*5.3. Spheroid Preparation and Culture*

Composite spheroids containing a mixture of tumor cells A549 and MRC-5 fibroblasts were prepared based on the hanging drop method as described earlier [13,35]. Sub-

confluent cells were trypsinized and resuspended in DMEM with 10% FBS to a concentration of 1 × 106/mL. Cell suspension of fibroblasts and tumor cells were prepared with or without 12.5 nM of CTX and then mixed at the ratio of 4:1 (4 fibroblasts per 1 tumor cell). Approximately 40 drops (25 mL/drop, 2.5 × 104 cells) were dispensed onto a lid of a cell culture dish. The lid was then inverted and placed over a cell culture dish containing a culture medium for humidity. For spheroid optimization, it was added to suspension cells 0.24% of methylcellulose so that it can contribute to more compact and circular spheroid morphology. The lid was incubated for 24 h, at 37 ◦C, and 5% CO2.
