Cystic fibrosis is caused by mutations in the CFTR gene and is highly variable between individuals, with over 1,900 CFTR mutations identified.1 CFTR (cystic fibrosis transmembrane conductance regulator) functions as an anion channel and is essential for normal fluid and electrolyte homeostasis at the epithelial surfaces of many organs. Although CFTR-specific drug targeting is promising, the degree of CFTR function restoration is still limited and would be facilitated by better in vitro drug compound screening cell models.2
Organoids are heterogeneous, self-organizing 3D cell culture models that are frequently derived from stem cells.3 Intestinal epithelium organoids, often referred to as enteroids or “mini-guts”, maintain physiological characteristics of the gastrointestinal system, and have been a useful in vitro tool for modeling intestinal diseases including cystic fibrosis.4 Human colon organoids can be effective as a phenotypic drug screening tool to monitor CFTR function using a rapid and quantitative water-uptake swelling assay induced by forskolin.5, 6, 7
Figure 1. Mechanism of organoid-based forksolin-induced swelling assays.CFTR function can be assayed using patient-derived intestinal organoids by adding forskolin, a CFTR activator that stimulates adenylate cyclase and increases intracellular cAMP. Elevated cAMP levels activate protein kinase A (PKA) which phosphorylates CFTR, opening the CFTR channel leading to ion (Cl-/Na+) and water uptake (organoid swelling). CFTR wild type intestinal organoids will swell with forskolin addition within 2-3 hours, while cystic fibrosis-derived intestinal organoids will remain at baseline volume.
Forskolin Forskolin (20 µM)
Figure 2. Time-course for forskolin-induced organoid swelling. Human colon organoids were incubated with/without forskolin (20 µM) and captured over a 36 hour time period.
Figure 3. Morphology of colon organoids after forskolin-induced swelling.Wild-type human colon organoids (CFTR+) incubated with forskolin (20 µM) begin to swell at hour three and reach maximal swelling volume at hour sixteen. Colon organoids derived from cystic fibrosis patients (CFTR-) do not swell at any timepoint when subjected to the same conditions.