Name: Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids
Uploaded: 2023-10-13T12:30:00
Description: Watch this Scientific Journal Video about Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids at JoVE.com

setting up an acoustic assembly system and cell culture harvesting for generating uniform cell spheroids

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

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.

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

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

3D in vitro culture models, like cell spheroids, have been widely used in the fields of tissue engineering, disease modeling, and drug screening. Our research focuses on developing and invest per engineering method to fabricate spheroids or other 3D in vitro culture models with high quality for diverse biological studies. In the current protocol, the spheroids grew within the GelMA scaffold and are retrieved at the final layback, dissolving the scaffold, causing additional procedures.We will try to acoustically assemble cell aggregates in a cultural medium to enable them to grow within spheroids without scaffold. Compared to magnetic assembly techniques, acoustic waves can directly assemble cells in a label-free manner without potential toxic effects on cells. Besides, our protocol enables cell spheroids to be assembled in a three-dimensional array, substantially increasing throughput.

Watch this Scientific Journal Video about Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids at JoVE.com

Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids  

To begin, sterilize the chamber of the acoustic device with 75%alcohol for five minutes. Then, clean the device with sterile PBS and irradiate it with UV light for one hour. Mount the sterile acoustic device onto a microscope stage for top-view observation of the chamber's interior.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. Independently connect the wires from the three PZT transducers in series to three power amplifiers and three output channels of function generators. Program the settings on the function generators for each PCT transducer, specifying parameters such as sinusoidal waveforms, frequency, and amplitude.Culture 3CA cells in DMEM supplemented with 10%FBS and 1%penicillin streptomycin in a T25 cell culture flask. When the cells become 80%confluent, wash the culture twice with PBS. After removing the PBS, add two milliliters of 0.05%trypsin-EDTA to the flask and incubate at 37 degrees Celsius for cell detachment.Add two milliliters of complete cell culture medium into the flask to stop the trypsinization. Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 G for five minutes.

Research

JoVE Journal

Bioengineering

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