Figure 5.

Application of an oxygen gradient across an in vitro crypt. a) Schematic diagram of a single in vitro crypt with application of a chemical and oxygen gradient. Stem/proliferative cells are depicted in green while the differentiated cells in red. A single layer of cells lines the surface of the crypt-shaped scaffold. b) Whole-mount fluorescence images of a side view of two in vitro crypts. In the left panel, the crypt under a chemical gradient, but no applied oxygen gradient, i.e aerobic conditions. In the right panel, the crypt under both a chemical and oxygen gradient. The crypts were stained for pimonidazole adducts (red), EdU incorporation (green), and Hoechst 33342 (blue). c) The normalized pimonidazole adduct formation plotted against the location of the cells along the crypt long axis under different oxygenation conditions. The Y-axis shows the cell location with 0 being the base of the crypt (i.e. basal side) and 10 representing the top of the crypt at the luminal reservoir. Total crypt length was 600 μm. A total 25 individual crypts from 3 different arrays were analyzed for each condition. * represents p<0.01. d) The normalized EdU incorporation plotted against cell location along the crypt axis for different oxygen environments. The normalized EdU incorporation was calculated by dividing the EdU fluorescence intensity by the Hoechst 33342 fluorescence intensity. The aerobic and gradient data were significantly different (p<0.01) for all distances between 0 and 0.8 of the crypt length. A total of 30 (for aerobic) and 32 (for gradient) individual crypts from 3 different arrays were analyzed. e) The relative distribution of the EdU-labeled cells with respect to the location of the cell along the crypt axis. Three different arrays were analyzed for the aerobic (30 crypts total) and the gradient (32 crypts total) samples. Between 0–0.2 and 0.4–0.7 of the crypt length, the p value for the data comparisons p<0.05. f) Growth of B. adolescentis at 0 and 24 h of culture above the in vitro crypts under the fully oxygenated or oxygen gradient environment from 2 technical replicates for the aerobic condition and 3 for the gradient condition.