Forskolin-Induced Swelling Assays

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}

Forskolin-Induced Organoid Swelling Assay Protocol

1. Culture and expand human colon organoids (SCC300) as per manufacturer’s recommendations.
2. Seed human colon organoids into 25 µL Matrigel^® GFR (Growth Factor Reduced) domes.
3. Allow clumps or fragments of human colon organoids to reform into organoids in the presence of ROCKi, 10 µM (SCM075) and 3dGRO^™ Human Colon Organoid Expansion Medium (SCM304) for 2 days.
4. Prepare concentrations of Forskolin (F6886) (2.5, 5.0, 10, and 20 µM) in 3dGRO^™ Human Colon Organoid Expansion Medium (SCM304).
5. Add 1 mL of the desired concentration of Forskolin-supplemented media (from step 4) into each well of a 24-well plate containing a 25 µL Matrigel^® GFR dome.
6. Monitor and image approximately once per hour to capture the swelling of organoids. Swelling will typically begin at about 3 hours post-treatment and continue for up to 36 hours.

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.