Peter van Mourik

70 Chapter 4 When the cells in the five T175 flasks containing DMEM +/+ are 90 -100% confluent (usually after 3 - 4 days), split the cells in 145 mm petri dishes: 12. Remove DMEM +/+ medium from the flasks and wash once with 5 mL PBS0. 13. Add 0.05 % Trypsin EDTA and leave at 18-23 ° C for 7 - 10 min. 14. Add 3.5 mL of DMEM +/+ per flask, pool the cells from all five T175 flasks, and resuspend until a homogenous cell suspension is achieved (total volume = 25 mL DMEM +/+ medium with cells). 15. Measure the cell concentration per mL and dilute the cell suspension needed for seeding in 50 145 mm tissue culture petridishes to create one (the first) batch with 1100 ml of WCM. For 50 145 mm (2 x 10 6 cells per 22 ml * 50=) 100 x 10 6 cells are needed in 1100 ml. o Determine the cell concentration (with e.g. hemocytometer) [in #cells/ mL] = Y o Calculate 100/ Y = X, the volume of the cell suspension to dilute in 1100 ml. 16. Make sure the cell suspension is homogeneous and add 22 mL of cell suspension per 145 mm tissue culture petridish. Note: When more than the necessary amount of cells is counted, a part of the cell suspension may remain unused or extra 145 mm petridishes can be seeded. 17. Incubate the cells at 37 °C, 5 % CO 2 for 8 days (do not harvest the WCM/ DMEM +/+ before day 8) 18. Harvest the WCM from the petridishes in sterile 50 mL tubes and discard the petridishes with cells. 19. Centrifuge for 5 min at 650 g to remove floating cells and debris. 20. Filter medium through a 0.22 µm filter (stericups are preferable). 21. Store the WCM in 50 mL tubes or in 0.5 – 1 L containers at 4 °C. 22. Repeat the splitting of a 1 x T175 flask for the next batch as described above for 2-3 batches per WCM production. 23. WCM can be stored at 4°C and used for 2 months. Testing and approving of the WCM batches: WCM quality can be tested by culturing organoids with this specific WCM batch. Differentiated, non- budding round, thick-walled organoid structures indicate low Wnt activity (also see figure 10C). Running human colon organoid cultures often need time to adjust to a new batch of WCM. Judge organoid cultures after 2 weeks before making conclusions on the WCM quality. Note: A running L-wnt3a cell line can be used for the production of multiple batches of WCM until passage 18 - 20.