Name: rectal organoid morphology analysis (roma): a diagnostic assay in cystic fibrosis
Uploaded: 2022-06-10T14:30:00
Description: Watch this Scientific Journal Video about Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis at JoVE.com

We thank the patients and parents who participated in this study. We thank Abida Bibi for all culturing work with the organoids. We thank Els Aertgeerts, Karolien Bruneel, Claire Collard, Liliane Collignon, Monique Delfosse, Anja Delporte, Nathalie Feyaerts, C&#233;cile Lambremont, Lut Nieuwborg, Nathalie Peeters, Ann Raman, Pim Sansen, Hilde Stevens, Marianne Schulte, Els Van Ransbeeck, Christel Van de Brande, Greet Van den Eynde, Marleen Vanderkerken, Inge Van Dijck, Audrey Wagener, Monika Waskiewicz, and Bernard Wenderickx for logistic support. We also thank the Mucovereniging/Association Muco, and specifically Stefan Joris and Dr. Jan Vanleeuwe, for their support and financing. We thank all collaborators from the Belgian Organoid Project: Hedwige Boboli (CHR Citadelle, Li&#232;ge, Belgium), Linda Boulanger (University Hospitals Leuven, Belgium), Georges Casimir (HUDERF, Brussels, Belgium), Benedicte De Meyere (University Hospital Ghent, Belgium), Elke De Wachter (University Hospital Brussels, Belgium), Danny De Looze (University Hospital Ghent, Belgium), Isabelle Etienne (CHU Erasme, Brussels, Belgium), Laurence Hanssens (HUDERF, Brussels), Christiane Knoop (CHU Erasme, Brussels, Belgium), Monique Lequesne (University Hospital Antwerp, Belgium), Vicky Now&#233; (GZA St. Vincentius Hospital Antwerp), Dirk Staessen (GZA St. Vincentius Hospital Antwerp), Stephanie Van Biervliet (University Hospital Ghent, Belgium), Eva Van Braeckel (University Hospital Ghent, Belgium), Kim Van Hoorenbeeck (University Hospital Antwerp, Belgium), Eef Vanderhelst (University Hospital Brussels, Belgium), Stijn Verhulst (University Hospital Antwerp, Belgium), Stefanie Vincken (University Hospital Brussels, Belgium).

For all procedures involving human tissue, approval by the Ethics Committee Research UZ/KU Leuven (EC research) was acquired. All research was performed with informed consent and/or assent from parents, representatives, and/or patients.
NOTE: All procedures involving rectal biopsies and organoids should be performed in a laminar flow to protect the researcher from any biological hazard and to minimize the risk of contamination of the cultures. As for any lab procedure, researchers should at al...

<ol>
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We provide a detailed protocol for rectal organoid morphology analysis (ROMA). The two indexes calculated with ROMA, IR, and CI, distinguished organoids from subjects with CF from those without CF with perfect accuracy. ROMA could thus function as a novel physiological CFTR assay complementary to SCC and other currently available tests13,14,15.
The protocol is dependent on the use of intestinal organo...

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The CFTR protein is a chloride and bicarbonate channel, ensuring hydration of several epithelia1. CF is a high-burden, life-shortening, multi-system disease, manifesting primarily as a respiratory disease, but also affecting the gastrointestinal tract, pancreas, liver, and reproductive tract2.
Disease-causing CFTR mutations lead to a decrease in the amount or function of CFTR, in turn causing mucus dehydration. More than 2,000 variants in the CFTR gene have been described3, of which only 466 have been thoroughly characterized4.
A diagnosis of CF can be made when either sweat chloride concentration (SCC) is above the threshold of 60 mmol/L or when two disease-causing CFTR mutations (according to the CFTR2 database) are identified4,5. In subjects with only intermediately elevated (30-60 mmol/L) SCC, which occurs in about 4%-5% of sweat tests6, and CFTR mutations of varying or unknown clinical consequence, the diagnosis cannot be confirmed nor ruled out, even when they have CF compatible symptoms or a positive neonatal screening test. For these cases, second-line physiological CFTR assays (nasal potential difference (NPD) and intestinal current measurements (ICM)) have been included in the diagnostic algorithm. These tests are not readily available at most centers nor feasible at all ages, especially in infants5.
Rectal organoids are 3D structures grown from Lgr5(+) adult intestinal stem cells from intestinal crypts obtained through rectal biopsy7. Organoids are being used increasingly in biomedical research, such as testing modulator treatment in CF8. A viable biopsy can be obtained by either suction or forceps biopsy, a procedure that causes only minimal discomfort and is safe even in infants, with low complication rates9. The crypts isolated from the rectal biopsies are enriched in stem cells, and under specific culturing conditions, these self-organize into rectal organoids. The morphology of these organoids is determined by the expression and function of CFTR, located at the apical membrane of epithelial cells. Functional CFTR allows chloride and water to enter the organoid lumen, thereby inducing swelling of non-CF organoids. CF organoids do not swell and have no visible lumen10,11.
Rectal organoid morphology analysis (ROMA) allows the discrimination between CF and non-CF organoids based on these differences in organoid morphology. Non-CF organoids are more round and have a visible lumen, while the opposite is true for CF organoids. For this assay, patient-specific organoids are plated in 32 wells of a 96-well plate. After 1 day of growing, the organoids are stained with calcein green and imaged in a confocal microscope. The non-CF organoids show a more circular shape and a less fluorescent central part, as the lumen contains fluid and calcein stains only cells. These differences in morphology are quantified using two ROMA indexes: the circularity index (CI) quantifies the roundness of organoids, while the intensity ratio (IR) is a measure of the presence or absence of a central lumen. In this report, we describe in detail the protocol to obtain these discriminative indexes, to allow replication of the technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Sorenson</td>
			<td>17040</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR</td>
			<td>525-0605</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Greiner</td>
			<td>655101</td>
			<td></td>
		</tr>
		<tr>
			<td>Brightfield microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Binder</td>
			<td>CB160</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Hewlett-Packard</td>
			<td>Z240</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo Fisher</td>
			<td>51025413</td>
			<td></td>
		</tr>
		<tr>
			<td>Material for organoid culture as detailed in previous protocol10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes (20, 200, and 1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000039, 3123000055, 3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>Microsoft Excel 2019 MSO 64-bit</td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>NIS-Elements Advanced Research Analysis Imaging Software&#160;</td>
			<td>Nikon</td>
			<td>v.5.02.00</td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Pipette tips (20, 200, and 1000 &#181;L)</td>
			<td>Greiner</td>
			<td>774288, 775353, 750288</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen Blue software&#160;</td>
			<td>Zeiss</td>
			<td>v2.6</td>
			<td>Imaging software</td>
		</tr>
	</tbody>
</table>

rectal organoid morphology analysis (roma): a diagnostic assay in cystic fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Organoids from 212 subjects were collected during routine clinical visits. No adverse events occurred during or after the rectal biopsy procedure. Organoids were imaged by one researcher blinded to subject characteristics such as genotype and clinical information. Due to low-quality images, 23 subjects were excluded. Examples of successful and failed organoid cultures and image acquisition can be seen in Figure 2.
Organoids of 167 subjects with CF and two disease-...

Watch this Scientific Journal Video about Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis at JoVE.com

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

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.

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

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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