Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis
June 10th, 2022
This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.
ROMA can discriminate between organoids from subjects with and without cystic fibrosis when less than two disease-causing CFTR mutations are found and when sweat fluoride is intermediate. ROMA can be performed at all ages and with low complication rates. The analysis is semi-automated and standardized and biopsies can be sent to a central laboratory for analysis.
Organoids used for ROMA can also be used to access CFTR modulatory efficacy, and ROMA could help with measuring the degree of restoration of CFTR function. One day after plating, stain the organoids with calcium green. Rotate and slightly tilt the plate with the lid on a few times to ensure homogenous distribution of the calcium green in the whole well.
Incubate the plate again, for 15 to 30 minutes at 37 degrees Celsius and 5%carbon dioxide to ensure staining of all organoids in the wells. To focus on the organoids, determine the optimal X and Y position, and focus on the organoids in each well manually, using the confocal microscope. And save these positions in the imaging software.
Next, using live cell imaging settings, set the emission at 488 nanometers and excitation at 515 nanometers, and a LD magnification objective at 5X. To acquire organoid pictures, take images in a unit directional way with a resolution of 1024 pixels by 1024 pixels, and a depth of 16 bits with the confocal microscope. Choose the laser intensity and master gain for optimal visualization of morphological differences between cystic fibrosis and non-cystic fibrosis organoids.
Start the experiment to capture the images. Afterward, save one picture per well for all 32 wells in the microscope format and export them as TIFF files. First, load the TIFF files in the image analysis software and then perform the first quality check based on exclusion criteria determined by the operator.
Exclude the set of pictures if many differentiated or dead structures or debris are present when plating density is inadequate, when too many or too few organoids are present, and in case of inadequate fluorescence distribution. To prepare images for analysis, create and open one network data file of all 32 pictures for each organoid culture, enabling simultaneous analysis of all 32 pictures per subject. And recalibrate images so one pixel corresponds to 2.5 by 2.5 micrometers.
Then, delineate structures using a lower intensity threshold of 4, 500, and an upper threshold of 65, 535, turning off the smooth and clean functions, turning on the fill holes function, and putting the separate function at 3X. To count the organoids, select all structures more than or equal to 40 micrometers. Next, click the update ND measurement button, and the counted organoids will be numbered.
To measure intensity and circularity for calculation, select all structures more than or equal to 60 micrometers and count. Then, remove all structures touching the borders of the picture. Next, erode one pixel from the border of each, more than, or equal to 60 micrometers structure, and measure the mean intensity of each structure.
Afterward, select all structures more than or equal to 60 micrometers again, and erode 10 pixels from the border of each, more than, or equal to 60 micrometers structure. Then, measure the mean intensity of each eroded structure and the circularity of each structure. Then, calculate the intensity ratio by dividing the intensity measurement after eroding 25 micrometers from the border of each, more than, or equal to 60 micrometers structure by the mean of the intensity measurement after eroding 2.5 micrometers from the border of each, more than, or equal to 60 micrometers structure.
Perform the second quality check based on exclusion criteria determined by the software as described in the manuscript. Organoids from 212 subjects were collected and imaged. After exclusion of 23 sets of pictures, organoid pictures from 167 subjects with cystic fibrosis and two disease causing CFTR mutations and from 22 non-cystic fibrosis subjects were analyzed, the mean number of organoids per culture was found to be 1, 519.
The intensity ratio and circularity index were determined for organoids from subjects with and without cystic fibrosis. Using these two indexes, perfect discrimination was obtained between organoids from subjects with and without cystic fibrosis not only when using data from all 32 wells, but also when eight wells were chosen randomly for each culture. Histograms for four illustrative cultures were obtained, showing the distribution of values for circularity, the intensity of the central part of the organoid, and the intensity of the whole organoid.
Quality control is essential, as high quality pictures are required for accurate calculation of the intensity ratio and circularity index. Organoid cultures established for ROMA can also be used for physics aids to test the effect of CFTR treatment. RNA, DNA, and protein can be extracted for further characterization of CFTR variants.
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Overview
This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). ROMA can effectively discriminate between organoids from subjects with and without CF, particularly when specific genetic mutations are present.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Genetics
Background
- Cystic fibrosis is a genetic disorder affecting the respiratory and digestive systems.
- CFTR function is critical for proper organoid morphology.
- ROMA utilizes morphological characteristics to assess CFTR function.
- The analysis is semi-automated and can be standardized across laboratories.
Purpose of Study
- To develop a reliable diagnostic tool for cystic fibrosis.
- To measure CFTR function through organoid morphology.
- To provide a method that can be used across all ages with low complication rates.
Methods Used
- Organoids are stained with calcium green one day after plating.
- The analysis involves measuring roundness (circularity index) and lumen presence (intensity ratio).
- Biopsies can be sent to a central laboratory for analysis.
- ROMA can also assess CFTR modulatory efficacy.
Main Results
- Analysis of 189 subjects showed perfect discrimination between CF and non-CF.
- ROMA is effective even with less than two disease-causing CFTR mutations.
- It can be performed at all ages with low complication rates.
- ROMA helps measure the degree of restoration of CFTR function.
Conclusions
- ROMA is a promising tool for diagnosing cystic fibrosis.
- The method provides a standardized approach to assess CFTR function.
- Further research could enhance its application in clinical settings.
Frequently Asked Questions
Executive Industry Relevance
Rectal Organoid Morphology Analysis (ROMA) provides a standardized, quantitative assay for functional CFTR assessment, addressing diagnostic ambiguity in cystic fibrosis when genetic or sweat chloride results are inconclusive. By enabling robust discrimination between CF and non-CF phenotypes using organoid morphology, ROMA strengthens predictive confidence at the diagnostic and early discovery inflection point. This platform supports enterprise-wide reproducibility and cross-site sample processing, enhancing portfolio-level diagnostic reliability.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables functional validation of CFTR activity in patient-derived organoids.
- Supports mechanistic de-risking by quantifying morphological correlates of CFTR dysfunction.
- Facilitates hypothesis testing for genotype-phenotype relationships in rare or ambiguous CF cases.
- Provides a reproducible readout for target engagement and biological effect.
Screening & Assay Development
- Delivers standardized, semi-automated quantification of organoid morphology for assay development.
- Enables high-throughput, plate-based imaging and analysis suitable for screening workflows.
- Supports reproducibility and cross-lab standardization through defined exclusion criteria and image analysis protocols.
- Prepares validated biological systems for downstream compound evaluation and efficacy testing.
Translational & Preclinical Research
- Aligns in vitro organoid phenotypes with disease-relevant functional biomarkers.
- Enables assessment of CFTR modulator efficacy in patient-derived systems.
- Supports continuity from diagnostic discovery to preclinical validation of therapeutic interventions.
- Provides a platform for extracting RNA, DNA, and protein for further molecular characterization.
Pipeline & Workflow Integration
ROMA integrates into the diagnostic-to-preclinical continuum, bridging functional target validation, assay development, and translational research for CFTR-related programs.
- Discovery Biology: Quantifies CFTR-dependent morphological changes, supporting hypothesis-driven validation.
- Screening: Offers reproducible, quantitative outputs (circularity index, intensity ratio) for assay readiness.
- Analytics: Provides standardized image analysis and statistical discrimination between disease states.
- Translational Research: Enables in vitro efficacy testing of CFTR modulators in disease-relevant organoids.
- Enterprise Reuse: Supports centralized sample processing and cross-site assay deployment for scalable diagnostics.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in CFTR functional assessment.
- Operational Value: Delivers standardized, semi-automated workflows with defined quality control checkpoints.
- Strategic Value: Improves go/no-go decision-making and reduces late-stage diagnostic uncertainty.
- Portfolio Impact: Enables risk-adjusted prioritization of diagnostic and therapeutic development programs.
Implementation Considerations
- Requires expertise in organoid culture, confocal imaging, and quantitative image analysis.
- Needs access to confocal microscopy and compatible image analysis software.
- Demands rigorous cross-team standardization of exclusion criteria and analysis thresholds.
- Adaptable to various patient-derived samples but dependent on biopsy quality and culture conditions.
- Quality control is critical to ensure reliable calculation of morphological indexes.
