Peter van Mourik

94 Chapter 4 density should be 15-30 structures per drop, see figure 9B). Always recheck a new test drop after further dilution. 3. While preventing bubbles, plate 4* 7.5 µL matrigel with a p20 tip per well in a (pre- warmed) 24-well plate (see Figure 9). Slightly tilting the plate helps to deposit the drops in the desired positions. Note: Organoid structures will quickly settle at the bottom of the tube within 30 seconds so frequently resuspend the organoid suspension with a p200. While resuspending keep the tube on ice to prevent the matrigel from solidifying in the tube. Figure 9. Add 4 drops of 7.5 µL containing organoids in a pre-warmed 24-wells plate. Slightly tilting the plate helps to deposit the drops in the designated location. 4. Incubate the plate for 15 - 20 min at 37 °C. 5. Add 500 µL of pre-warmed of CM +/+ per well. 6. Incubate the organoid cultures for 7 days at 37 °C, CO 2 5% and refresh the wells with 500 µL of CM +/ + per well, every 2 - 3 days (Monday, Wednesday, Friday). Note: Human colon organoids can usually be passaged every 7 days. Therefore, it is important to regularly check the quality of the organoid cultures. Good quality organoid cultures show a stem cell phenotype which is characterized by efficient budding structures. For wild type (or very mild CF genotype) colon organoids a clear pre-swollen lumen can be seen with bright field imaging compared to colon organoids with a CF genotype where no lumen is visible (see Figure 10A and B).When colon organoids lose their stem cell phenotype (usually due to insufficient Wnt-3A activity in the WCM) the organoids appear as thick-walled, differentiated structures (see Figure 10C). These bad quality organoid cultures are not appropriate for CFTR- related experiments since this leads to unreliable results.