Name: Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids
Uploaded: 2023-10-13T12:30:00
Description: Watch this Scientific Journal Video about Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids at JoVE.com

bioink preparation, acoustic device assembly, and viability analysis of cell spheroids

3D音響アセンブリデバイスを用いた均一細胞スフェロイドのスケーラブルな生成

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.

著者スポットライト:高品質3D細胞スフェロイド培養のためのスキャフォールドフリー音響アセンブリ法の開発 

細胞スフェロイドの量産のための3次元音響組立装置

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 Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids at JoVE.com

Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of Cell Spheroids  

バイオインク調製、音響デバイスアセンブリ、および細胞スフェロイドの生存率分析

バイオインク溶液を調製するには、適量のC3A細胞懸濁液を滅菌したゲルMA溶液と混合します。C3A細胞スフェロイドを2マイクロモルのセルトラッカー溶液で37度で20分間染色します。細胞を洗浄するには、まず遠心分離によってスーパーナチンを除去し、次に新しい細胞培養培地を加えます。滅菌した音響装置チャンバーに少なくとも2ミリリットルのバイオインクを追加します。関数発生器とパワーアンプをオンにして、各PZTトランスデューサの作動を開始し、細胞凝集体の3Dアレイを生成します。青色光を使用してバイオインクを30秒間架橋し、3Dハイドロゲル足場を作成し、組み立てられた細胞凝集体を音響的にカプセル化します。次に、3Dハイドロゲルスキャフォールドをチャンバーからペトリ皿に慎重に移します。きれいなカミソリで細かく切ってから、ペトリ皿に細胞培養培地を入れます。1マイクロリットルのカルシウムamと2マイクロリットルのヨウ化プロピジウムを含む1ミリリットルのPBS中で、C3A細胞スフェロイドを異なる培養時点で摂氏37度で15分間インキュベートします。ハイドロゲル足場に埋め込まれたサンプルをPBSで2回すすぎます。回収したスフェロイドについては、200 Gで5分間遠心分離を行い、PBSで2回洗浄します。蛍光顕微鏡を使用して、回転楕円体を観察し、画像を取得します。細胞凝集体を、緑色蛍光シグナルを有する規則的な3Dドットアレイパターンで配置した。組み立てられたC3A gelMA凝集体は徐々に統合され、スフェロイド直径の増加を伴って3日目までにタイトなスフェロイドを形成しました。細胞スフェロイドの生存率は3日目までは良好であったが、1週間の培養後にわずかに低下した。放出されたスフェロイドは、アルブミンの望ましい生存率および発現とともに、狭いサイズ分布で良好な球形形態を維持しました。

Research

JoVE Journal

Bioengineering

Bioink Preparation, Acoustic Device Assembly, and Viability Analysis of 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 ...