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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 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,

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 .......

Representative 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 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.......

Acknowledgements

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).

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Materials

NameCompanyCatalog NumberComments
0.22-μm filterMerckSLGSM33SSUsed for GelMA solution sterilization
35 mm-cell culture dishCorning430165Used for culturing cells
Confocal microscopeNikonA1RHD25Fluorescent cell observation
DiO dyeBeyotimeC1038Dye used to stain cells
DMEMGibco12430054Cell culture media
FBSGibco10099141CCell culture media supplement
Function generatorRigolDG5352For RF signal generation
GelMARegenovononeUsed to prepare bioink
GelMA lysis bufferEFLEFL-GM-LS-001Used to dissolve GelMA scaffolds
Inverted microscopeNikonTi-UCell observation
LAPSigma-Aldrich900889Used as photoinitiator
Live-Dead kitBeyotimeC2015MCell vability analysis
PBSGibco10010002Used as buffer
Penicillin-streptomycinGibco15070063Prevent cell culture contamination
Power ampliferMinicircuitLCY-22+Increase the voltage amplitude of the RF signal
PZT transducersYantai Xingzhiwen Trading Co.,Ltd.PZT-41Functional units for acoustic assembly device
T25 cell culture flaskCorning430639Used for culturing cells
Trypan blue Gibco15250061Cell counting
Trypsin-EDTA Gibco25200056Cell dissociation enzyme

References

  1. Eiraku, M., et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature. 472 (7341), 51-56 (2011).
  2. Lancaster, M. A., Knoblich, J. A. Organogenesis in a dish: modeling develop....

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