Peter van Mourik

90 Chapter 4 E. First week of culture Note: Crypts close quickly and start to proliferate (visible as “budding”) after several days (see Figure 6). 1. Often check wells for infections. When any of the wells shows clear signs of infection, aspirate the medium from the well and add 1 mL ethanol for 30 s. Aspirate ethanol and matrigel drops and re-add 1 mL of ethanol for 30 s. Aspirate and leave well empty (important: leaving ethanol creates ethanol vapour in the plate, which leads to cell death in the other wells). This approach prevents cross contamination of other wells from this patient sample. 2. Refresh the wells with 500 µL colon organoid medium +/+ with additional gentamicin (50 µg/mL) and vancomycin (50 µg/mL) every 2 - 3 days (e.g. Monday, Wednesday, Friday). If crypts are isolated on a Friday, add 1 mL CM +/+ instead of 500 µL (with additional gentamicin and vancomycin) to ensure optimal growth during the weekend Note: To remove the media, use a new tip for every well to decrease infection and well to well contamination risks. 3. Once the crypt structures are budding, closed and proliferated (see figure 6), passage them as described in 5.1. This can usually be performed around day 6 - 8. Note: FIS experiments should only be performed after a minimum of two passages after isolation. Figure 6. Development of crypt into budding organoid structure on day 1; 3; 4 and 7 (passage 0). Arrows point to proliferating crypt and subsequent organoid structure (bright field, 4 x magnification).