Peter van Mourik

102 Chapter 4 Therefore, the organoid pellet can be resuspended in approximately 370 (80*4 + 50) µL of 50% matrigel. In case the organoids are too densely seeded, organoids can be diluted by adding more matrigel. 3. Check the amount of organoids in a 4 µL test drop of matrigel under a light microscope, to determine whether the organoid suspension should be further diluted (the density should be 30-50 structures per drop). Always recheck a new test drop after further dilution. Note: when the organoid density is too low, matrigel can be resuspended in 10 mL of Ad-DF+++ and centrifuged at 130 g for 5 min at 4 °C. Next, aspirate medium and resuspend the organoid pellet in less matrigel. 4. Transfer the organoid suspension to a cold microcentrifuge tube. 5. While preventing bubbles add a 4 µL drop to a (warm) flat-bottom 96-well plate (see Figure 11). This can be performed with a regular p20 pipet. Figure 11. Add 4 µl drops containing 20 - 50 organoids per drop to every well in a pre-warmed 96-wells plate. Note: Organoid structures will quickly settle at the bottom of the microcentrifuge tube within 30 s so resuspend the organoid suspension with a p200 regularly. While resuspending keep the tube on ice to prevent the matrigel from solidifying in the tube. It is advisable to use a repetitive pipet (like a Viaflo||, Integra) since the organoid seeding is performed more efficient, faster and homogeneously. 6. Incubate the 96-wells plate for 5-10 min at 37 °C. 7. Add 50 µL of CM +/+ to each well. 8. Incubate 20 -24 h (overnight) at 37 °C, CO 2 5 %.