Peter van Mourik

112 Chapter 4 Expected Outcomes Sometimes after 3-4 days the colon organoid concentration will be so dense that it is advisable to reseed the structures from 1 well into 3-4 new wells of a 24-wells plate. Take up the small organoids in 1 mL Ad-DF+++ in a microcentrifuge tube, centrifuge at a table top centrifuge and directly plate out in 100 µL fresh matrigel into 3-4 new wells of a 24-wells plate. This allows the small organoid structures to have more space to expand and start budding before they are ready for the first mechanical disruption/ shearing of the organoids. After 7 days some organoid structures are already efficiently budding and ready for mechanical disruption for splitting. When organoid structures are still very small and not yet efficiently proliferating but the matrigel is fading: do not yet disrupt but take up the small organoids from four wells in 1 mL Ad-DF+++ in a microcentrifuge tube, centrifuge with a table top microcentrifuge and plate the organoids into 100 - 120 µL of fresh matrigel into 3-4 new wells of a 24-wells plate. This allows the small organoid structures to have more time to expand and start budding before they are ready for the first mechanical disruption/ shearing of the organoids. Quantitative Analysis and Statistics Raw image data software analysis The goal of this procedure is to quantify the relative increase in total organoid area (organoid swelling) after stimulation of CFTR function by fsk. The raw data of the FIS assay consists of the confocal images of calcein green stained organoids that are generated at 10 minute intervals, which are digitally stored as a time-series-clip of each well. Image analysis software (e.g. Zen blue (Zeiss), Cell profiler (open-source)) is used to identify the perimeter of each closed organoid structure (X,Y plane), which is defined as a confined region-of-interest. The image software is set to ‘fill objects’ (region-of-interest). Each region-of-interest is expressed as area µm 2 per time point per well. Total area µm 2 associated with all regions-of-interest (organoids) per time point per well is calculated and compared to t = 0 min. The area µm 2 over a time series of 60 min (with 10 minute intervals creating 7 time points (t=0; 10; 20; 30; 40; 50; 60 min) represents the relative swelling increase from baseline. The total increase in area µm 2 during the 7 time points is calculated as percent area increase per well. Hereby t=0 is set as a baseline of 100% and time point t=10 – t=60 min are normalized to time point 0 (see table 4). This data can be used for further calculations with the following readouts: