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Background: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications., Methods: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay., Results: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %., Conclusions: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Patient-derived intestinal organoids (PDIOs) show great potential as in vitro drug testing platform for personalised medicine in Cystic Fibrosis and oncology. PDIOs can be generated by culturing adult stem cells obtained through rectal forceps biopsy or suction biopsy, but the safety of these procedures and the success rates of generating organoids after shipment to a centralized lab using these procedures has not been studied in this context. We here report the safety and success rates of both biopsy procedures and the subsequent generation of PDIOs in the international multicentre HIT-CF Organoid Study., Methods: 502 biopsy procedures were conducted, on 489 adult people with Cystic Fibrosis from 33 different hospitals across 12 countries. Depending on the preference of the hospital, either rectal forceps biopsies or suction biopsies were obtained and internationally shipped to a central laboratory for organoid generation., Results: No adverse events were reported for 280 forceps biopsy procedures, while 222 rectal suction biopsy procedures resulted in 2 adverse events, namely continued bleeding and a probably nonrelated gastroenteritis. The success rate of organoid generation from all biopsies was 95%, and the main reason for failure was insufficient sample viability (3.2%)., Conclusion: Our results indicate that both rectal suction biopsy and forceps biopsy procedures are safe procedures. The high success rates of PDIO generation from the obtained tissue samples demonstrate the feasibility of the organoid technology for personalised in vitro testing in an international setting., Competing Interests: Declaration of competing interest No conflict., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Organoid technology is emerging rapidly as a valuable tool for precision medicine, particularly in the field of Cystic Fibrosis (CF). However, biobank storage and use of patient-derived organoids raises specific ethical and practical challenges that demand sound governance. We examined the perspectives of professionals affiliated with CF or organoids on the ethical aspects of organoid biobanking for CF precision medicine. By conducting this study parallel to the process of innovation and development of organoid biobanking, its findings are valuable for the design of responsible governance frameworks., Methods: To identify relevant themes and attitudes we conducted 21 semi-structured qualitative interviews with professionals in the field of organoid technology, biobanking, or CF research and care., Results: We identified three key challenges, as well as the suggestions of professionals on how to address them: (1) The challenges associated with commercial involvement, trust, and ownership, (2) Navigating the blurring boundary between research and clinical care, (3) Appropriate approaches to the informed consent procedure., Conclusion: Sound governance of organoid biobanks aimed at precision medicine requires coming to terms with the fact that its stakeholders no longer belong to separate domains. Responsible governance should be aimed at finding a sound, context-sensitive balance between integration of ongoing co-operation and mutual consideration of interests, and maintaining a feasible and sustainable research climate., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Variability in disease severity and CFTR modulator responses exists between patients with identical CFTR genotypes. Here, we characterized transcription, translation and function of R117H-CFTR using intestinal organoids and correlated them with in vitro responses to ivacaftor (VX-770)., Methods: Organoids were generated from individuals possessing at least one R117H-CFTR allele. The forskolin-induced swelling (FIS) assay was used to measure CFTR function and response to VX-770 treatment. R117H-CFTR protein and mRNA expression levels were determined in parallel and Pearson's correlation coefficients were assessed., Results: Variability in R117H-CFTR FIS responses was observed and correlated significantly with mRNA and protein expression. Response to VX-770 treatment in organoids correlated with mRNA and protein expression as well., Conclusions: Our results indicate that gene expression, protein expression and CFTR function are strongly correlated in organoids from people with CFTR-R117H-7T/9T, which may suggest that CFTR gene expression may have consequences for CF diagnosis, prognosis and therapeutic benefit., Competing Interests: Declaration of Competing Interest Cornelis K. van der Ent reports grants from GSK, Nutricia, TEVA, Gilead, Vertex, ProQR, Proteostasis, Galapagos NV, and grants from Eloxx outside the submitted work and has a patent 10006904 with royalties paid. Prof. Dr. Beekman has a patent on organoid swelling for personalized diagnosis in CF with royalties paid to Hubrecht Organoid Technology and received financial support to cover travel costs and preparation time of scientific lectures from Vertex Pharmaceuticals. He also received funding from Galápagos NV for CFTR modulator studies., (Copyright © 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: CFTR function measurements in intestinal organoids may help to better characterise individual disease expression in F508del homozygous people. Our objective was to study correlations between CFTR function as measured with forskolin-induced swelling in rectal organoids with clinical parameters in adult patients with homozygous F508del mutations., Methods: Multicentre observational study. Thirty-four adults underwent rectal biopsy, pulmonary function tests (FEV 1 and FVC), chest X-ray and chest CT. Body-mass index (BMI) was assessed at study visit and exacerbation rate was determined during five years prior to study visit. Organoids were cultured and measured after stimulation with 5 µm forskolin for three hours to quantitate CFTR residual function., Findings: FIS was positively correlated with FEV 1 (r = 0.36, 95% CI 0.02-0.62, p = 0.04) and BMI (r = 0.42, 95% CI 0.09-0.66, p = 0.015). FIS was negatively correlated with PRAGMA-CF CT score for% of disease (r = -0.37, 95% CI -0.62- -0.03, p = 0.049). We found no significant correlation between FIS and chest radiography score for CF (r = -0.16, 95% CI -0.48-0.20, p = 0.44). We observed a trend between higher FIS and a lower mean number of exacerbations over the last 5 years of observation, but this was not statistically significant (Poisson regression, p = 0.089)., Interpretation: FIS of intestinal organoids varied between subjects with homozygous F508del and correlated with pulmonary and nutritional parameters. These findings suggest that differences at low CFTR residual function may contribute to clinical heterogeneity in F508del homozygous patients and small changes in CFTR residual function might impact long-term disease expression., (Copyright © 2019. Published by Elsevier B.V.)

This protocol describes the isolation, handling, culture of, and experiments with human colon stem cell organoids in the context of cystic fibrosis (CF). In human colon organoids, the function of cystic fibrosis transmembrane conductance regulator (CFTR) protein and its rescue by CFTR modulators can be quantified using the forskolin-induced swelling assay. Implementation procedures and validation experiments are described for six CF human colon organoid lines, and representative CFTR genotypes are tested for basal CFTR function and response to CFTR-modulating drugs. For complete details on the use and execution of this protocol, please refer to Dekkers et al (2016) and Berkers and van Mourik (2019)., Competing Interests: J.F.D. is an inventor on a patent related to the organoid technology. M.A.A. reports grants from Gilead Pharmaceuticals, Vertex Pharmceuticals, Proteostasis Therapeutics and personal fees from Vertex Pharmceuticals and PTC Pharmaceuticals outside the submitted work. K.B. reports consultancy work for CHIESI, grants from VERTEX, advisory board meetings for PROTEOSTASIS and ELOXX, grants from and advisory board meetings for GALAPAGOS, and advisory board meetings for TRANSLATE BIO outside the submitted work. R.V. is CEO of Hubrecht Organoid Technology, which provides organoid services to companies. C.K.E. reports grants from GSK, Nutricia, TEVA, Gilead, Vertex, ProQR, Proteostasis, Galapagos NV, and Eloxx outside the submitted work and has a patent 10006904 with royalties paid. J.M.B. is inventor on a patent related to CFTR function measurements in organoids and has received financial royalties. He also participates in industry-sponsored clinical trials (Galapagos NV, Vertex Pharmaceuticals, Proteostasis, Eloxx Pharmaceuticals) and has received travel reimbursements and speaker’s fees. The other authors have nothing to disclose., (© 2020 The Authors.)

Competing Interests: Conflict of interest: P. van Mourik has nothing to disclose. Conflict of interest: J.M. Beekman has a patent on organoid swelling for personalised diagnosis in CF with royalties paid by Hubrecht Organoid Technology. He received financial support to cover travel costs and preparation time of scientific lectures from Vertex. He also received funding from Galapagos for CFTR modulator studies. Conflict of interest: C.K. van der Ent reports grants from Vertex Pharmaceuticals Inc, Gilead Sciences and Galapagos, grants and other funding from ProQR, and other funding from TEVA pharmaceutical industries, outside the submitted work.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease., (© 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r= -0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study., Competing Interests: Conflict of interest: J.M. Beekman reports a licensed patent, number CA2859614 A1, for a rapid quantitative assay to measure CFTR function in a primary intestinal culture model. Conflict of interest: J.F. Dekkers reports a licensed patent, number CA2859614 A1, for a rapid quantitative assay to measure CFTR function in a primary intestinal culture model. Conflict of interest: R. Vries reports support from Vertex for drug screening and from CZ for development of diagnostics, outside the submitted work. Conflict of interest: H.A.W.M. Tiddens reports fees/grants from Roche for an industry symposium on treatment in CF, from Novartis for lectures and advisory boards, from CFF for Lung Analysis, from Vertex for Lung Analysis and advisory boards, from Gilead for lectures and advisory boards, and from Chiesi for an investigator-initiated trial, all outside the submitted work. He also reports a licensed combined patent from Vectura on specific targeting with DNase, and an issued patent from the PRAGMA-CF scoring system. He heads the Erasmus Medical Center/Sophia Children's Hospital core laboratory Lung Analysis. Conflict of interest: K.M. de Winter-de Groot has nothing to disclose. Conflict of interest: H.M. Janssens has nothing to disclose. Conflict of interest: R.T. van Uum has nothing to disclose. Conflict of interest: G. Berkers has nothing to disclose. Conflict of interest: A. Vonk has nothing to disclose. Conflict of interest: E. Kruisselbrink has nothing to disclose. Conflict of interest: H. Oppelaar has nothing to disclose. Conflict of interest: H. Clevers has nothing to disclose. Conflict of interest: R.H.J. Houwen has nothing to disclose. Conflict of interest: J.C. Escher has nothing to disclose. Conflict of interest: S.G. Elias has nothing to disclose. Conflict of interest: H.R. de Jonge has nothing to disclose. Conflict of interest: Y.B. de Rijke has nothing to disclose. Conflict of interest: C.K. van der Ent has nothing to disclose., (Copyright ©ERS 2018.)

Background: Organoid technology enables the cultivation of human tissues in a dish. Its precision medicine potential could revolutionize the Cystic Fibrosis (CF) field. We provide a first thematic exploration of the patient perspective on organoid technology to set the further research agenda, which is necessary for responsible development of this ethically challenging technology., Methods: 23 semi-structured qualitative interviews with 14 Dutch adult CF patients and 12 parents of young CF patients to examine their experiences, opinions, and attitudes regarding organoid technology., Results: Four themes emerged: (1) Respondents express a close as well as a distant relationship to organoids; (2) the open-endedness of organoid technology sparks hopes and concerns, (3) commercial use evokes cautiousness. (4) Respondents mention the importance of sound consent procedures, long-term patient engagement, responsible stewardship, and stringent conditions for commercial use., Conclusions: The precision medicine potential of organoid technology can only be realized if the patient perspective is taken adequately into account., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The potentiator VX-770 (ivacaftor/KALYDECO™) targets defective gating of CFTR and has been approved for treatment of cystic fibrosis (CF) subjects carrying G551D, S1251N or one of 8 other mutations. Still, the current potentiator treatment does not normalize CFTR-dependent biomarkers, indicating the need for development of more effective potentiator strategies. We have recently pioneered a functional CFTR assay in primary rectal organoids and used this model to characterize interactions between VX-770, genistein and curcumin, the latter 2 being natural food components with established CFTR potentiation capacities. Results indicated that all possible combinations of VX-770, genistein and curcumin synergistically repaired CFTR-dependent forskolin-induced swelling of organoids with CFTR-S1251N or CFTR-G551D, even under suboptimal CFTR activation and compounds concentrations, conditions that may predominate in vivo. Genistein and curcumin also enhanced forskolin-induced swelling of F508del homozygous organoids that were treated with VX-770 and the prototypical CFTR corrector VX-809. These results indicate that VX-770, genistein and curcumin in double or triple combinations can synergize in restoring CFTR-dependent fluid secretion in primary CF cells and support the use of multiple potentiators for treatment of CF., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Identifying subjects with cystic fibrosis (CF) who may benefit from cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs is time-consuming, costly, and especially challenging for individuals with rare uncharacterized CFTR mutations. We studied CFTR function and responses to two drugs-the prototypical CFTR potentiator VX-770 (ivacaftor/KALYDECO) and the CFTR corrector VX-809 (lumacaftor)-in organoid cultures derived from the rectal epithelia of subjects with CF, who expressed a broad range of CFTR mutations. We observed that CFTR residual function and responses to drug therapy depended on both the CFTR mutation and the genetic background of the subjects. In vitro drug responses in rectal organoids positively correlated with published outcome data from clinical trials with VX-809 and VX-770, allowing us to predict from preclinical data the potential for CF patients carrying rare CFTR mutations to respond to drug therapy. We demonstrated proof of principle by selecting two subjects expressing an uncharacterized rare CFTR genotype (G1249R/F508del) who showed clinical responses to treatment with ivacaftor and one subject (F508del/R347P) who showed a limited response to drug therapy both in vitro and in vivo. These data suggest that in vitro measurements of CFTR function in patient-derived rectal organoids may be useful for identifying subjects who would benefit from CFTR-correcting treatment, independent of their CFTR mutation., (Copyright © 2016, American Association for the Advancement of Science.)

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air–liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA. [ABSTRACT FROM AUTHOR]

The article presents a research study and comparison of organoid swelling and In Vivo Biomarkers of cystic fibrosis transmembrane conductance regulator (CFTR) function. Topics include its aim to determine effects of drug, Lumacaftor–Ivacaftor in patients with cystic fibrosis homozygous for the F508del mutation.

As highly effective CFTR modulator therapies (HEMT) emerge, there is an unmet need to find effective drugs for people with CF (PwCF) with ultra-rare mutations who are too few for classical clinical trials and for whom there are no drug discovery programs. Therefore, biomarkers reliably predicting the benefit from CFTR modulator therapies are essential to find effective drugs for PwCF through personalized approaches termed theranostics. Here, we assess CFTR basal function and the individual responses to CFTR modulators in primary human nasal epithelial (pHNE) cells from PwCF carrying rare mutations and compare these measurements with those in native rectal biopsies and intestinal organoids, respectively, in the same individual. The basal function in pHNEs shows good correlation with CFTR basal function in rectal biopsies. In parallel, CFTR rescue in pHNEs by CFTR modulators correlates to that in intestinal organoids. Altogether, results show that pHNEs are a bona fide theranostic model to assess CFTR rescue by CFTR modulator drugs, in particular for PwCF and rare mutations. [ABSTRACT FROM AUTHOR]

• Treatment of people having CFTR-S1251N with ivacaftor, but not genistein or curcumin, led to clear clinical improvement. • Plasma concentrations of curcumin and genistein treatment were below the micromolar range. • Plasma of people that received ivacaftor strongly potentiated CFTR function in organoids. • Drug testing using physiological dosing likely improves the ability to predict clinical outcomes using organoid testing. The natural food supplements curcumin and genistein, and the drug ivacaftor were found effective as CFTR potentiators in the organoids of individuals carrying a S1251N gating mutation, possibly in a synergistic fashion. Based on these in vitro findings, we evaluated the clinical efficacy of a treatment with curcumin, genistein and ivacaftor, in different combinations. In three multi-center trials people with CF carrying the S1251N mutation were treated for 8 weeks with curcumin+genistein, ivacaftor and ivacaftor+genistein. We evaluated change in lung function, sweat chloride concentration, CFQ-r, BMI and fecal elastase to determine the clinical effect. We evaluated the pharmacokinetic properties of the compounds by evaluating the concentration in plasma collected after treatment and the effect of the same plasma on the intestinal organoids. A clear clinical effect of treatment with ivacaftor was observed, evidenced by a significant improvement in clinical parameters. In contrast we observed no clear clinical effect of curcumin and/or genistein, except for a small but significant reduction in sweat chloride and airway resistance. Plasma concentrations of the food supplements were low, as was the response of the organoids to this plasma. We observed a clear clinical effect of treatment with ivacaftor, which is in line with the high responsiveness of the intestinal organoids to this drug. No clear clinical effect was observed of the treatment with curcumin and/or genistein, the low plasma concentration of these compounds emphasizes that pharmacokinetic properties of a compound have to be considered when in vitro experiments are performed. [ABSTRACT FROM AUTHOR]

Background New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis. Methods CFTR-mediated ion transport was measured in rectal organoid-derived monolayers grown from subjects with various CFTR mutations and compared to donor-matched intestinal current measurements (ICM) in rectal biopsies and forskolin-induced swelling of rectal organoids. Results Rectal organoid-derived monolayers were generated within four days. Ion transport measurements of CFTR function using these monolayers correlated with ICM and organoid swelling (r = 0.73 and 0.79 respectively). Culturing the monolayers under differentiation conditions enhanced the detection of mucus-secreting cells and was accompanied by reduced CFTR function. Conclusions CFTR-dependent intestinal epithelial ion transport properties can be measured in rectal organoid-derived monolayers of subjects and correlate with donor-matched ICM and rectal organoid swelling. [ABSTRACT FROM AUTHOR]