We gratefully acknowledge the contributions of all the participants who donated HNE brush biopsies to develop this protocol. We thank Latona Kersh and Children&#39;s Research Unit staff for coordinating study volunteer recruitment and sample collections. We thank Lily Deng, Johnathan Bailey, and Stephen Mackay, former trainees in our laboratory, for technical assistance. We thank Zhong Liu and Rui Zhao for their technical help. Steven M. Rowe, Director of the CF Research Center at UAB, provides leadership and resources, without which this work would not be possible. We also would like to thank Sarah Guadiana at Biotek for assistance with instrument training, Robert Grabski for confocal microscopy assistance at the UAB High-Resolution Imaging Facility, and Dezhi Wang for histological assistance at the UAB Histology Core. This work was supported by the National Institutes of Health (NIH.) Grant K23HL143167 (to JSG), Cystic Fibrosis Foundation (CFF) Grant GUIMBE18A0-Q (to JSG), the Gregory Fleming James Cystic Fibrosis Center [NIH Grants R35HL135816 and DK072482 and the CFF University of Alabama at Birmingham (UAB) Research and Development Program (Rowe19RO)], and the UAB Center for Clinical and Translational Science (NIH Grant UL1TR001417).

HNE samples were collected at the Children&#39;s of Alabama hospital. All procedures and methods described here have been approved by the IRB University of Alabama at Birmingham (UAB IRB #151030001). To facilitate the expansion and improve the function of human nasal epithelial cells (HNEs), the present culturing methods are adapted from the well-known air-liquid interface (ALI) culture method28,34. HNEs were initially collected by brush biopsy as previously described12,14, with the only difference being the use of a cytology brush. All sample processing steps and cell culture were performed in the biosafety cabinet.
1. Cell culture and expansion of nasal epithelial cells
<ol>
	<li>After brushing, store the nasal biopsy sample in 8 mL of RPMI media in a 15 mL conical tube on ice and transfer it to the lab within 4 h (not more than 24 h).</li>
	<li>Dissociate the nasal brush biopsy into 8 mL of RPMI media in a 15 mL conical tube by passing the cytology brush through a 1 mL large bore pipette tip (cut off the tip) several times until the brush is clear of tissue.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C, remove the supernatant, and then re-suspend the cell pellet in 3 mL of cell detachment solution (see Table of Materials) and incubate at room temperature for 16 min to digest.</li>
	<li>Use 5 mL of expansion media (Table 1) to wash the cells twice. Then, add cells to a T75 flask pre-seeded with irradiated and inactivated 3T3 fibroblasts22 (50%-60% confluence) to co-culture the cells and expand in the expansion media for 7-14 days (see Figure 1 for the appropriate colony). Before use, test the irradiated 3T3 fibroblasts to ensure they cannot proliferate, which will negatively influence the epithelial cell expansion. 
	NOTE: Discard the cells if the nasal epithelial cell confluence does not reach 70% within 14 days.</li>
	<li>For the cells derived from patients with CF, introduce four antibiotics (100 &#181;g/mL of tobramycin, 2.5 &#181;g/mL of amphotericin B, 100 &#181;g/mL of ceftazidime, 100 &#181;g/mL of vancomycin, see Table of Materials) into the expansion media for disinfection of the cells for the first 3 days of culture, and then replace the media with antibiotic-free expansion media changing the media every 2 days. 
	NOTE: This limited use of antibiotics is intended to reduce culture loss due to bacterial colonization while encouraging proliferation after the additional antibiotics are removed. These antibiotics can be tailored to the patient&#39;s specific microbiology results, with sensitivities, if needed.</li>
	<li>Harvest HNEs from co-culture using the double trypsinization method22 once the cells reach approximately 80% confluence. This method ensures that the irradiated and inactivated 3T3 fibroblasts are removed from the flask, and they do not contaminate subsequent organoid seeding.
	<ol>
		<li>Wash the cells with 1x DPBS, and then add 1.5 mL of 0.05% trypsin-EDTA into the T75 flask for 4 min at 37 &#176;C to remove the irradiated and inactivated 3T3 fibroblast from culture.</li>
		<li>Rinse the T75 flask with 1x DPBS twice to thoroughly wash away any remaining 3T3 fibroblasts; add 1.5 mL of 0.05% trypsin-EDTA into the T75 flask for 10 min at 37 &#176;C to detach HNEs.</li>
		<li>Neutralize the trypsin with soybean trypsin inhibitor (see Table of Materials) at a 1:1 ratio. Centrifuge the cells at 500 x g for 5 min, and then remove the supernatant. After washing the cells with 5 mL of expansion media once, the cells are ready for seeding to grow organoids. 
		&#8203;NOTE: HNEs with a passage number of three are recommended to be used for further experiments.</li>
	</ol>
	</li>
</ol>
2. Growth and differentiation of organoids in slides and culture inserts
<ol>
	<li>Thaw the organoid culture extracellular matrix (ECM) overnight at 4 &#176;C. Cool pipette tips to 4 &#176;C and 15-well angiogenesis slides (see Table of Materials) overnight at 4 &#176;C. 
	NOTE: ECM should be put at 4 &#176;C the night before the cell harvesting needs to be done.</li>
	<li>Coat the slides with 5 &#181;L of cold 100% ECM on ice (pipette 5 &#181;L of cold 100% ECM with a cold pipette tip into each well of the 15-well slide), and then place them into a cell culture incubator at 37 &#176;C for at least 30 min for consolidation.</li>
	<li>Count the HNEs harvested from co-culture using a hemocytometer and dilute the cells to 500 cells/&#181;L in total number with 20% ECM diluted by differentiation media (Table 2) on ice. Then, seed 5 &#181;L of the cold cell/ECM mixture into each well of the slides coated with ECM.</li>
	<li>Immediately transfer the slides into a culture incubator at 37 &#176;C for 1 h to consolidate the cell/ECM mixture.</li>
	<li>Feed the cells in each well of the 15-well angiogenesis slides with 50 &#181;L of differentiation media. Change the media every other day until the organoids are ready for further experiments. 
	NOTE: Organoids can usually be visualized after 1-2 days. There are 20-90 organoids commonly formed in each well of the slides. The organoids can typically survive for 40-60 days in ECM with feeding every other day when kept in a humidified incubator at 37 &#176;C.</li>
	<li>Culture the organoids in the culture inserts (see Table of Materials) for greater quantity for specific applications (such as sectioning for histology or immunofluorescence) as per the steps mentioned below.
	<ol>
		<li>To grow organoids in the culture inserts, prepare the organoid culture ECM, pipette tips, and inserts as mentioned in steps 2.1-2.2. Coat the inserts with 100 &#181;L of cold 100% ECM.</li>
		<li>Seed 60 &#181;L of cell/ECM mixture (500 cells/&#181;L with 20% ECM in differentiation media) on top of the ECM coating in the insert. 
		NOTE: All the other steps for making organoids in the inserts are the same as those conducted in the slides, steps 2.4-2.5.</li>
		<li>Add 600 &#181;L of the differentiation media into the bottom part of the insert. Change the media every alternate day until the organoids are used for experiments, typically 2-3 weeks.</li>
	</ol>
	</li>
</ol>
3. Preparation and isolation of organoids for whole-mount immunofluorescence
<ol>
	<li>Isolation and fixation of organoids
	<ol>
		<li>Pre-treat 8-well glass-bottom chamber slides with cell adhesive (see Table of Materials) for 30 min following Reference35. After discarding the solution, air-dry the wells for 30 min.</li>
		<li>To harvest the organoids, remove the media from the top of the ECM, and then add 50 &#181;L of cold 1x PBS into each well of the 15-well slides on ice (1:1 with ECM volume).</li>
		<li>Pipette up and down 3-5 times using 200 &#181;L of the large-bore pipette tip, and then dispense the solution onto the center of a well of the 8-well chamber slides.</li>
		<li>Immediately remove excess liquid from the wells by a fine-tip pipette. Then, place the chamber slide into a 37 &#176;C incubator for 40 min to enhance the organoid adhering to the glass bottom.</li>
		<li>After gently washing with 1x PBS twice, fix the organoids with 300 &#181;L of 4% paraformaldehyde in each well for 30 min at room temperature (RT).</li>
		<li>Wash twice with 1x PBS and store the organoids in 1x PBS at 4 &#176;C for immunostaining for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>To reduce auto-fluorescence, add 250 &#181;L of 50 mM NH4Cl in 1x PBS into each well of the slides at RT for 30 min while gently shaking on a shaker at 20 rpm.</li>
		<li>After washing with 1x PBS twice, permeabilize the cells with 0.1% Triton X-100 for 30 min at RT while gently shaking at 20 rpm.</li>
		<li>After washing with 1x PBS twice, add 300 &#181;L of blocking solution, including 5% BSA and 0.1% Triton X-100 in 1x PBS, into each well for 1 h at RT.</li>
		<li>Following washing, add primary antibody into the appropriate wells followed by secondary antibodies (see Table of Materials). Prepare primary and secondary antibody solutions in 2% BSA and 0.3% Triton X-100 in 1x PBS. Incubate all the primary antibodies at 4 &#176;C for 2 days and all the secondary antibodies at 4 &#176;C for 1 day. 
		NOTE: The final concentrations are dependent on the protein desired for the experiment. Please check the stock concentration on the manufacturer&#39;s datasheet. Then, calculate the final concentrations based on the dilutions used in the Table of Materials.</li>
		<li>After incubation, wash the wells thoroughly with 1x PBS and add DAPI in 2% BSA and 0.3% Triton X-100 into each well for nuclear staining.</li>
		<li>Image the organoids using a confocal laser scanning microscope, with a 20-60x oil immersion objective (see Table of Materials). Use Z-stack mode to set the upper and lower bounds of the image and use the recommended optimal Z-step size determined by the confocal software. 
		&#8203;NOTE: The following four confocal laser excitation wavelengths were used: 408.7 nm, 489.1 nm, 561.7 nm, and 637.9 nm.</li>
	</ol>
	</li>
</ol>
4. Preparation and isolation of organoids for histological sectioning
<ol>
	<li>To harvest the organoids for histological studies, remove the media from the culture and add 50 &#181;L of cold 1x PBS into each well of the slides on ice.</li>
	<li>Pipette up and down three to five times using a 200 &#181;L large-bore pipette tip, combine all the solutions from the 15-well slide or culture inserts into a 15 mL conical tube on ice. Adjust the total solution volume in the tube to 10 mL by adding additional cold 1x PBS.</li>
	<li>Centrifuge the tube at 4 &#176;C, 300 x g for 5 min; aspirate out the supernatant and add 60 &#181;L of warm histogel (see Table of Materials) to mix with the organoid pellet using a 200 &#181;L of the large-bore pipette tip.</li>
	<li>Immediately transfer the suspension into a histology mold. After the consolidation of the histogel at room temperature, put the mold block into 4% paraformaldehyde for fixation overnight at 4 &#176;C.</li>
	<li>After embedding in paraffin, cut the histogel block into 5 &#181;m cross-sections (for example, with a microtome), fix the sections onto glass slides, and stain using hematoxylin and eosin (H&#38;E) or immunofluorescence-labeled antibodies. Take images using a bright-field microscope or inverted epi-fluorescence microscope (see Table of Materials).</li>
</ol>
5. Imaging of live organoids
NOTE: The following steps are carried out using an automated imaging system (see Table of Materials). Different imaging systems need to adapt these steps following their specific manufacturer&#39;s instructions. Regardless of the equipment utilized, imaging live organoids require a temperature-controlled and humidified environmental chamber with an accompanied CO2 gas controller.
<ol>
	<li>To monitor the differentiation of organoids before performing a functional swelling assay36, capture the whole slide images manually with any bright-field microscope or with an automated imaging system, as detailed below.
	<ol>
		<li>Turn on the power to the automated imaging system and the CO2/O2 gas controller and allow the system to complete the automated calibration (~30 min).</li>
		<li>After completion, set the imaging system temperature to 37 &#176;C; add 15 mL of sterile water to the humidification reservoir, open the CO2 valves, close the lids, and let the imaging system pre-incubate for a minimum of 30 min before imaging.</li>
		<li>Open the automated imaging software to set up a protocol for imaging and choose the location to save the raw experimental data. 
		NOTE: An example protocol file (Supplementary File 1), specific to the imaging system, has been provided as a template for the automated imaging of organoids to monitor organoid differentiation.</li>
		<li>Once the environmental settings are met, immediately transfer up to two 15-well slides with lids into the environmental chamber in the automated image system&#39;s slide holder insert (see Table of Materials).</li>
		<li>Select the wells to be imaged and start imaging to complete imaging of the desired wells. 
		NOTE: The basic recommended settings are: 4x and 10x air objective, bright-field channel, 2 x 2 montage for 4x objective to cover the whole well area, 4 x 4 montage for a 10x objective. Z-stack settings: three to six Z-stack slices, Z-step size = 50-100 &#181;m, one to two slices below autofocus point, and three to five slices above.</li>
	</ol>
	</li>
	<li>To image live organoids for use in a forskolin-induced swelling (FIS) assay, use a microscope with an automated stage equipped with an environmentally controlled imaging chamber that allows temperature and CO2 control.
	<ol>
		<li>Begin with steps 5.1.1-5.1.4, substitute the protocol file in step 5.1.3 with the one provided in the example protocol file (Supplementary File 2) containing settings specific for performing a FIS assay.</li>
		<li>Before each experiment, adjust the exposure settings by evaluating at least three wells for each slide (far left, middle, and far right). Apply the X and Y coordinate offsets to ensure that the objective will be in the center of the well for all the wells. 
		NOTE: The basic recommended settings are: 4x air objective, Channel 1 = Bright Field, Channel 2 = DAPI, 2 x 2 montage (four image tiles). Z-stack settings: three to four Z-stack slices, Z-step size = 50-100 &#181;m, one slice below autofocus point and two to three slices above. Image acquisition time: 8 h with 20 min intervals (Total reads = 25; Read 1 is T = 0).</li>
		<li>Once all the settings are appropriately selected, choose the wells to image for both slides, or select all, and begin the run as per the equipment&#39;s instructions.</li>
		<li>After running, save the experiment, close the imaging software, and shut down the imaging system37.</li>
	</ol>
	</li>
</ol>
6. Baseline lumen measurements
NOTE: This is done using manual imaging analysis software (see Table of Materials). A similar methodology can be followed using an open-source software38 or any software that can measure the area of a region on an image.
<ol>
	<li>In the software, open the Automated Measurement Panel by right-clicking the bottom of the screen, select Measurement, and then Automated Measurement Results. The area of each region of interest (ROI) measured will appear there.</li>
	<li>Open the organoid image in the software and select 5-10 organoids with visible lumens. 
	NOTE: Lumens will be a circular area in the middle of the organoid that is visibly different in color than the rest of the organoid (Figure 2A).</li>
	<li>Using the polygon ROI measurement feature, hold right click on the image to open the menu and select Polygonal ROI to outline the full organoid to obtain the organoid&#39;s total surface area (TSA). Then using the same feature, outline the lumen area (LA) (Figure 2B).</li>
	<li>Repeat for the remaining organoids in the well and all the wells in the assay.</li>
	<li>Export the data to excel. Divide the LA by the TSA and average all the organoids from the sample to get the Baseline Lumen Ratio (BLR)11,13. 
	&#8203;NOTE: Typically, ~87% of non-CF organoids will have a BLR over 0.6, and 97% will be over 0.5, while only 14% of CF organoids will have a BLR over 0.6, and 31% will be over 0.5.</li>
</ol>
7. Pre-treatment and automated imaging of HNE organoids
NOTE: All pre-treatment steps are carried out in a clean biosafety cabinet. Pre-setup the automated imaging system and the software for recording the assay before step 7.1. The incubation with DAPI is optional but is recommended as a fail-safe if the quality of bright field images is compromised. In this case, the DAPI channel (377 nm) can be analyzed instead.
<ol>
	<li>Pre-incubate the organoids in a well of 15-well slides with 50 &#181;L of differentiation media containing DAPI with or without 100 &#181;M of CFTRinh-172 (see Table of Materials) in a 37 &#176;C incubator for 1 h. While the organoids incubate, perform step 5.2.1 using a swelling assay custom protocol following Supplementary File 2.</li>
	<li>Remove the pre-incubation medium using a glass Pasteur pipette with aspiration. Add 10 &#181;M of forskolin and 100 &#181;M of IBMX (stimulation cocktails) (see Table of Materials) for a total volume of 50 &#181;L differentiation media into each well. 
	NOTE: Ensure no bubbles are introduced into the wells. Bubbles on the images will affect the automated analysis.</li>
	<li>Without delay, begin the FIS imaging protocol following step 5.2.2 to 5.2.4. Acquire images every 20 min in each well, with a total runtime of 8 h.</li>
</ol>
8. Automated analysis of forskolin-induced swelling assay on HNE organoids
<ol>
	<li>Open the automated imaging analysis software, find the experiment previously saved from step 7.3, and bring up the images of the experiment.</li>
	<li>Choose the vessel window to be evaluated and select the option containing the processed images that have been stitched and z-projected. This step should provide a picture of the entire well with all organoids within the imaging frame for masking assessment and measurements.</li>
	<li>Choose the well image to assess. Select Analyze. Repeat this process for other images to ensure appropriate masking for all images included in the automated measurements. Save the setting parameters.</li>
	<li>After completing the analyzing settings, apply the changes. The software will change the measurements based on the settings. 
	NOTE: After the initial image pre-processing is complete, quality control (QC) measures should be performed to ensure consistent masking. These include manually reviewing all wells to ensure organoids are within the imaging frame, bubbles or debris are not inappropriately masked, and checking the masking in bright field and DAPI channels.</li>
	<li>Export the data for the summary analysis (including graphing and statistical analysis).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+keratinocytes+are+efficiently+immortalized+by+a+Rho+kinase+inhibitor.">Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.</a> Journal of Clinical Investigation. 120 (7), 2619-2626 (2010).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells.">ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.</a> The American Journal of Pathology. 180 (2), 599-607 (2012).</li><li>Palechor-Ceron, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+induces+diffusible+feeder+cell+factor(s)+that+cooperate+with+ROCK+inhibitor+to+conditionally+reprogram+and+immortalize+epithelial+cells.">Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells.</a> The American Journal of Pathology. 183 (6), 1862-1870 (2013).</li><li>Scudieri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionocytes+and+CFTR+chloride+channel+expression+in+normal+and+cystic+fibrosis+nasal+and+bronchial+epithelial+cells.">Ionocytes and CFTR chloride channel expression in normal and cystic fibrosis nasal and bronchial epithelial cells.</a> Cells. 9 (9), (2020).</li><li>Marthin, J. K., Stevens, E. M., Larsen, L. A., Christensen, S. T., Nielsen, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+three-dimensional+explant+spheroids+derived+from+human+nasal+airway+epithelium:+a+simple+methodological+approach+for+ex+vivo+studies+of+primary+ciliary+dyskinesia.">Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia.</a> Cilia. 6, 3 (2017).</li><li>Blouquit, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+and+fluid+transport+properties+of+small+airways+in+cystic+fibrosis.">Ion and fluid transport properties of small airways in cystic fibrosis.</a> American Journal of Respiratory Cell and Molecular Biology. 174 (3), 299-305 (2006).</li><li>Birket, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+with+cystic+fibrosis+transmembrane+conductance+regulator+modulators+augment+the+airway+functional+microanatomy.">Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy.</a> The American Journal of Physiology - Lung Cellular and Molecular Physiology. 310 (10), 928-939 (2016).</li><li>Birket, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+anatomic+defect+of+the+cystic+fibrosis+airway.">A functional anatomic defect of the cystic fibrosis airway.</a> American Journal of Respiratory and Critical Care Medicine. 190 (4), 421-432 (2014).</li><li>Chu, K. 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In vivo imaging of airway cilia and mucus clearance with micro-optical coherence tomography.</a> Biomedical Optics Express. 7 (7), 2494-2505 (2016).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+quantitative+study+of+airway+functional+microanatomy+using+micro-optical+coherence+tomography.">Method for quantitative study of airway functional microanatomy using micro-optical coherence tomography.</a> PLoS One. 8 (1), 54473 (2013).</li><li>Tuggle, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+defects+in+ion+transport+and+tissue+development+in+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)-knockout+rats.">Characterization of defects in ion transport and tissue development in cystic fibrosis transmembrane conductance regulator (CFTR)-knockout rats.</a> PLoS One. 9 (3), 91253 (2014).</li><li>McCravy, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalised+medicine+for+non-classic+cystic+fibrosis+resulting+from+rare+CFTR+mutations.">Personalised medicine for non-classic cystic fibrosis resulting from rare CFTR mutations.</a> European Respiratory Journal. 56 (1), 2000062 (2020).</li><li>Mutyam, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+benefit+observed+with+the+CFTR+potentiator,+ivacaftor,+in+a+CF+patient+homozygous+for+the+W1282X+CFTR+nonsense+mutation.">Therapeutic benefit observed with the CFTR potentiator, ivacaftor, in a CF patient homozygous for the W1282X CFTR nonsense mutation.</a> Journal of Cystic Fibrosis. 16 (1), 24-29 (2017).</li><li>Corning Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CORNING CELL-TAK CELL AND TISSUE ADHESIVE. , (2013).</li><li>Anderson, J. D., Liu, Z., Odom, L. V., Kersh, L., Guimbellot, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+function+and+clinical+response+to+modulators+parallel+nasal+epithelial+organoid+swelling.">CFTR function and clinical response to modulators parallel nasal epithelial organoid swelling.</a> The American Journal of Physiology - Lung Cellular and Molecular Physiology. 321 (1), 119-129 (2021).</li><li>Biotek Instruments, Incorporated. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lionheart FX Live Cell Imager Operator's Manual. , (2016).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. 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JSG is listed as an inventor on a patent application 20170242033 from the University of North Carolina that describes a similar model. When licensed technology from UNC produces royalties, the inventors receive a share of the revenue. Otherwise, the authors declare no conflicts of interest. The funders had no role in the study's design, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

This manuscript provides detailed methodologies for comprehensive live and fixed imaging of the airway epithelial organoids derived from HNE brush biopsy. It describes functional assays that can determine CFTR activity in an individual. HNEs provide a minimally invasive, primary tissue for a variety of applications. The expansion techniques offered here can be used for modeling airways disease, including organoids. Organoids can be used for precision therapeutic approaches and to monitor the stability of gene or mRNA-bas...

Introduction to the technique 
Ex vivo culture-based assays are an increasingly utilized tool for precision medicine and the study of disease pathophysiology. Primary human nasal epithelial (HNE) cell culture has been used in numerous studies of cystic fibrosis1,2,3,4,5,6,7,8,9,10,11,12,13, an autosomal recessive disease that affects epithelial cell function in multiple organs. HNE culture provides a renewable source of airway epithelia that may be obtained prospectively and recapitulates electrophysiological and biochemical qualities to test Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity. HNE cells can be sampled with minimal side effects14, similar to common viral respiratory swabs. Research work describing a model for cystic fibrosis study derived from HNE brush biopsies has been recently published11,13. While similar to other models using primary HNE2,3 and intestinal tissue15,16,17,18,19, detailed characterization of the differentiation and imaging of this model are described here for use in CF research and for aiding in the studies of other airways diseases13. The organoid model is not unlimited like immortalized cell lines but can be expanded by conditional reprogramming (using irradiated and inactivated feeder fibroblasts and Rho-kinase inhibitors) to a more stem cell-like state20,21,22,23. The processing of HNE brush biopsies using this method yields large numbers of epithelial cells for use in multiple applications at higher throughput while still retaining the ability to differentiate fully. While this protocol was developed using feeder cells, other methodologies may be used by investigators wishing to avoid feeder cell technology14,24.
Importance of the technique to pulmonary biology 
A significant study has been devoted to understanding how the absence of regular, functioning CFTR in the cell membrane of epithelial cells results in dysfunction in the lungs, pancreas, liver, intestine, or other tissues. Dysfunctional epithelial ion transport, particularly that of chloride and bicarbonate, results in a decreased volume of the epithelial lining fluids and changes in mucous secretions, leading to mucous stasis and obstruction. In other airway diseases, such as primary ciliary dyskinesia, altered ciliary motion impairs mucociliary clearance and leads to mucous stasis and obstruction25. Therefore, the current HNE organoid model has been developed for various applications, depending on the investigator&#39;s experimental design and resources. This includes live-cell imaging using live-cell stains; fixation and sectioning to characterize the morphology; immunofluorescence staining with antibodies and whole-mount confocal imaging to avoid disrupting intraluminal structures; and bright-field imaging and micro-optical coherence tomography for quantitative measurements of ciliary beat frequency and mucociliary transport13. To facilitate expansion to other investigators, commercially available reagents and supplies were used for culturing. A functional assay was developed that used common microscope techniques and more specialized equipment. Overall, while the present model was designed to assess CFTR activity at baseline or in response to therapeutics, the techniques described in this protocol can be applied to other diseases involving epithelial cell function, especially epithelial cell fluid transport.
Comparison to other methodologies 
Recently the utility of this organoid model was developed by correlating in vitro CFTR modulator responses of patients&#39; organoids with their clinical response11. Notably, it is also demonstrated that the present model paralleled short-circuit current responses, the current gold standard for assessing CFTR function, in the same patients. Short-circuit current differs from the swelling assay because the former measures CFTR function via ion transport26. In contrast, this assay measures a more downstream effect with fluid transport, providing additional information about the overall function of CFTR27,28,29,30,31,32. Short-circuit current measurements have continued to be a common and reliable method for determining CFTR chloride channel activity1,33. These electrophysiological assays require specialized, expensive equipment, require many times more cells for each experimental replicate than the organoid assay, cannot be easily automated, and are not amenable to scaling up for higher throughput applications. Another organoid model derived from intestinal epithelia has additional advantages15,16,17,18, such as more excellent replicative capability, but is neither derived from an airway tissue nor is universally available. HNE brushings are obtained with inexpensive cytology brushes without the need for sedation and at minimal risk. Getting the brushing does not require a clinician and can be performed by trained research coordinators and other research staff14. The HNE organoid model can be cultured by any laboratory with primary cell culture capabilities, and some of the applications can be performed with standard microscopy techniques. Altogether, these advantages provide additional access to technology for assessing airway epithelial function that might otherwise be unavailable to some laboratories. Furthermore, HNE organoids can be utilized to study other disease states that affect the airway, such as primary ciliary dyskinesia25 or viral infection, which intestinal organoids cannot.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nasal brush</td>
			<td>Medical Packaging CYB1</td>
			<td>CYB-1</td>
			<td>Length: 8 inches, width approximately 7 mm</td>
		</tr>
		<tr>
			<td>Large-Orifice Pipette Tips</td>
			<td>ThermoFisher Scientific</td>
			<td>02-707-141</td>
			<td>Large bore pipette tips</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ThermoFisher Scientific</td>
			<td>A1110501</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>0.05% trypsin -EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from soybean</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td>Working solution: 1mg/mL in 1XDPBS</td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning</td>
			<td>356255</td>
			<td>Extracellular matrix (EM)</td>
		</tr>
		<tr>
			<td>&#181;-Slide Angiogenesis</td>
			<td>Ibidi</td>
			<td>81506</td>
			<td>15-well slide</td>
		</tr>
		<tr>
			<td>24-Well Transwell</td>
			<td>Corning</td>
			<td>7200154</td>
			<td>Culture insert</td>
		</tr>
		<tr>
			<td>Chambered Coverglass</td>
			<td>ThermoFisher Scientific</td>
			<td>155409</td>
			<td>8-well glass-bottom chamber slides</td>
		</tr>
		<tr>
			<td>Cell-Tak Cell and Tissue Adhesive</td>
			<td>ThermoFisher Scientific</td>
			<td>354240</td>
			<td>Cell adhesive</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue</td>
			<td>ThermoFisher Scientific</td>
			<td>R37605</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Eclipse Ts2-FL (Inverted Routine Microscope)</td>
			<td>Nikon</td>
			<td></td>
			<td>Inverted epi-fluorescence microscope or bright-field microscope</td>
		</tr>
		<tr>
			<td>Nikon A1R-HD25</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>NIS Elements- Basic Research</td>
			<td>Nikon</td>
			<td></td>
			<td>manual imaging analysis software</td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>ThermoFisher Scientific</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Base Molds</td>
			<td>ThermoFisher Scientific</td>
			<td>41-740</td>
			<td></td>
		</tr>
		<tr>
			<td>Lionheart FX</td>
			<td>BioTek</td>
			<td>BTLFX</td>
			<td>Automated image system</td>
		</tr>
		<tr>
			<td>Lionheart Cover</td>
			<td>BioTek</td>
			<td>BT1450009</td>
			<td>Environmental Control Lid</td>
		</tr>
		<tr>
			<td>Humidity Chamber</td>
			<td>BioTek</td>
			<td>BT1450006</td>
			<td>Stage insert (environmental chamber)</td>
		</tr>
		<tr>
			<td>Gas Controller for CO2 and O2</td>
			<td>BioTek</td>
			<td>BT1210013</td>
			<td>Gas controller</td>
		</tr>
		<tr>
			<td>Microplate/Slide Stage Insert</td>
			<td>BioTek</td>
			<td>BT1450527</td>
			<td>Slide holder</td>
		</tr>
		<tr>
			<td>Gen5 Imaging Prime Software</td>
			<td>BioTek</td>
			<td>BTGEN5IPRIM</td>
			<td>Automated imaging analysis software</td>
		</tr>
		<tr>
			<td>4x Phase Contrast Objective</td>
			<td>BioTek</td>
			<td>BT1320515</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Phase Contrast Objective</td>
			<td>BioTek</td>
			<td>BT1320516</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Cube</td>
			<td>BioTek</td>
			<td>BT1225007</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cube (DAPI)</td>
			<td>BioTek</td>
			<td>BT1225100</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>CFTRinh-172</td>
			<td>Selleck Chemicals</td>
			<td>S7139</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Expansion Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11965</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 Nutrient mix</td>
			<td>ThermoFisher Scientific</td>
			<td>11765</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;16140-071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;PHG0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone (HC)</td>
			<td>Sigma</td>
			<td>&#160;H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>&#160;I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>&#160;A2786</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stemgent</td>
			<td>&#160;04-0012-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ceftazidime</td>
			<td>Alfa Aesar</td>
			<td>&#160;J66460-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Alfa Aesar</td>
			<td>&#160;J67340</td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Alfa Aesar</td>
			<td>&#160;J67251</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>&#160;A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Differentiation Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 (1:1)</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;11330-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultroser-G</td>
			<td>Pall</td>
			<td>&#160;15950-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Clone II</td>
			<td>Hyclone</td>
			<td>&#160;SH30066.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Brain Extract</td>
			<td>Lonza</td>
			<td>&#160;CC-4098</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>&#160;I-9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>&#160;H-0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma</td>
			<td>&#160;T-6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>&#160;T-0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma</td>
			<td>&#160;E-0135</td>
			<td></td>
		</tr>
		<tr>
			<td>Epinephrine</td>
			<td>Sigma</td>
			<td>E-4250</td>
			<td></td>
		</tr>
		<tr>
			<td>O-Phosphorylethanolamine</td>
			<td>Sigma</td>
			<td>P-0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human CFTR antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1660</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>Thermo Fisher</td>
			<td>MA3-39100-A647</td>
			<td>Dilution: 1000x</td>
		</tr>
		<tr>
			<td>Anti-MUC5B antibody</td>
			<td>Sigma</td>
			<td>HPA008246</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>Anti-acetylated tubulin</td>
			<td>Sigma</td>
			<td>T7451</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>Anti-beta IV Tubulin antibody</td>
			<td>Abcam</td>
			<td>Ab11315</td>
			<td>Dilution: 100x</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td>Dilution: 2000x</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L), Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A21207</td>
			<td>Dilution: 2000x</td>
		</tr>
	</tbody>
</table>

culture and imaging of human nasal epithelial organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

HNEs expansion is essential for a thriving organoid culture. HNEs from a successful sample collection should expand to over 70% confluence around 10 days. An example of successful and unsuccessful samples is shown in Figure 1A and Figure 1B, respectively. The cells must be discarded if they cannot reach 70% confluence by 14 days after co-culture with irradiated 3T3 cells. Any contaminated cells are to be immediately discarded if unable to rescue...

Watch this Scientific Journal Video about Culture and Imaging of Human Nasal Epithelial Organoids at JoVE.com

Culture and Imaging of Human Nasal Epithelial Organoids

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

culture-and-imaging-of-human-nasal-epithelial-organoids

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Medicine

3.5K Views.  University of Alabama at Birmingham (UAB). A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Video: Culture and Imaging of Human Nasal Epithelial Organoids

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...