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 ...

Hangzhou Dianzi University

Blood tests have been a powerful tool for the clinical analysis of many diseases. With the advances in microfluidic technology, two more specific indicators from the circulation system, namely, emerging "liquid biopsy" of circulating tumor cells (CTCs) and fetal nucleated red blood cells (fNRBCs), can be screened and analyzed as a simple blood test for the noninvasive diagnosis of cancers as well as fetal disorders. The unique feature of precisely manipulating a trace of fluid endows microfluidic devices with the ability to isolate CTCs or fNRBCs from numerous blood cells with high performance, which undoubtedly facilitates biomedical applications of these two kinds of rare cells. In this review, advanced developments in microfluidic technologies focusing on the detection and sorting of rare CTCs and fNRBCs from peripheral blood are summarized. The development of microfluidic devices incorporated with various multifunctional microstructures and nanomaterials for enhancing the sensitivity, purity, and viability of CTC or fNRBC detection enables CTC molecular analysis and fNRBC-based noninvasive prenatal diagnosis (NIPD). These microfluidics-based approaches provide great potential opportunities in noninvasive cancer diagnosis or NIPD applications.

Emerging Microfluidic Technologies for the Detection of Circulating Tumor Cells and Fetal Nucleated Red Blood Cells.

Pancreatic ductal adenocarcinoma (PDAC) having features of dense fibrotic stromal and extracellular matrix (ECM) components has poor clinical outcome.  construction of relevant preclinical PDAC models recapitulating the tumor-stroma characteristics is therefore in great need for the development of pancreatic cancer therapy. In this work, a three-dimensional (3D) heterogeneous PDAC microtissue based on a dot extrusion printing (DEP) system is reported. Gelatin methacryloyl (GelMA) hydrogel beads encapsulating human pancreatic cancer cells and stromal fibroblasts were printed, which demonstrated the capacity of providing ECM-mimetic microenvironments and thus mimicked the native cell-cell junctions and cell-ECM interactions. Besides, the spherical structure of the generated hydrogel beads, which took the advantage of encapsulating cells in a reduced volume, enabled efficient diffusion of oxygen, nutrients and cell waste, thus allowing the embedded cells to proliferate and eventually form a dense pancreatic tumor-stroma microtissue around hundred microns. Furthermore, a tunable stromal microenvironment was easily achieved by adjusting the density of stromal cells in the hydrogel beads. Based on our results, the produced heterogeneous pancreatic microtissue recapitulated the features of cellular interactions and stromal-like microenvironments, and displayed better anti-cancer drug resistance than mono-cultured pancreatic cancer spheroids. Together, the DEP system possesses the ability to simply and flexibly produce GelMA hydrogel beads, providing a robust manufacturing tool for the pancreatic cancer drug screening platform fabrication. In addition, the engineered pancreatic tumor-stroma microtissue based on bioprinted GelMA hydrogel beads, other than being ECM-biomimetic and stroma-tunable, can be used for observation  and may serve as a new drug screening platform.

Bioprinting of hydrogel beads to engineer pancreatic tumor-stroma microtissues for drug screening.

Acoustic cell assembly devices are applied in cell spheroid fabrication attributed to their rapid, label-free and low-cell damage production of size-uniform spheroids. However, the spheroids yield and production efficiency are still insufficient to meet the requirements of several biomedical applications, especially those that require large quantities of cell spheroids, such as high-throughput screening, macro-scale tissue fabrication, and tissue repair. Here, we developed a novel 3D acoustic cell assembly device combined with a gelatin methacrylamide (GelMA) hydrogels for the high-throughput fabrication of cell spheroids. The acoustic device employs three orthogonal piezoelectric transducers that can generate three orthogonal standing bulk acoustic waves to create a 3D dot-array (25 × 25 × 22) of levitated acoustic nodes, enabling large-scale fabrication of cell aggregates (>13,000 per operation). The GelMA hydrogel serves as a supporting scaffold to preserve the structure of cell aggregates after the withdrawal of acoustic fields. As a result, mostly cell aggregates (>90%) mature into spheroids maintaining good cell viability. We further applied these acoustically assembled spheroids to drug testing to explore their potency in drug response. In conclusion, this 3D acoustic cell assembly device may pave the way for the scale-up fabrication of cell spheroids or even organoids, to enable flexible application in various biomedical applications, such as high-throughput screening, disease modeling, tissue engineering, and regenerative medicine.

High-throughput fabrication of cell spheroids with 3D acoustic assembly devices.

3D cell culture models replicating the complexity of cell-cell interactions and biomimetic extracellular matrix (ECM) are novel approaches for studying liver cancer, including in vitro drug screening or disease mechanism investigation. Although there have been advancements in the production of 3D liver cancer models to serve as drug screening platforms, recreating the structural architecture and tumor-scale microenvironment of native liver tumors remains a challenge. Here, using the dot extrusion printing (DEP) technology reported in our previous work, we fabricated an endothelialized liver lobule-like construct by printing hepatocyte-laden methacryloyl gelatin (GelMA) hydrogel microbeads and HUVEC-laden gelatin microbeads. DEP technology enables hydrogel microbeads to be produced with precise positioning and adjustable scale, facilitating the construction of liver lobule-like structures. The vascular network was achieved by sacrificing the gelatin microbeads at 37 °C to allow HUVEC proliferation on the surface of the hepatocyte layer. Finally, we used the endothelialized liver lobule-like constructs for anti-cancer drug (Sorafenib) screening, and stronger drug resistance results were obtained when compared to either mono-cultured constructs or hepatocyte spheroids alone. The 3D liver cancer models presented here successfully recreate liver lobule-like morphology, and may have the potential to serve as a liver tumor-scale drug screening platform.

Keke Chen

School of Automation,
Key Laboratory of Medical Information and 3D Bioprinting of Zhejiang Province

Hangzhou Dianzi University

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

Keke Chen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Automation,Key Laboratory of Medical Information and 3D Bioprinting of Zhejiang Province

Hangzhou Dianzi University

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

School of Automation,
Key Laboratory of Medical Information and 3D Bioprinting of Zhejiang Province