Resurrection and cellular characterisation of 2D enteroids after cryopreservation. Immunolocalisation of 2D enteroids grown from cryopreserved intestine cells on Matrigel-coated transwells, on day 4 of culture. Z stack of co-immunolocalisation of F-actin (apical brush border, red) with intestinal epithelial cells (green); A ZO1 (Tight Junctions), B Villin (enterocytes), C Lysozyme (Paneth cells), D Muc5AC (Goblet cells) and E Chromogranin A (enteroendocrine cells). F Magnification of Z-stack of Muc5AC showing DAPI stained nuclei in basal cells (white arrow heads). Scale bars: 15 µm. G Vimentin expression (green) in basal mesenchymal cells in the absence of epithelial cells. Scale bar: 50 µm. Nuclei are stained with DAPI. The data represents three independent experiments. H, I Cells were thawed and grown as 2D enteroids in transwells; CHIR99021 was added to Maintenance Media for H 24 h and I 72 h. Data is the median of 3–4 independent experiments and 95% CI.