Microfluidic Control of Tumor and Stromal Cell Spheroids Pairing and Merging for Three-Dimensional Metastasis Study

Three-dimensional cell culture provides an efficient way to simulate the in vivo tumorigenic microenvironment where tumor-stroma interaction intrinsically plays a pivotal role. Conventional three-dimensional (3D) culture is inadequate to address precise coexistential heterogeneous pairing and quantitative measurement in a parallel algorithm format. Herein, we implemented a set of microwell array microfluidic devices to study the cell spheroids-based tumor-stromal metastatic process in vitro. This approach enables accurate one-to-one pairing between tumor and fibroblast spheroid for dissecting 3D tumor invasion in the manner of high-content imaging. On one single device, 240 addressable tumor-stroma pairings can be formed with convenient pipetting and centrifugation within a small area of 1 cm². Consequential confocal imaging analysis disclosed that the tumor spheroid could envelop the fibroblast spheroid. Specific chemicals can effectively hamper or promote this 3D metastasis. Due to the addressable time-resolved measurements of the merging process of hundreds of doublets, our approach allows us to decipher the metastatic phenotype between different tumor spheroids. Compared with traditional protocols, massive heterogeneous cellular spheroids pairing and merging using this method is well-defined with microfluidic control, which leads to a favorable high-content tumor-stroma doublet metastasis analysis. This simple technique will be a useful tool for investigating heterotypic spheroid-spheroid interactions.