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. 2008. CFTR Function and Prospects for Therapy. Annu. Rev. Biochem.. 77(1):701-726. https://doi.org/10.1146/annurev.biochem.75.103004.142532

2. Verkman A, Lukacs G, Galietta L. 2006. CFTR Chloride Channel Drug Discovery - Inhibitors as Antidiarrheals and Activators for Therapy of Cystic Fibrosis. CPD. 12(18):2235-2247. https://doi.org/10.2174/138161206777585148

3. Clevers H. 2016. Modeling Development and Disease with Organoids. Cell. 165(7):1586-1597. https://doi.org/10.1016/j.cell.2016.05.082

4. van Mourik P, Beekman JM, van der Ent CK. 2019. Intestinal organoids to model cystic fibrosis. Eur Respir J. 54(1):1802379. https://doi.org/10.1183/13993003.02379-2018

5. Boj SF, Vonk AM, Statia M, Su J, Vries RRG, Beekman JM, Clevers H. Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients. JoVE.(120): https://doi.org/10.3791/55159

6. Dekkers JF, Wiegerinck CL, de Jonge HR, Bronsveld I, Janssens HM, de Winter-de Groot KM, Brandsma AM, de Jong NWM, Bijvelds MJC, Scholte BJ, et al. 2013. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med. 19(7):939-945. https://doi.org/10.1038/nm.3201

7. Berkers G, van Mourik P, Vonk AM, Kruisselbrink E, Dekkers JF, de Winter-de Groot KM, Arets HG, Marck-van der Wilt RE, Dijkema JS, Vanderschuren MM, et al. 2019. Rectal Organoids Enable Personalized Treatment of Cystic Fibrosis. Cell Reports. 26(7):1701-1708.e3. https://doi.org/10.1016/j.celrep.2019.01.068