Effect of Matrigel concentration on in vitro expansion of colonic crypts into 3-D colonoids. (A) Serial overlaid brightfield and eGFP fluorescence images of the same colonoid over 1 week in culture. Scale bar = 50 μm. (B) Effect of Matrigel concentration on the percentage of crypts growing into colonoids over 1 week of culture. Squares, triangles, circles and diamonds represent 25%, 50%, 75% and 100% Matrigel, respectively. 50% Matrigel provides the optimum 3-D growth environment for the colonoids. (C) Quantification of the cell properties in the colonoids. Shown is the colonoid volume (left y axis, black) staining positive for Muc-2, ChgA or EdU divided by that positive for Hoechst 33342 when colonoids were cultured in 100% (filled bars) or 50% (open bars) Matrigel. The volume of the colonoid expressing eGFP relative to that expressing dsRed is shown on the right y-axis (grey) for colonoids cultured in 100% (filled bars) or 50% (open bars) Matrigel. (D-E) Colonoids were cultured for 1 week and then stained by immunohistochemistry for: (D) mucin-2 (goblet cell marker: green) and (E) chromogranin-A (enteroendocrine marker: green). (F) A crypt obtained from a Sox9 eGFP-CAGDsRed mouse was cultured for 1 week and then imaged for eGFP fluorescence. (G) Fluorescence image of a colonoid (1 week culture) after an 8-hour EdU pulse (red). Hoescht 33442 was used as a nuclear stain (blue) in panels C-G. Scale bar = 75 μm.