Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts: A Method to Characterize the Gastrointestinal Stem Cell Niche

Figure 6

Cardia organoid cultures and cocultures with neurons as a model to investigate the gastrointestinal stem cell niche. (a) IHC for H&E, PAS, Ki-67, and α-SMA for further characterization of the method, arrowheads indicating positive stained cells, scale bars, 100 μm. (b) Light microscopy of Lgr5-GFP-positive organoid, scale bar, 50 μm. (c) Immunofluorescence of Dclk1-labeled neurons in cultured conditions (NBM). (d) Cultured neurons (NBM) stained with beta-III Tubulin, scale bar, 100 μm. (e) Cocultured neuron with cardia organoid, light microscopy and immunofluorescence, Dclk1-labeled neuron (green fluorescence) neighboring crypt wall, scale bar, 100 μm. (f) Ca-imaging of neuron in cocultured conditions (NBM), different points in time beginning from application of nicotinic acid to Fluo-4-labeled neurons, brightening of neuron indicating Ca-influx. Scale bar, 50 μm. (g) Analysis of the RLI of stimulated neuron, ROI I, ROI II, and ROI III measuring different regions of the neuron as indicated in (f), picture 2 (regions labeled 1, 2, and 3). ((h), (i)) Analysis of organoids and cocultured neurons in different conditions, distribution of mean diameters and circumferences per group and point in time and standard error of the mean (SEM) (CM = crypt medium, CaM = Wnt-conditioned cardia medium, NBM = cocultured neurons in neurobasal complete medium, groups compared at 7 d and 10 d), measured in μm; comparison of average organoid growth between groups applying two-tailed -test (level of statistical significance, ), experiments per group and point in time. (j) Representative light microscopic image of each group and point in time (arrowheads indicating neurons), scale bar, 100 μm.

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