture, remove the culture medium and thoroughly wash the hydrogel scaffolds twice with PBS.</li>
	<li>Incubate the scaffolds with more than 2 mL of GelMA lysis buffer at a 1: 200 dilution with cell culture medium for 30 min in a cell incubator. This step aims to dissolve the hydrogel scaffolds.</li>
	<li>Transfer the solution from the previous step into a tube and then centrifuge it at 200 &#215; g for 5 min at room temperature to obtain the cell spheroid pellet.</li>
	<li>Discard the supernatant and resuspend the pellet in fresh culture medium for downstream applications.</li>
</ol>
7. Spheroid viability analysis
<ol>
	<li>Assess the viability of cell spheroids either within the hydrogel scaffolds or in the retrieved spheroids using a live/dead staining kit (see Table of Materials).</li>
	<li>Incubate the samples at different culture time points with 1 mL of PBS solution containing 1 &#181;L of Calcein-AM and 2 &#181;L of Propidium Iodide (PI) for 15 min at 37 &#176;C.</li>
	<li>For samples within the hydrogel scaffolds, wash them directly with PBS twice. For retrieved spheroids, centrifuge at 200 &#215; g for 5 min at roomtemperature to obtain a spheroid pellet, and wash it twice before observation.</li>
	<li>Observe the samples using a fluorescence microscope and capture the fluorescence images.</li>
	<li>Calculate the ratio of the area stained with Calcein-AM to the total area stained with both Calcein-AM and PI using ImageJ to determine spheroid viability.</li>
</ol>

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

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The authors have nothing to disclose.

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 of standing waves is essential for constructing a 3D-dot array acoustic field. In this device, only one PZT transducer is present in each dimension, serving as a sound excitation source. Therefore, for each dimension, it&#39;s crucial that the face-to-face distance between the opposite walls of the PMMA chamber or PMMA-water surface is an integer multiple of half the wavelength. The primary frequency of the PZT transducers we used is 1 MHz, which results in a wavelength in the water or bioink of about 500 &#181;m. Consequently, for the horizontal dimension, the face-to-face distance between the two opposite walls of the PMMA chamber in the acoustic device was set to 21 mm, accounting for the size of the PZT transducers. For the vertical dimension, the face-to-face distance between the bottom PMMA wall and the water surface can be adjusted by varying the bioink volume to meet the conditions for standing wave formation. During experimental operation, slight frequency adjustments may be necessary to compensate for distance deviations. Additionally, it&#39;s important to keep the input voltage at a moderate level to prevent PZT transducer overheating, which could heat up the bioink and potentially damage the cells. In the experiments, a peak-to-peak voltage of 10 Vpp was used, completing cell aggregate assembly within 30 s while maintaining the bioink temperature below 30 &#176;C.
One limitation of this approach is the reliance on the GelMA scaffold26, which plays a crucial role in maintaining the structural integrity of the acoustically assembled cell aggregates as they mature into cell spheroids when the acoustic field is removed. However, the step involving the dissolution of the hydrogel scaffolds for spheroid retrieval may lead to reduced cell viability and potential spheroid loss. Additionally, the volume of the GelMA scaffold needs to be within the range of several millimeters to allow for nutrient penetration and exchange for the encapsulated cells. The limited scaffold size in the millimeter range can constrain the production yield of cell spheroids, even if larger scaffolds are initially produced and then cut into smaller pieces. Future efforts may focus on maintaining acoustically assembled cell aggregates in situ, suspended in the culture medium, to enable them to grow into spheroids without the need for an external scaffold.
In summary, the acoustic assembly device described in this protocol can be easily assembled and operated with relatively simple steps, offering the advantage of efficiently fabricating cell spheroids. Moreover, this acoustic assembly device is compatible with various cell types, including the fabrication of organoids, showcasing its significant potential for a wide array of biomedical applications.

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><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

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.

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 (Figure 1C).
The optimal resonant frequencies used to actuate the three PZT transducers attached to the X/Y/Z planes of the chamber were 3.209 MHz, 3.283 MHz, and 3.215 MHz, respectively. The optimal amplitude for all three PZT transducers was 10 peak-to-peak output voltage (Vpp), measured by an oscilloscope. Figure 2A illustrates the working mechanism of cell aggregates generated using the 3D acoustic assembly device. When the signal is applied, cells are driven to the acoustic nodes under the influence of acoustic radiation force (ARF). To visualize cell spheroids, cells were pre-stained with 2 &#181;M DiO (green fluorescence). After acoustic cell assembly, a confocal microscope was used to observe the 3D acoustically assembled cell aggregates. These cell aggregates were observed to be arranged in a regular 3D-dot array pattern with uniform green fluorescent signals (Figure 2B). Different top views of bright-field images also demonstrated that the formed aggregates in each layer were arranged in a 2D-dot array pattern (Figure 2C).
The growth of cell aggregates within the hydrogel at different time points was observed. Results showed that the assembled aggregates gradually integrated and formed tight spheroids by day 3, accompanied by an increase in spheroid diameter (Figure 3A,B). A live/dead staining was conducted to evaluate the viability of the cell spheroids. Good cell viabilities (&#62;90%) were achieved before day 3, while viability decreased slightly after a week of culture (Figure 3C,D).
For the retrieval of spheroids, a GelMA lysis buffer was used to dissociate the hydrogel scaffolds, releasing the encapsulated cell spheroids (Figure 4A). Consequently, after three days of cultivation, the small pieces of hydrogel scaffolds were treated with GelMA lysis buffer at 37 &#176;C for 30 min. The released spheroids maintained a good spherical morphology with a narrow size distribution, along with the expression of albumin and desirable viability (Figure 4B-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66078/66078fig01.jpg" /> 
Figure 1: 3D acoustic assembly device. (A) Schematic diagram depicting the top view of the 3D acoustic assembly device, consisting of a PMMA chamber attached with three PZT transducers. (B) Photograph displaying the actual 3D acoustic assembly device. (C) Photograph showing the 3D acoustic assembly device connected with two function generators and three power amplifiers. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66078/66078fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66078/66078fig02.jpg" /> 
Figure 2: Acoustically assembled cell aggregates. (A) Schematic illustrating the working mechanism of cell aggregates generated by the 3D acoustic assembly device, where cells are driven to the acoustic nodes by acoustic radiation force. (B) Confocal images showcasing the 3D acoustically assembled cell aggregates from different perspectives. (C) Bright-field images displaying the formed cell aggregates at various layers within the hydrogel scaffold. The scale bar represents 250 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66078/66078fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66078/66078fig03.jpg" /> 
Figure 3: Growth of cell aggregates into spheroids within the GelMA scaffold. (A) Bright-field images showing the formation of compact cell spheroids after a 3-day culture period. (B) Quantification of spheroid sizes. (C) Live/dead staining of spheroids within the hydrogel scaffold after one week of culture. (D) Quantification of cell spheroid viability. The scale bar represents 250 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66078/66078fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66078/66078fig04.jpg" /> 
Figure 4: Retrieval of acoustically fabricated cell spheroids. (A) Illustration depicting the steps for retrieving acoustically fabricated cell spheroids. (B) Bright-field images displaying the retrieved spheroids at different magnifications. The scale bar represents 250 &#181;m. (C) Analysis of viability and functionality of the retrieved spheroids. The scale bar represents 100 &#181;m. (D) Size distribution of the spheroids after retrieval. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66078/66078fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

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.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

1.1K Views.  Hangzhou Dianzi University. 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.

Video: Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

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.

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

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.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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.

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

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.

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

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using 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 ...

Biochemistry

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.

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.

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

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

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.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

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.

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

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Biology

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

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

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