Abstract
Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the “Lab-on-a-Cloud” scenario for routine study.
Highlights
In the cellular biological study, it is generally recognized that the flask-based cell culture approaches are intrinsically not able to recreate cellular architectures found in organisms[1,2,3]
Four different assays have been developed based on 3D-printed hanging drop dripper (3D-phd) platform: (i) Specific components for drug screening can be added through device, and cell toxicity imaging was directly acquired by dripping down treated cell spheroid with specific staining; (ii) 3D metastasis of cell spheroid on/in matrix could be performed on a matrix gel-coated plate or dropping off with cell-matrix gel solution directly; (iii) The 3D tumor spheroid based transendothelial migration analysis; (iv) Heterotypical spheroids interaction by using the double nozzles 3D-phd
Conventional ways to generate 3D tumor spheroids focused on creating an effective method to produce a significant amount of size-controllable cell aggregates
Summary
In the cellular biological study, it is generally recognized that the flask-based cell culture approaches are intrinsically not able to recreate cellular architectures found in organisms[1,2,3]. We developed a desktop 3D-printed hanging-drop dripper (3D-phd) device that allows riding on 96/384-well plate for uniformly generating cell spheroids, long-term culturing, drug testing, and in situ analysis of tumor migration and invasion in ECM niche. Due to the dripping-like collection of cultured spheroids, our platform is seamlessly compatible with many assays, such as drug-induced cell death by inverted confocal microscopy, metastasis on ECM surface or embedded in ECM gel, and tumor cell transendothelial migration within ECM microenvironment To our knowledge, this is the first demonstration of a 3D printed device for hanging drop generating cell aggregates and subsequently used for a variety of tumor-based assays without recovery
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