A functional CFTR assay using primary cystic fibrosis intestinal organoids

Abstract

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

High-fat diet modifies the PPAR-γ pathway leading to disruption of microbial and physiological ecosystem in murine small intestine

“…Previous studies have showed that in mouse models of cystic fibrosis, the lack of CFTR functionality is correlated to Ppar-γ down-regulation and its decreased activity, leading to steatorrhea (59)(60)(61). Interestingly, in Cftr −/− mice, Ppar-γ is down-regulated and when mice are treated with rosiglitazone, effects of the CFTR defect (i.e., mucus accumulation) disappeared in Cftr −/− Ppar-γ IEC+/+ mice but not in Cftr…”

Microfluidics platform for measurement of volume changes in immobilized intestinal enteroids

“…In addition, efficient trapping of optimal-size enteroids was an important design criteria as the number of enteroids can be limited, for example, in preparations obtained from human intestinal biopsies. 7,11 The post-pair design and layout were optimized for efficient trapping and exclusion of small enteroids, and the pre-filter region effectively excluded large enteroids and debris. Another challenge was the measurement of absolute volume without assumptions about enteroid shape and swelling geometry, which was overcome by an extracellular dye exclusion method.…”

“…Enteroids generated from human rectal biopsies from cystic fibrosis patients have been used to study the phenotype of mutant CFTR (cystic fibrosis transmembrane conductance regulator) chloride channels causing cystic fibrosis and the potential efficacy of CFTR-targeted drugs and gene therapy. 7,8 Another major transport application of enteroids is in studies of the pathophysiology of secretory diarrheas and testing of potential antisecretory therapeutics. [7][8][9] a) Author to whom correspondence should be addressed.…”

“…7,8 Another major transport application of enteroids is in studies of the pathophysiology of secretory diarrheas and testing of potential antisecretory therapeutics. [7][8][9] a) Author to whom correspondence should be addressed. The principal readout in membrane transport studies is enteroid volume, which changes with osmotic water movement driven by active transepithelial ion or solute transport.…”

“…The current approach to follow enteroid volume changes involves confocal imaging of enteroids in response to addition of membrane transport agonists or inhibitors, or changes in solution composition. 7 Enteroids are immobilized in an extracellular matrix such as Matrigel and images are acquired following manual solution changes. Limitations of the current methodology include: (i) restrictions of the embedding extracellular matrix on access of extracellular components and enteroid swelling; (ii) inefficient solution mixing; (iii) the need to label enteroids with a fluorescent dye for confocal imaging; (iv) challenges in making parallel measurements on many enteroids; and (v) inaccuracies in deducing enteroid volume changes from confocal images.…”