Peter van Mourik

88 Chapter 4 3. Place the tube on a roller mixer for 90 - 120 min at 4 °C. The incubation time is dependent on the time between the biopsy collection and the start of the crypt isolation procedure. The longer the interval between biopsy collection and isolation procedures, the longer it is required to incubate. Crypts can be isolated from colon biopsies up until 48 h after biopsy collection but crypt yield decreases over time. C. Isolate intestinal crypts from biopsies 1. Allow the colon biopsies to settle at the bottom of the tube. 2. Discard the supernatant (PBS0 + EDTA). 3. Add 3 mL cold PBS0 to the tube containing the biopsies and pipet the biopsies up and down vigorously 10 - 20 times. Crypts are released from the biopsies and float in the PBS0, which is visible by eye through the observation of the solution becoming more cloudy. If no crypts emerge from the colon biopsies after 90-120 min of EDTA incubation add new PBS0 + EDTA and incubate for another 60 min. 4. Allow the biopsies to settle at the bottom and transfer the supernatant with the crypts to a clean 15 mL tube. 5. Add fresh 3 mL of cold PBS0 to the biopsies again and repeat previous steps until no more crypts detach or until a sufficient amount of crypts are transferred to the clean tube. 6. Add Ad-DF+++ to top up the solution containing the crypts in the 15 mL tube and centrifuge the crypts at 130 g for 5 min at 4 °C. 7. Gently remove the supernatant, the crypt cell pellet is vulnerable and can easily be lost by aspiration. 8. Add 10 mL of Ad-DF+++ to the crypt pellet for a second washing step and centrifuge at 130 g for 5 min at 4 ºC. D. Plating intestinal crypts in matrigel 1. For the first passage (p.0) make sure to use a separate 24-wells plate per patient sample since isolated crypts from primary intestinal material during the first passage are most susceptible to infections. 2. Keep the intestinal crypt pellet on ice. 3. Analyze the size of the pellet by eye to determine the appropriate volume of 50% matrigel. o Usually, 4 - 6 wells per biopsy is sufficient for p.0. However, a higher number of wells can be used if the crypt yield is very high (7 – 12 wells) or a lower number when the yield is low (1-3 wells). 4. To ensure the crypt density is not too low, first resuspend the crypt pellet in 100 ml 50 % matrigel.