Simultaneous 2D and 3D cell culture array for multicellular geometry, drug discovery and tumor microenvironment reconstruction

Cell culture systems are indispensable in vitro tools for biomedical research. Although conventional two-dimensional (2D) cell cultures are still used for most biomedical and biological studies, the three-dimensional (3D) cell culture technology attracts increasing attention from researchers, especially in cancer and stem cell research. Due to the different spatial structures, cells in 2D and 3D cultures exhibit different biochemical and biophysical phenotypes. Therefore, a new platform with both 2D and 3D cell cultures is needed to bridge the gap between 2D and 3D cell-based assays. Here, a simultaneous 2D and 3D cell culture array system was constructed by microprinting technology, in which cancer cells exhibited heterozygous geometry structures with both 2D monolayers and 3D spheroids. Cells grown in 3D spheroids showed higher proliferation ability and stronger cell-cell adhesion. Spheroids derived from various types of cancer cell lines exhibited distinct morphologies through a geometrical confinement stimulated biomechanical transduction. Z-projected images of cancer cell aggregates were used to analyze 3D multicellular architecture features. Notably, by using a support vector machine classifier, we distinguished tumor cells from normal cells with an accuracy greater than 95%, according to the geometrical features of multicellular spheroids in phase contrast microscopy images. Cancer cells in multicellular spheroid arrays exhibited higher drug resistance of anticancer drug cisplatin than cells grown in 2D cultures. Finally, we developed a co-culture system composed of tumor spheroid arrays, fibroblast cells and photo-crosslinkable gelatin methacryloyl hydrogel to mimic tumor microenvironment which consisted of solid tumor massed, surrounding stromal cells and extracellular matrix. Together, our newly developed simultaneous 2D and 3D cell culture array has great potential in comprehensive evaluation of cellular events in both 2D and 3D, rapid production of spheroid arrays and multicellular geometry-based tumor cell detection.