Peter van Mourik

72 Chapter 4 17. Harvest the Ad-DF+++ (NCM) on day 8 -10. Never harvest NCM before day 8 or when cells are not at least 100 % confluent. 18. Pool the NCM from the T175 flasks in sterile 50 mL tubes and discard the T175 flasks. 19. Centrifuge the NCM 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 NCM in 50 mL tubes or in 0.5 – 1 L containers at -20 °C . Note: A running HEK293T-Noggin-hFc cell line can be used for the production of multiple batches of NCM until passage 18 - 20. Note: No properly validated NCM quality/ quantity assay has yet been developed. After testing the functionality (on human colon organoid cultures) the NCM batches can be thawed, pooled and refrozen in aliquots and are stable at -20 ºC for at least one year. Try to avoid unnecessary freeze-thaw cycles. Note: Different NCM batches can show much variation in Noggin activit. It is advisable to produce multiple batches and mix an NCM pool to ensure a minimal efficiency level of active NCM.