Intestinal organoid culture, fixation, and paraffin embedding

Intestinal organoid culture was performed as described previously (Sato et al, 2009; Heuberger et al, 2014). Briefly, jejunal crypts were isolated by filtration (70 μm) and centrifugation (400 g/3 min) of selected fractions after mechanical dissociation (shaking) of the villi and crypts after 5-min incubation at room temperature with 8 mM and 2 mM EDTA and at 25-min rotation at 4°C, respectively. We embedded 400 crypts in 50 μl Matrigel (BD, 356231) and cultured them in DMEM/F12 medium (12634; Life Technologies) supplemented with N2 and B27 (17502-040 and 17504-044, respectively; Life Technologies), mNoggin (Cat. No. 250-38, final concentration 100 ng/ml; PeproTech), mEGF (mouse epidermal growth factor, PMG 8041, final concentration 50 ng/ml; Life Technologies), hrSpo1 (human rSpo1, Cat. No. 120-38, final concentration 100 ng/ml; PeproTech), and acetylcysteine (A9165, final concentration 1.25 mM; Sigma-Aldrich). Cre-mediated recombination was induced by administering 800 nM 4-OHT for 2 consecutive days.

Growth of individual organoids was tracked with a Leica DIM6000 microscope equipped with an NPlan 10× NA 0.25 objective and a motorized LMT200 V3 High precision Scanning Stage to relocate multiple times previously stored positions. Growth of organoids by total cell numbers was measured with the NucleoCounter NC-200 from Chemotec. Defined cell numbers of organoids were seeded and cultured for 4 d. Cells of organoids were harvested directly from the Matrigel using buffer A100 (4 min incubation and repeated trituration) and buffer B according to the manufacturer’s description.

Organoids containing Matrigel were disintegrated by trituration and transferred to 5 ml of cold DMEM/F12 medium. After centrifugation, the organoids were resuspended for 3 h in 4% PFA/PBS. The fixative was exchanged with PBS, and the organoids were embedded in 2% agarose/PBS and transferred to 70% ethanol, followed by paraffin embedding. Paraffin sections (5–10 μm) were obtained for histological analysis.