Figure 1.

Experimental overview and growth of colonoids cultured in high and low oxygen. (A) General graphic representation of the experimental design. Colonoids were dissociated into single cells before plated at a density of 8000-10000 cells/50 ml matrigel and cultured in parallel in two incubators with 2% or 20% oxygen. Complete growth medium (CGM) was added every other day from establishment until 9 days, including ROCK inhibitor Y-27632 for the first two changes. On day nine (unless specified otherwise), cell differentiation was induced in half of the wells by replacing CGM with differentiation medium, while the other half continued with CGM. The colonoids were kept in culture until day 14 unless they were treated with pro-inflammatory TNF + poly(I:C). The blue line represents colonoids cultured in a 2% oxygen environment, while the red line represents cultivation in a 20% oxygen environment. As illustrated, stem cells began as single cells, proliferated into spheroids, and ultimately differentiated into 3D structured colonoids with crypts and a central lumen. For the stimulation assays, the colonoids were cultured as described until day 14. Subsequently, they were treated with TNF + poly(I:C) for 24 hours before the material (RNA and conditioned medium) was collected. (B) Representative brightfield images (10x magnification, EVOS microscope) of colonoid growth. The four image rows represent the four different conditions (undifferentiated or differentiated in 2% and 20%) cultured in parallel. The day the image was captured is indicated above the images. The white arrows follow a cell from a single cell to a 3D colonoid. Scale bar = 100 μm.