Protocol for generating airway organoids from 2D air liquid interface-differentiated nasal epithelia for use in a functional CFTR assay

Summary We present a protocol to generate organoids from air-liquid-interface (ALI)-differentiated nasal epithelia. We detail their application as cystic fibrosis (CF) disease model in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent forskolin-induced swelling (FIS) assay. We describe steps for isolation, expansion and cryostorage of nasal brushing-derived basal progenitor cells, and their differentiation in ALI cultures. Furthermore, we detail the conversion of differentiated epithelial fragments into organoids of healthy controls and CF subjects for validating CFTR function and modulator responses. For complete details on the use and execution of this protocol, please refer to Amatngalim et al.1


MATERIALS AND EQUIPMENT Stock preparation of medium compounds
Timing: 16-20 h Preparation of basic BC medium, BC isolation and BC expansion medium Basic basal cell (BC) medium is first prepared and can be used to make BC isolation medium or BC expansion medium by addition of fresh supplements.Basic BC medium can be stored at À20 C for a maximum of 6 months, if desirable as 10 mL or 40 mL aliquots.BC isolation and expansion medium can be stored at 4 C for 2 weeks.BC isolation medium is used for the culturing of basal progenitor cells the first 7 days after isolation.BC expansion medium is used for further expansion of basal progenitor cells.

Protocol
The following variations of ALI-diff medium are used for different stages of differentiation.

Preparation of airway organoid medium
Airway organoid (AO) medium is the basic medium for airway organoids.It can be stored at À20 C for 6 months or at 4 C for 2 weeks.
The following variations of AO medium are used.

STEP-BY-STEP METHOD DETAILS
Workflow to culture nasal brushing-derived basal progenitor cells

Timing: 1-2 months
This part describes the workflow to culture nasal epithelial basal progenitor cells and includes the isolation, expansion and cryostorage of nasal brushing-derived basal progenitor cells (Figure 1).

Final concentration Amount
Advanced Obtain nasal cells by nasal brushing, according to the section ''obtaining nasal cells by nasal brushing''.2. Expand human nasal epithelial cells (HNEC) passage 0 (p0) in one well of a 12-well plate until a 90% confluent cell layer exists.3. Passage HNEC p0 cells to 2 wells of a 6-well plate according to the section ''passaging and expansion of nasal epithelial basal progenitor cells'' for further expansion as HNEC p1 cells.4. When confluent, freeze HNEC p1 cells as a master cell bank (MCB) according to the section ''freezing nasal epithelial basal progenitor cells''.
Note: All cells can be frozen as a MCB or 0.5 3 10 6 cells can be passaged to another collagencoated well on a 6-well plate for further expansion.

Expand HNEC p2 cells.
Note: Use cells from the previous step or thaw a MCB vial according to the section ''thawing and further expansion of cryostored nasal epithelial basal progenitor cells''.
Note: All cells can be frozen as a WCB or 0.5 3 10 6 cells can be passaged to another collagencoated well on a 6-well plate for further expansion.
Note: Use cells from the previous step or thaw a WCB vial according to the section ''thawing and further expansion of cryostored nasal epithelial basal progenitor cells''.
8. Once confluent, these cells are ready for differentiation as ALI-cultures.

Timing: 15 min
This section describes the collection of nasal cells through brushing of the inferior nasal turbinates.
Preparations 9. Prepare a 15 mL tube with 7 mL collection medium.10.Prepare a 15 mL tube with 5 mL PBS0.
Note: Smaller nasal brushes may be needed for younger children.These can be created by connecting a p200 non-filter tip to an interdental brush with parafilm (Figure 2).

Nasal brushing
11. Inform the nasal cell donor about the procedure and intended use of the cells.Explain that the lacrimal gland will be stimulated by the brushing procedure, which might cause tearing eyes.
Infrequently, it causes a nose bleed.12. Make sure informed consent is documented through signing of appropriate informed consent forms.13.Ask the donor to blow their nose.14.Ask the donor to sit straight and sideways to the person taking the brush.15.Wet the nasal brush in the tube with PBS0.16.Hold one hand at the back of the head to prevent head movements during the procedure.17.Insert the brush horizontally into the nostril and the inferior turbinate until mild resistance.Turn the brush twice and remove the brush from the nose.18. Place the brush in the 15 mL tube with collection medium, properly labeled, and keep on ice.19.Repeat the procedure for the other nostril with a new brush and place in the same 15 mL tube with collection medium.20.Cut the top of the brushes and close the 15 mL tube.21.Maintain the collection tube with brushes on ice or in a refrigerator at 4 C until further processing, which should be performed within 6 h.

Timing: 45-60 min
This part describes the isolation of nasal epithelial basal progenitor cells from nasal brushes.Half of the cells will be plated for expansion and the other half will be cryopreserved.
CRITICAL: All laboratory procedures with nasal cells should be performed in a laminar flow and sterile compounds should be used to prevent contamination.

Preparations
22. Coat a well in 12-well plate according to the section ''preparation: precoating of well plates for basal progenitor cell expansion''.23.Prepare and pre-warm BC isolation medium in a water bath at 37 C. 24.Prepare freezing medium, according to the section ''overview of media''.

Isolation of epithelial cells from nasal brushes
25. Cut the tip (approximately 0.5 cm) of a non-filter p1000 pipette tip with scissors to allow the nasal brush to fit through the opening of the tip (Figure 3).
Note: This procedure can be performed in a sterile environment with ethanol-cleaned scissors.Another option is to autoclave tips after cutting.
26. Place one of the brushes temporarily in an empty 15 mL tube to provide space for scraping cells from the brush.27.Dissociate nasal cells in the tube with collection medium by scraping the brush through the opening of the p1000 pipette tip.Move the brush approximately ten times up and down through the p1000 pipette tip (Figure 4).Discard the brush afterwards.28.Repeat the previous step for the second brush.The cut tip used for the first brush can be re-used.29.Centrifuge the 15 mL tube with nasal cells at 400 g for 5 min at 4 C. 30.Aspirate the supernatant and resuspend the pellet in 4 mL TrypLE express enzyme + 20 mL Sputolysin (0.5% (v/v)).
CRITICAL: Be careful with aspiration when a lot of mucus is present in the pellet to prevent unintended loss of cells.
31.Incubate the cell suspension for 5 min at 37 C (water bath or incubator).32.Resuspend the cells by pipetting up and down approximately 5 times with a p1000 pipette to dissolve mucus.33.Incubate the suspension for an additional 5 min at 37 C (water bath or incubator).34.Add 8 mL of Ad-DF to inactivate the TrypLE express enzyme by dilution.35.Strain the cell suspension through a 100 mm cell strainer.36.Wash the strainer with 2 mL Ad-DF.37. Transfer half of the cell suspension to a new 15 mL tube.38.Centrifuge both 15 mL tubes at 400 g for 5 min at 4 C. 39.Use one 15 mL tube with cells for freezing: a. Aspirate the supernatant and resuspend the cell pellet in 500 mL CryoStor CS10 freezing medium (supplemented with 5 mM Y-27632).b.Transfer the cell suspension to a sterile cryovial, labeled with donor number.c.Gradually freeze the cells by placing the cryovials in a cell freezing container at À80 C. d.Transfer the vials to a liquid nitrogen freezer after 24 h.e. Refresh the medium three times a week, with a maximum of 2 consecutive non-refreshing days, with 1 mL BC isolation medium.f.After 7 days, change BC isolation medium for BC expansion medium.g.Culture the cells until $90% confluence (Figure 5).This will usually take 7-14 days.
CRITICAL: It is recommended to culture only one donor per plate because of the risk for infections in HNEC p0 cells.

Timing: 45-60 min
This part describes the procedure of passaging and further expansion of basal progenitor cells in 12or 6-well plates.This step is applied as indicated in the section ''workflow to culture nasal brushingderived basal progenitor cells''.Preparations 39.Coat wells in 12-or 6-well plates according to the section ''preparation: precoating of well plates for basal progenitor cell expansion''.40.Prepare and pre-warm BC expansion medium in a water bath at 37 C.

Passaging procedure
41. Proceed with passaging when the cell culture is approximately 90% confluent (Figure 5).42.Remove medium and wash the cells with PBS0.Protocol 49.Remove the collagen solution or PBS0 from the precoated wells and transfer the cells dissolved in BC expansion medium to these wells: a.For one well in a 12-well plate: add 0.2 3 10 6 cells to 1 mL BC expansion medium.b.For one well in a 6-well plate: add 0.5 3 10 6 cells to 2 mL BC expansion medium.50.Refresh the medium three times a week with BC expansion medium, until approximately 90% confluence (Figure 5).a.For one well in a 12-well plate: refresh with 1 mL BC expansion medium b.For one well in a 6-well plate: refresh with 2 mL BC expansion medium

Timing: 45-60 min
This part describes the general procedure of freezing nasal brushing-derived basal progenitor cells after expansion.
Preparations 51.Prepare freezing medium, according to the section ''overview of media''.
Freezing procedure 52.After expansion, detach basal progenitor cells from the well plate according to step 41-46 from the section ''passaging and expansion of nasal epithelial basal progenitor cells''.53.Determine cell concentration with a cell counter (e.g., BioRad Automated Cell Counter).54.Centrifugate the cell suspension at 400 g for 5 min at 4 C. 55.After centrifugation, aspirate the supernatant and resuspend the cells in CryoStor CS10 freezing medium (containing Y-27632) to obtain a cell suspension with a final concentration of 1-2 3 10 6 cells per mL.56.Divide 0.5 mL of the cell suspension over sterile cryovials.Depending on the dilution at step 55, each cryovial should contain 0.5-1 3 10 6 cells.Label the cyrovials, indicating passage number and donor number.57.Gradually freeze the cells by placing the cryovials in a cell freezing container at À80 C and subsequently transfer them to a liquid nitrogen freezer.
CRITICAL: Cells are first placed in a À80 C freezer for gradual cooling.For optimal cell viability after thawing, cells should be transferred to the liquid nitrogen freezer as soon as possible after reaching a temperature of À80 C.

Timing: 15-30 min
This part describes the general procedure of thawing and further expansion of cryostored nasal brushing-derived basal progenitor cells.
Preparation 58.Coat wells in 12-or 6-well plates according to the section ''preparation: precoating of well plates for basal progenitor cell expansion''.59.Prepare and pre-warm BC expansion medium.60.Prepare a 15 mL tube containing 5 mL Ad-DF.

Protocol
Thawing procedure 61.Take a cryovial with cells from the liquid nitrogen freezer and store on dry ice.If not processed immediately, store in the À80 C freezer for a maximum of 14 days.62. Place the cryovial with cells in a 37 C water bath until the cell suspension is almost thawed and rapidly transfer the cell suspension to the 15 mL tube.63.Wash the cryovial with 1 mL of Ad-DF and add to the 15 mL tube.64.Centrifuge at 400 g for 5 min at 4 C. 65.Remove the collagen solution or PBS0 from the precoated wells and transfer the cells dissolved in BC expansion medium to these wells: a.For one well in a 12-well plate: add 1 mL BC expansion medium.b.For one well in a 6-well plate: add 2 mL BC expansion medium.66. Refresh cell cultures times a week with BC expansion medium.
a.For one well in a 12-well plate: refresh with 1 mL BC expansion medium.b.For one well in a 6-well plate: refresh with 2 mL BC expansion medium.

Timing: 3-6 weeks
This section describes the 2D ALI differentiation of HNEC.Basal progenitor cells are transferred to Transwell inserts and cultured in submerged conditions with BC expansion medium to obtain a confluent cell layer.Medium is then changed to ALI-diff medium supplemented with the TGF-b inhibitor A83-01 for habituation to a new medium but without directly starting differentiation.In the next step, the apical medium is removed to culture the cells air-exposed to stimulate differentiation towards a pseudostratified airway epithelium, including secretory and ciliated cells.The day of airexposure is called t = 0. Neuregulin-1b is added to the medium to boost CFTR expression (Table 1).
Transfer basal progenitor cells to transwell inserts 69.Dissociate basal progenitor cells from a well plate according to step 41-46 from "passaging and expansion of nasal epithelial basal progenitor cells''.70.Centrifuge the cell suspension at 400 g for 5 min at 4 C. 71.Remove the coating solution from the Transwell inserts.72.Seed basal progenitor cells on the apical side of the Transwell inserts: a.For 6.5 mm inserts (24-well plate): 0.2 3 10 6 cells in 200 mL BC expansion medium.b.For 12 mm inserts (12-well plate): 0.5 3 10 6 cells in 200 mL BC expansion medium.73.Add BC expansion medium to the basolateral side: a.For 6.5 mm inserts (24-well plate): 800 mL BC expansion medium.b.For 12 mm inserts (12-well plate): 1 mL BC expansion medium.74.Refresh the medium three times a week and culture the cells under submerged conditions until full confluence is reached.This takes approximately 4-7 days.75.When confluent, change apical and basolateral medium for ALI-diff phase 1 medium (ALI-diff medium supplemented with A83-01 (500 nM)).76.Refresh the medium three times a week until a homogeneous monolayer exists with all cells having a similar size and morphology (Figure 7).This takes approximately 4-10 days.

Note:
The moment of air-exposure is called t = 0 of differentiation.
78.After 3-5 days, replace ALI-diff phase 2 medium for ALI-diff phase 3 medium (ALI-diff medium supplemented with neuregulin-1b (0.5 nM)).Refresh the basolateral medium twice a week.79.Wash the cells once a week at the apical surface, by incubation with PBS0 in a tissue incubator at 37 C for 5-10 min.a.For 6.5 mm inserts (24-well plate): wash with 125 mL PBS0.Note: A high abundance of ciliated cells might cause the unwanted formation of inside-out oriented organoids during the conversion of ALI-differentiated nasal epithelial cells into airway organoids.It is therefore recommended to proceed to the next step as soon as beating cilia are visible and not wait too long before proceeding.

Timing: 2-3 h
This section describes the conversion of ALI-differentiated nasal epithelial cells into airway organoids.The ALI-differentiated epithelial cell layer is detached from the Transwell insert with collagenase.Epithelial sheets are then manually disrupted and strained to generate 30-100 mm fragments to be plated in Matrigel droplets.The complete workflow from ALI-differentiated cell cultures towards airway organoids is shown in Figure 8.
Preparations 81.Pre-warm 24-well suspension plates in a tissue incubator for at least 24 h.82.Thaw Matrigel on ice or at 4 C. 83.Prepare and pre-warm AO culture medium.

Conversion of ALI-differentiated nasal epithelial cells into airway organoids
84. Wash the differentiated ALI-cultures at the apical surface with PBS0.Incubate for 5 min in a tissue incubator.a.For 6.5 mm inserts (24-well plate): 125 mL PBS0.b.For 12 mm inserts (12-well plate): 200 mL PBS0.85. Remove the PBS0 or medium at the apical and basolateral side of the ALI-culture.Add collagenase type II solution (1 mg/mL in Ad-DF) to the basolateral side.a.For 6.5 mm inserts (24-well plate): 600 mL collagenase solution.b.For 12 mm inserts (12-well plate): 1000 mL collagenase solution.86.Incubate for 45-60 min in a tissue incubator.Note: Epithelial fragments might stick to the edge of the Transwell membrane, which can be checked with a light microscope.In this case, a P200 pipet can be used to scrape the epithelial layer off the membrane.
89. Disrupt the epithelial layer into smaller fragments with a P1000 pipet tip.Evaluate the size of the fragments by eye or a light microscope.The majority of the epithelial fragments should have a diameter of approximately 30-100 mm (Figure 9).
Optional: When having difficulties to obtain small fragments, an additional P200 non-filter tip can be placed on top of the P1000 pipet tip.
90. Strain the epithelial fragments with a combination of a 100 mm pluriStrainerâ placed on top of a 30 mm pluriStrainerâ.a. Add an additional 4 mL Ad-DF to the 15 mL tube with epithelial fragments, and transfer the fragments through the strainers (Figure 10A).b.Discard the 100 mm strainer (Figure 10B).c.Flip the 30 mm strainer upside down on top of a new 50 mL tube (Figures 10C and 10D).d.Add 5 mL Ad-DF to the reversed 30 mm pluriStrainerâ to collect the epithelial fragments (size between 30-100 mm) in the 50 mL tube (Figure 10E).
Note: Wash both sides of the 100 mm and 30 mm pluriStrainersâ before use with 1 mL Ad-DF to enable efficient flow-through.CRITICAL: This step should be performed on ice to prevent solidification of Matrigel.
CRITICAL: Avoid formation of bubbles during resuspension of the epithelial fragments in Matrigel.
93. Place a pre-warmed 24-well suspension plate on top of a T175 flask filled with handwarm tap water (Figure 11).Apply 30 mL droplets per well.After 2 min, flip the plate upside down and solidify the droplets in a tissue incubator for 15-30 min.

Note:
The plate is flipped upside down to distribute the epithelial fragments equally through the Matrigel, enabling equal access to nutrients.
94. Add 500 mL pre-warmed AO culture medium to the epithelial fragments embedded in Matrigel (Figure 12A).95.Refresh the medium twice a week.
Note: Gently add the medium to avoid disruption of the Matrigel droplets.
Organoids can be passaged to a 96-well plate after lumen formation.

Passaging of nasal organoids to a 96-well plate
Timing: 1-2 h    Alternatives: Cell recovery solution is recommended to dissolve the Matrigel efficiently.As an alternative, ice cold Ad-DF can be used, in which case the 4 C incubation step can be skipped.a.With good quality organoid cultures, one 30 mL Matrigel droplet from a 24-well plate can be seeded into 16 wells of a 96-well plate.b.To avoid too sparsely seeded wells, it is recommended to first resuspend the organoids in a Matrigel volume sufficient to seed half of the wells.Plate a 4 mL test droplet and check density under a light microscope.In case the organoid density is too high, organoids can be diluted by adding more Matrigel (Figure 13).

Note:
The Matrigel-embedded organoids can be transferred to an Eppendorf tube for easier handling.
106.Take a pre-warmed 96-well plate and add 4 mL Matrigel droplets with organoids in each well.
CRITICAL: Regularly resuspend the organoids in the Eppendorf tube to keep a homogeneous suspension.
CRITICAL: Tap the 96-well plate regularly on a flat surface during plating (G after every 16-24 wells) to distribute the organoids in the same plane.
107.Solidify the Matrigel droplets by placing the plate in a tissue incubator for 15-25 min.108.Add 100 mL AO culture medium to each well and place the plate in a tissue incubator at 37 C and 5% CO2.109.Refresh the medium twice a week.

FIS assay for nasal organoids
Timing: 2-2.5 h This section describes the FIS assay which can be used to visualize and quantify the functional rescue of CTFR protein function by CFTR modulator treatment in nasal organoid swelling experiments.
Preparations: 5-10 days prior to the FIS assay 110.Replace organoid medium with 100 mL pre-warmed AO FIS assay medium, 5-10 days prior to the FIS assay.
Note: Organoids display complete loss of lumen formation 2-5 days after the media change to AO FIS assay medium (Figure 12C).
Preparations: Similar day as the FIS assay 112.Turn on the environmental control of the confocal microscope at least 1 h before start of the experiment at 37 C and 5% CO 2 .113.Dissolve calcein green (50 mg) in 6 mL DMSO, and prepare calcein green solution (1:750 v/v) in Ad-DF (10 mL/well).114.Prepare 23 concentrated dilutions of compounds to be added acute: i.e., forskolin (10 mM), CFTR potentiators (VX-770; 10 mM) or vehicle in Ad-DF.Dispense 125 mL of the compound solutions per well in a new U-bottom 96-well suspension plate.
Note: The stimuli will be further diluted 1:1 when added to the well plate with organoids, already containing 100 mL medium.So final concentrations of both forskolin and CFTR potentiators are 5 mM.

FIS assay
115.Dispense 10 mL calcein green solution to each well of the 96-well plate with organoids.Mix the calcein green solution through gentle resuspension (2-3 times pipetting) with a multichannel.
Alternatives: Instead of adding the calcein green solution with a multichannel, an automatic repetitive pipet (such as the Eppendorf Multipette E3) with a high dispensing speed could be used.
116.Incubate the plate in a tissue incubator for 30 min.117.Position the 96-well plate with organoids in the place holder of a confocal microscope and ensure the plate is fixed in position.118.Set the live cell imaging settings: a. Use the 2.53 objective.b.Calcein green staining can be visualized with an emission at 488 nm and excitation at 515 nm.c.Set the laser intensity, ensuring that the calcein green signal in the organoid structures is slightly oversaturated.d.Set the right position (X, Y) and focus (Z) for each well to obtain a good view of the organoids, or use autofocus when possible.e. Set a time lapse for 1 h measurement: i. Interval = 10 min.
Optional: If possible, include brightfield/DIC imaging to obtain a better view of organoid morphology.
119.Add 100 mL from the 96-well plate with acute stimuli to the well plate with organoids, using a multichannel.Immediately start imaging after addition of the acute stimuli.
CRITICAL: It is important to start imaging quickly after addition of the acute stimuli, as organoids might directly start swelling.

EXPECTED OUTCOMES
The FIS assay with intestinal organoids is already an established and widely used assay to predict CFTR modulator efficacy on individual basis. 2,3However, it is proposed that an airway model might be more convenient as people with CF are mostly suffering from respiratory symptoms.Furthermore, a nasal brushing is less invasive compared to an intestinal biopsy.Here we describe a protocol to perform the FIS assay in nasal brushing-derived airway organoids, of which we recently showed their ability to predict CFTR modulator responses. 1To guarantee evenly differentiated organoids, we differentiated basal progenitor cells as ALI-cultures before their transformation into organoids. 1his will diminish the variation in organoid morphology and in FIS between individual organoids in a single well.
By use of this protocol, investigators should be able to isolate, expand and differentiate basal progenitor cells from nasal brushes, to generate nasal airway organoids and to conduct the FIS assay to determine CFTR function and CFTR modulator response efficacy.
From nasal brushings of one donor, approximately 5 3 10 6 cells can be isolated and cryostored as a MCB p1 (10 vials containing 5 3 10 5 cells).Each MCB vial can be further expanded in the next passage (p2) to a yield of approximately 5 3 10 6 cells, indicating that in total approximately 5 3 10 7 basal progenitor cells (p2) can be generated from the MCB and 5 3 10 8 cells at a higher passage (p3).These basal progenitor cells (p3) are transferred to Transwell filters for ALI-differentiation. 5 3 10 5 basal progenitor cells are seeded on a 12 mm 12-well insert, which yields approximately 48-96 wells with organoids in a 96-well plate.When smaller Transwell inserts are used, 2 3 10 5 basal progenitor cells are seeded on a 6.5 mm 24-well insert, which yields approximately 16-32 wells with organoids in a 96-well plate.
Figures 14 and 15 illustrate example results of a FIS assay experiment.In non-CF nasal organoids, forskolin stimulation induces organoid swelling (Figure 14).CF nasal organoids lack swelling in response to forskolin, but the swelling response is restored upon treatment with CFTR modulators (Figure 15).

QUANTIFICATION AND STATISTICAL ANALYSIS
The output of the FIS assay is a time series of confocal images containing calcein green stained organoids.For quantification and interpretation of the results, organoid swelling is calculated as the relative increase of total organoid area over time, according to the following steps.
1. Determination of total organoid area in each image (Table 2): a.Total organoid area is determined in each individual image using image analysis software, such as Zen blue (Zeiss), or open-source software, such as ImageJ or CellProfiler. 4Further processing can be performed with Excel, Graphpad or R. b.Particles with a size smaller than 6.000 mm 2 are excluded from analysis.
2. Normalization of total organoid area to baseline (Table 3): a. Organoid swelling is usually expressed as relative increase to baseline (t = 0), which is set at 100%.The formula to calculate normalized organoid area for individual wells at a specific time point x is: Normalized organoid area at t = x = Total organoid surface area at t = x Total organoid surface area at t = 0 3 100% b.The average normalized organoid area (%) of technical replicates within a plate can be plotted against time (minutes) to visualize the kinetics of organoid swelling (Figure 16A).
Note: Individual wells can be excluded from analysis when less than 10 organoids are present in a well, or when out of focus organoids are not recognized in all time points of the experiment.

LIMITATIONS
This protocol describes the use of primary nasal cell cultures.It is important to realize that working with primary cell cultures is different compared to cell lines.Primary cell cultures have an increased risk of microbial outgrowth during the first day of cell isolation, especially in cell cultures derived from individuals with CF.Furthermore, donor to donor variation exists, such as differences in the expansion rate of basal progenitor cells, in differentiation time of ALI-cultures and in the organoid yield.This variation might be related to donor characteristics, to the yield of cells from the nasal brushing, to environmental conditions or to the freeze/thawing procedure.It is recommended to prevent changes in medium batches and guarantee correct freeze/thawing procedures.Be aware that changing manufacturer of medium compounds or using alternative cultureware may affect the quality of the cell and organoid cultures.

TROUBLESHOOTING Problem 1
Fungal infection of basal cell cultures, related to the section ''isolation of nasal epithelial basal progenitor cells from nasal brushes''.

Potential solution
Add the antifungal reagent Fungin (50 mg/mL) to the cell culture medium, wrap the plate in foil, keep the cell culture isolated from other cell cultures and check if the infection disappears in  the coming 3 days.If not, the cell culture should be thrown away to prevent contamination of other cell cultures.

Problem 2
Squamous cell differentiation in basal cell cultures or ALI-cultures related to the section ''passaging and expansion of nasal epithelial basal progenitor cells'' or ''2D ALI differentiation of nasal epithelial cells'' (Figure 17).

Potential solution
In general, basal progenitor cells displaying squamous cell differentiation cannot be used for ALI-differentiation or organoid formation.
When squamous cell differentiation occurs in multiple donors cultured in parallel, it might be caused by incorrect preparation of the culture medium, e.g., the use of compounds past their expiring date, or incorrect storage or preparation of stock compounds or media batches.Renew compounds or medium when needed.
The occurrence of squamous cell differentiation might also relate to a specific donor.Thaw and expand cells of other vials from this donor and determine whether squamous cell differentiation occurs.If this is the case, the cells of this donor cannot be further used.Consider to repeat the isolation of nasal cells from the frozen back up vial or to collect new nasal brushes.

Problem 3
Cells have difficulty starting up after thawing, related to the section ''thawing and further expansion of cryostored nasal epithelial basal progenitor cells''.Total organoid area is normalized to baseline (t = 0, 100%).

Potential solution
Thaw cells in a smaller wells, or thaw cells from a lower passage number to create a new working cell bank.

Problem 4
Inside out-oriented organoids, related to the section ''conversion of ALI-differentiated nasal epithelial cells into airway organoids''.

Potential solution
When differentiated ALI cultures display high numbers of ciliated cells, this may impede formation of organoids with the lumen facing inward.Cilia beating in highly ciliated epithelial fragments prevents solidification of the Matrigel, leading to formation of inside out-oriented organoids.Therefore, cells should not be differentiated too long at the ALI and should be converted into organoids as soon as cilia appear during differentiation.

Problem 5
Nonoptimal organoid density in 96-well plates for a FIS assay experiment, related to the section ''passaging of nasal organoids to a 96-well plate''.

Potential solution
For optimal performance of the FIS assay, it is important to seed organoids in the right density in 96-well plates (Figure 13).A high seeding density might cause problems for organoid recognition during image analysis, especially when organoids are in different planes.A low seeding density will provide unreliable results.

Problem 6
Organoids swell during the experiment without any stimulation, related to the section ''FIS assay for nasal organoids''.

Potential solution
Check temperature and CO 2 settings of the microscope incubator as organoid swelling might be pH dependent.

3 0
,5-Triiodo-L-thyronine sodium salt Dissolve 100 mg in 100 mL NaOH (0.2 M) and filter sterilize (0.22 mm) 1 mg/mL À80 C for 1 year (G)-Epinephrine hydrochloride Dissolve 25 mg in 5 mL PBS0 and filter sterilize (0.22 mm) 5 mg/mL À20 C for 1 year Hydrocortisone Dissolve 5 mg in 1 mL Ethanol 100% and subsequently in 49 mL Ad-DF.Filter sterilize (0.22 mm) 100 mg/mL À80 C for 1 year N-Acetyl-L-cysteine Dissolve 3.26 g in 40 mL PBS0 MQ and filter sterilize (0.22 mm) -epidermal growth factor (EGF) Dissolve 100 mg in 2 mL PBS0-BSA 0.1% 50 mg/mL À20 C for 1 year Recombinant human Fibroblast growth factor 10 (FGF-10) Dissolve 1 mg in 1 mL PBS0-BSA 0.1% 1 mg/mL À20 C for 1 year Recombinant human Fibroblast growth factor 7 (FGF-7) Dissolve 100 mg in 1 mL PBS0-BSA 0.1% 100 mg/mL À20 C for 1 year Recombinant human Hepatocyte growth factor (HGF) Dissolve 100 mg in 0.4 mL PBS0-BSA 0.1% 250 mg/mL À20 C for 1 year Recombinant Neuregulin-1b Dissolve 100 mg in 1333 mL PBS-BSA 0.1 % 10 mM À20 C for 1 year Recombinant Interleukin-1b Dissolve 10 mg in 1000 mL PBS-BSA 0.1 % 10 mg/mL 20 C for 1 year RSPO3-Fc Fusion Protein conditioned medium (R-spondin 3) Ready to use -À80 C or À20 C for 3 months Collagen IV Dissolve 10 mg in filter sterilized (0.22 mm filter) 10 mL acetic acid (0Overview of all different media used in the protocol Name Abbreviation Preparation Application Collection medium N/A Section ''preparation of collection medium'' Collection and storage of nasal brushings (Continued on next page) STAR Protocols 4, 102337, September 15, 2023 Protocol Preparation of collection medium Collection medium is used for the collection and preservation of nasal brushings.It can be stored at 4 C for a maximum of one year.

Figure 1 .
Figure 1.Workflow to culture nasal brushing-derived basal progenitor cells

Figure 2 .
Figure 2. Nasal brushes (A) Nasal brush for younger children, created from an interdental brush connected to a p200 non-filter tip.(B) Cytological brush used for adults.

Figure 3 .
Figure 3.The tip of a non-filter p1000 pipette tip is cut off to make the opening large enough for passing of a nasal brush

Figure 4 .
Figure 4. Scraping cells off the brush (A) The brush is moved up and down through the pipette tip to scrape cells from the brush and collect them in the tube with collection medium.(B) A close-up of how to move the brush up and down through the pipette tip.The procedure must be carried out in the collection medium inside the 15 mL tube.

Figure 5 .
Figure 5. 90% confluent nasal epithelial basal progenitor cell culture, ready for passaging (A and B) The cell layer is shown in a low (A) and high (B) magnification.Scale bar equals 200 mm.

Figure 6 .
Figure 6.Detached cells after incubation with TrypLE express enzyme Scale bar equals 200 mm.

Figure 7 .
Figure 7. Transwell insert with a 100% confluent cell layer with homogeneous morphology, ready for the switch to air-exposed conditions Scale bar equals 200 mm.

Figure 9 .
Figure 9. Correct size of epithelial fragments in a 15 mL tube after disruption Scale bare equals 200 mm.

Figure 10 .
Figure 10.The steps (A-E) of the straining procedure to obtain epithelial fragments with a size between 30-100 mm.A 100 mm (yellow) and 30 mm pluriStrainerâ (purple) are used.

Figure 11 .
Figure 11.Epithelial fragments embedded in Matrigel are plated in a 24-well plate placed on top of a T175 flask filled with warm water (A) Side view.(B) Top view.

Figure 12 .
Figure 12.Formation of airway organoids (A) Epithelial fragments embedded in Matrigel, 30 min after plating and solidification of the Matrigel in the 24-well plate.(B) Airway organoids acquired intrinsic lumen, 3 days after plating.(C) Airway organoids lose their intrinsic lumen by changing medium to AO FIS medium, 5 days after medium change and after passaging to a 96-well plate.Scalebar equals 1000 mm.
101.Resuspend the Matrigel droplet once in cell recovery solution.102.Incubate the well plate at 4 C in the fridge for 5-10 min until the Matrigel is dissolved.
103.Transfer the organoids to a 15 mL tube with 7 mL ice cold Ad-DF to inactivate the cell recovery solution.Keep the tube on ice.104.Centrifuge the organoids at 400 g and 4 C for 5 min.105.Remove supernatant and resuspend the organoid pellet in 100% Matrigel.Keep the tube on ice.The plating volume is 4 mL * amount of wells in 96-well plate with 10% extra volume.

Figure 13 .
Figure 13.Organoids plated in different densities (A) Well containing an appropriate number of organoids.(B) Well with too many organoids and in different Z planes.(C) Well with too few organoids.

Figure 14 .
Figure 14.Confocal images of calcein green stained organoids from a non-CF donor, unstimulated or stimulated with forskolin Scale bar equals 500 mm.

Figure 15 .
Figure 15.Confocal images of calcein green stained organoids from a CF donor (F508del/F508del) unstimulated or stimulated with either forskolin alone or with forskolin + CFTR modulator treatment (48h pre-incubation with VX-661 and VX-445 and acute stimulation with VX-770) Scale bar equals 500 mm.

Figure 16 .
Figure 16.Quantification of a FIS assay with the experimental data from Table 2 and Table 3 Panel A shows normalized organoid area (%) over time and panel B shows the corresponding AUC values.Data is shown as mean G SD from 4 technical replicates per condition.Fsk = forskolin.

Figure 17 .
Figure 17.Squamous cell differentiation of basal progenitor cells Scale bar equals 200 mm.
3. Calculation of area under the curve (AUC) values as a measure for organoid swelling:a.AUC values are calculated as a measure for organoid swelling.This creates a single data point per well which can be used to compare organoid swelling in response to different stimuli.b.Mean AUC values of technical replicates within a plate can be visualized with bar graphs (Fig-

Table 2 .
Example dataset showing total organoid area (mm 2 ) per well and per time point for a FIS experiment Quadruplicates are used as technical replicates within an experimental plate.