Spheroid Formation: Cell Co-culture Protocol

Spheroid preparation was performed according to Augustin and Korff 1998 [31 (link)]. Six g methocel was dissolved in 500 mL M199 medium (supplemented with 1% L-glut, 1% PS and 10% FCS). The combined solution was stirred overnight at 4 °C and centrifuged at 3500× g for 3 h. Meanwhile, the cells were labelled with Cell Tracker™ according to the manufacturer’s instructions (hOBs with Cell Tracker™ Green and HUVECs with Cell Tracker™ Red). One part of the methocel solution (2.4 mL/96-well plate) was diluted with four parts medium (9.6 mL/96-well plate) and the cell suspension (for 1 × 96-well: 6 × 10⁴ cells per mono-culture or 3 × 10⁴ of each cell type per co-culture) was added. After gently mixing the cells, they were seeded in a 96-well plate with U-bottom (100 μL/well corresponding to 500 cells/well) and incubated overnight. On the next day, the spheroids had formed.