3D Organotypic Culture of Spheroids

MDA-MB-231 and MCF-10A spheroids were generated as previously described^{31 (link)}. Briefly, spheroids were generated by seeding approximately 1 × 10^{3 (link)} cells in each of the 96 wells of an ultra-low attachment plate (Corning, No. 7007) and allowed to form for 48 h in the presence of 2.5% v/v Matrigel. Once formed, individual spheroids surrounded by 5 µl of media were transferred onto coverslips inside PDMS wells (9 mm in diameter) created in 35 mm glass-bottom petri dishes (one spheroid per dish). Each spheroid was covered by 195 µl of ice-cold, rat-tail collagen I solution to achieve a total volume of 200 µl and a specific collagen concentration in each well. Collagen solutions were prepared by mixing acid-solubilized collagen I (Corning, No. 354249) with equal volumes of a neutralizing solution (100 mM HEPES buffer in 2 × PBS). The desired collagen concentration was reached by adding adequate volumes of 1 × PBS. Collagen solutions at different concentrations (1 and 4 mg/ml) polymerized for 1 h at 37 °C. The cell culture plates were rotated every minute for the first 10 min of polymerization to guarantee full embedding of the spheroid within the 3D collagen matrix. Finally, 2 ml of culture media (phenol-free, 50:50 v/v of MDA-MB-231 media to MCF-10A media) was added and the 3D organotypic culture was placed inside the incubator until taken out for FLIM measurements.