Peter van Mourik

9 General Introduction to quantify CFTR function in intestinal organoids that rely on the luminal chloride secretion and coupled water transport. First, incubation of organoids with forskolin leads to rapid luminal fluid secretion through CFTR activation that causes whole organoid swelling within 60 minutes. The forskolin-induced swelling (FIS) phenotype is absent in human and mouse organoids lacking functional CFTR gene products (e.g. two class I mutations or Cftr knock out), and is inhibited by chemical CFTR inhibitors, supporting full CFTR-dependency of the FIS readout 10 . The resulting swelling of organoids is quantified by live confocal microscopy after organoid labelling with calcein-green. The relative size increase of all organoids in a well is calculated over time using 10 minute intervals. A relative swelling curve is generated from these data and an area-under-the-curve of this relative swelling is calculated to compare conditions (e.g. different drugs, different donors) in a single graph 11 . Second, steady-state differences in luminal phenotype exist between healthy control and CF organoids independent of forskolin. Healthy control rectal organoids have large fluid-filled lumens, suggesting the presence of functional CFTR and physiological cAMP signalling during standard culture conditions leading to luminal salt and fluid transport 12 . CF organoids do not have lumens that are easily recognized upon visual inspection. This phenotypical difference can be quantified before a FIS assay is performed by manually drawing in the lumen of calcein-labeled organoids in the baseline images, and subsequently expressing the lumen area as percentage of total area. Whereas CF organoids have steady-state lumen areas (SLA) between 0-10 percent of total organoid area, healthy control SLA is between 40-80%. While SLA is mostly CFTR-driven, some variability in this phenotype exists at the 0-10% range independent of CFTR genotype. FIS and SLA are complementary assays. FIS has considerable throughput and quantifies CFTR function at levels associated with CF disease and treatment thereof with current CFTR modulators 7,10 . As FIS measures relative size increase, it requires that the initial starting sizes are comparable. This is true for CF organoids, but at larger SLA (~>25%), relative size increases are underestimated when 2D area measurements are performed 7 . FIS of CF and healthy controls are therefore not directly comparable. SLA facilitates comparison between CF and healthy control organoids, but has limited resolution to discriminate at lower CFTR function levels associated with severe CF disease 7 . 1