Transwell chamber with a 0.4 μm pore size permeable membrane

A Transwell® chamber with a 0.4-μm pore size permeable membrane (Corning) was used to co-culture the MCF-7 cells, MDA-MB-453, or MDA-MB-231 with the hAD-MSCs at a ratio of 1:1. The cells were continuously passaged when needed.
To detect the secretion of interleukin-6 (IL-6), TGF-β, and VEGF, hAD-MSCs with or without co-culture with MCF-7 for 7 days were plated in 6-well plates at a cell density of 2 × 10⁵/ml. After incubation in serum-free medium for 24 h, the supernatant was collected for analysis using enzyme-linked immunosorbent assay (ELISA) kits (NeoBioscience, China) according to the manufacturer’s instructions.

A Transwell chamber with a 0.4 μm pore size permeable membrane (Cat. #3450, Corning, Corning, USA) was used to co-culture MSCs with colorectal cancer cells (HCT-8, HCT-116 and LOVO). The MSCs were plated in the lower chamber of a six-well transwell plate at a density of 2 × 10⁵/ml, and colorectal cancer cells (2 × 10⁵/ml) were plated in the upper chamber. All cells were incubated at 37 °C in a 5% CO₂ humidified incubator. After 7 day of co-culture, the MSCs could be induced to CAFs for subsequent experiments.
To avoid the effect of the HIF-1α selective protein inhibitor on tumor cells during co-culture, conditioned medium was used instead of co-culture. The culture medium of colorectal cancer cells after 24 h of culture was collected as the conditioned medium for the following experiment. Previous studies suggested that the effect of conditioned medium was similar to the effect of direct co-culture [22 (link), 23 (link)].