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.
