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

Summary

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.

Abstract

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.

Introduction

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 screening^1,2,3. 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 interactions^4,5,6. 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 size^10,11. 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 technique^12,13,14, have shown potential for mass manufacturing of cell spheroids with high quality and throughput, based on the principle of enhancing cell aggregation through external forces^15,16,17,18. Unlike electromagnetic or magnetic forces, acoustic-based cell manipulation techniques are non-invasive and label-free, enabling spheroid formation with excellent biocompatibility^19,20.

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 fields^21,22,23. Particularly, acoustic assembly devices based on BAWs, with the merits of convenient manufacture, easy operation, and excellent scalability, have gained attention for fabricating cell spheroids^24,25. 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 ANs^27,28,29, 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.

Protocol

1. Fabrication of the 3D acoustic assembly device

1. 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.
2. Next, attach another 1 mm thick PMMA sheet to the bottom of the chamber to serve as a holder for the bioink.
3. 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.
4. Solder wires to the two conductive areas of each PZT transducer.
5. Finally, secure the device onto a hollow base to prevent the bottom PZT from coming into contact with other countertops.

2. Setting up the acoustic assembly system

1. Begin by mounting the acoustic device onto a microscope stage, allowing for top-view observation of the chamber's interior.
2. 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.
3. 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).
4. Program the settings on the function generators for each PZT transducer, specifying parameters such as sinusoidal waveform, frequency, and amplitude.
5. 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.

3. Cell culture and harvest procedure

1. Begin by culturing C3A cells, a human hepatocellular carcinoma cell line, in a T25 cell culture flask using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (see Table of Materials).
2. 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 °C to facilitate cell detachment. Stop the trypsinization process by adding 2 mL of complete culture medium.
3. Transfer the cell solution to a 15 mL tube, and centrifuge it at 200 × g for 5 min at room temperature to obtain a cell pellet.

4. Preparation of the bioink

1. 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 °C water bath for 1 h.
2. Before mixing with cells, pass the GelMA solution through a 0.22 µm filter (see Table of Materials) for sterilization.
3. 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.
4. 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 × 10⁶ cells/mL.
5. For visualization of the assembled cell spheroids, pre-stain C3A cells by incubating them with 2 µM cell tracker (DiO dye, see Table of Materials) at 37 °C for 20 min. Subsequently, wash the labeled cells with fresh cell culture medium three times before use.

5. Assembling the cell spheroids using the acoustic device

1. 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 (λ) can be estimated using the formula λ = c/f, where c represents the speed of sound in the medium, and f is the frequency of the PZT transducer.
2. 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.
3. 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.
4. Carefully transfer the 3D hydrogel scaffold from the chamber into a Petri dish and cut it into small pieces (e.g., 2 mm × 2 mm × 2 mm) using a clean razor.
5. Add cell culture medium for cultivation.
   ​NOTE: Remember to change the culture medium every day.

6. Retrieving cell spheroids

1. Begin by observing spheroid formation at different layers of the scaffolds using an inverted microscope.
2. After 3 days of culture, remove the culture medium and thoroughly wash the hydrogel scaffolds twice with PBS.
3. 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.
4. Transfer the solution from the previous step into a tube and then centrifuge it at 200 × g for 5 min at room temperature to obtain the cell spheroid pellet.
5. Discard the supernatant and resuspend the pellet in fresh culture medium for downstream applications.

7. Spheroid viability analysis

1. 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).
2. Incubate the samples at different culture time points with 1 mL of PBS solution containing 1 µL of Calcein-AM and 2 µL of Propidium Iodide (PI) for 15 min at 37 °C.
3. For samples within the hydrogel scaffolds, wash them directly with PBS twice. For retrieved spheroids, centrifuge at 200 × g for 5 min at roomtemperature to obtain a spheroid pellet, and wash it twice before observation.
4. Observe the samples using a fluorescence microscope and capture the fluorescence images.
5. 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.

Results

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'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 (

Discussion

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 screening^1,2,3. 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...

Materials

Name	Company	Catalog Number	Comments
0.22-μm filter	Merck	SLGSM33SS	Used for GelMA solution sterilization
35 mm-cell culture dish	Corning	430165	Used for culturing cells
Confocal microscope	Nikon	A1RHD25	Fluorescent cell observation
DiO dye	Beyotime	C1038	Dye used to stain cells
DMEM	Gibco	12430054	Cell culture media
FBS	Gibco	10099141C	Cell culture media supplement
Function generator	Rigol	DG5352	For RF signal generation
GelMA	Regenovo	none	Used to prepare bioink
GelMA lysis buffer	EFL	EFL-GM-LS-001	Used to dissolve GelMA scaffolds
Inverted microscope	Nikon	Ti-U	Cell observation
LAP	Sigma-Aldrich	900889	Used as photoinitiator
Live-Dead kit	Beyotime	C2015M	Cell vability analysis
PBS	Gibco	10010002	Used as buffer
Penicillin-streptomycin	Gibco	15070063	Prevent cell culture contamination
Power amplifer	Minicircuit	LCY-22+	Increase the voltage amplitude of the RF signal
PZT transducers	Yantai Xingzhiwen Trading Co.,Ltd.	PZT-41	Functional units for acoustic assembly device
T25 cell culture flask	Corning	430639	Used for culturing cells
Trypan blue	Gibco	15250061	Cell counting
Trypsin-EDTA	Gibco	25200056	Cell dissociation enzyme