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}

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-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.

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.

References

1. Riordan JR., et al. 2008. CFTR function and prospects for therapy. Annu Rev Biochem. 2008;77:701-26. RJea2CfapftARB2.

2. Galietta LJ., et al. 2006. CFTR chloride channel drug discovery--inhibitors as antidiarrheals and activators for therapy of cystic fibrosis. Curr Pharm Des. 2006;12(18):2235-47. GLea2CccddaaaaftocfCPD2.

3. Clevers H. et al. 2016. Modeling Development and Disease with Organoids. Cell. 2016 Jun 16;165(7):1586-1597. CHea2MDaDwOC2J1.

4. Van der Ent CK., et al. 2019. Intestinal organoids to model cystic fibrosis. Eur Respir J. 2019 Jul 4;54(1). VdECea2IotmcfERJ2J4.

5. Hans Clevers, et al. 2017. Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients. J Vis Exp. 2017; (120): 55159. HCea2FSiIOAIVAfADRiCFPJVE2(5.

6. Beekman JM., et al. 2013. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med. 2013 Jul;19(7):939-45. BJea2AfCaupcfioNM2J.

7. Van der Ent CK., et al. 2019. Rectal Organoids Enable Personalized Treatment of Cystic Fibrosis. Cell Rep. 2019 Feb 12;26(7):1701-1708.e3 VdECea2ROEPToCFCR2F1.