Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Summary

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Abstract

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 °C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Introduction

CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an epithelial anion channel. CF affects approximately 85,000 people worldwide¹. Over 2,000 CFTR mutations have been identified (www.genet.sickkids.on.ca). This diversity partially explains a wide spectrum of observed disease phenotypes (www.CFTR2.org)^2,3. Six classes of CFTR mutations are defined based on their effect on CFTR protein expression and function: (I) no synthesis, (II) impaired trafficking, (III) defective channel gating, (IV) altered conductance, (V) reduced levels of normally functioning CFTR, and (VI) impaired cell surface stability⁴. Although the common CFTR mutations are well studied, CFTR function and relation with clinical status remain poorly understood at the level of the individual, particularly for the large group of rare "orphan" mutations (www.CFTR2.org)^1,3.

Recently, drugs have been developed that target the CFTR protein in a mutation-specific fashion. Two classes of CFTR protein-targeting drugs are currently in clinical use and have distinct modes of action. Potentiators, such as VX-770, enhance the open-probability of apically-localized mutant CFTR and act directly upon their addition to cells⁵. Correctors, such as VX-809, restore the trafficking of endoplasmic reticulum-localized misfolded CFTR and require pre-incubation with cells before the effects are observed⁶. The CFTR potentiator, VX-770, has been registered for subjects with the G551D mutation^7,8, as well as for eight other CFTR gating mutations, including S1251N⁹; together, these mutations are carried by 5% of all CF subjects. Other clinical trials have indicated that VX-770, combined with the corrector VX-809, has limited yet significant effects on lung function and causes a decrease in exacerbation rates in subjects homozygous for the F508del mutation carried by 45-50% of patients^10,11.

Conventional clinical trials to identify drug-responsive subjects within the remaining 50% of CF patients are costly and time-consuming and are not feasible for individuals with extremely rare CFTR genotypes. Novel, cost-effective, personalized methods are crucial to match the increasing number of CFTR modulators to individuals carrying any type of CFTR mutation. Until now, the trial inclusion of patient groups carrying specific CFTR mutations has been guided by studies using mutant CFTR gene transfection in heterologous cell systems, followed by electrophysiological studies in Ussing chambers^5,6,12. Due to a lack of adequate CF animal models, drug efficacy studies in air-liquid-interface-differentiated bronchial epithelial cells derived from CF lung explant materials have been used for drug development^13,14,15. However, the limited availability of lung explant tissues and the invasive procedures to obtain bronchial cells from subjects without end-stage disease hamper the analysis of less common CFTR mutations and prevent drug testing in a personalized fashion. To overcome these limitations, "easy access" tissues, such as colorectal organoids, nasal airway cells, and airway cells derived from induced pluripotent stem cells, are currently being explored for personalized drug treatments.

Previously, we established protocols to culture epithelial stem cells from any gastrointestinal organ in the form of 3D organoids^16,17. For the human colon/rectum, the culture conditions involve defined growth factors (Epithelial Growth Factor (EGF), Gastrin, Wnt-3A, R-spondin 3 (Rspo3), and Noggin) combined with small molecules (Nicotinamide, A83-01, and SB202190) in a basement membrane matrix. Under these conditions, single stem cells or small tissue fragments grow out into closed, cystic, 3D structures formed by highly-polarized epithelium with its basal side oriented towards the outside. All cell types typically appear in their normal ratios and positions. Organoids can be expanded over long time periods by weekly mechanical disruption and re-plating. They are genetically and phenotypically stable and can be stored, allowing long-term expansion and bio-banking¹⁷. They are amenable to all standard cell-biological/genetic manipulations and analytical techniques developed for 2D cell lines¹⁸.

We recently demonstrated that CFTR function can be readily measured in colorectal organoids in a forskolin-induced swelling (FIS) assay^19,20. When exposed to forskolin (Fsk) or, alternatively, to cholera toxin, organoids rapidly increase their cyclic adenosine monophosphate (cAMP) levels, which in turn results in the opening of the CFTR channel¹⁹. Organoids from healthy individuals, or from subjects with CFTR mutations associated with residual function, will subsequently swell as a consequence of ion and water transport to the organoid lumen, the in vitro equivalent of secretory diarrhea. The FIS response of colorectal organoids was previously shown to be fully CFTR-dependent, as indicated by organoids derived from CFTR-null individuals and by the use of specific pharmacological CFTR inhibitors¹⁹. Large subject-specific data sets can be derived within several weeks after taking a biopsy.

For the FIS assay described in detail here, organoids are cultured from rectal biopsies that can be obtained at any age and with only limited discomfort²¹. Organoids are passaged weekly by mechanical disruption into single crypts that easily reseal and form new organoids. For running the FIS assay, ~30-80 of these disrupted little organoids are plated in each well of a 96-well plate¹⁹. At the day of the assay, the organoids are stained with calcein green, a fluorescent cell-permeable dye that it is retained within living cells, facilitating live imaging. Then, Fsk, which raises intracellular cAMP and thereby activates CFTR, is added in order to stimulate organoid swelling. Potentiators that act upon apical CFTR are added simultaneously with the forskolin, whereas correctors that restore CFTR trafficking are added 24 hr before the addition of Fsk. The organoid swelling is quantified by an automated image analysis that calculates the relative increase in the total area of all fluorescent objects for each time point upon forskolin addition.

3D organoid swelling provides advantages and disadvantages over existing electrophysiological CFTR readouts in 2D cultured airway cells in Ussing chambers. A major advantage is the throughput of the swelling assay. Cells are cultured and assayed using a single type of culture medium, and an experienced technician can culture up to 25 organoid samples on a weekly basis while quantifying approximately 1,200 data points per week in 12 patient samples. We conventionally type a single experimental condition by duplicate or triplicate measurements per plate and repeat such measurements at three independent incubation time points. In total, approximately 300-500 single organoid structures are then measured per experimental condition, which leads to very precise measurements of CFTR function with limited technical variability. This precision allows us to clearly define differences in residual function and response to CFTR modulators and allows us to readily pick up genetic background effects between patients carrying identical CFTR mutations^{19,22,23,24,25}. Data quality can be easily assessed from microscope images. While FIS is fully CFTR-dependent, it is an indirect outcome measure for CFTR function, its read-out caused by the coupling of ion transport to fluid transport. This contrasts with direct CFTR function measurements in Ussing chambers, which measure transepithelial ion currents²⁶. Ussing chambers allow the select stimulation of apical or basolateral compartments (which the organoid assay does not allow); by permeabilizing basolateral membranes, the apical CFTR-dependent anion secretion can be selectively measured²⁷.

Protocol

All experimentation using human tissues described herein was approved by the ethical committee at University Medical Center Utrecht (UMCU; TcBio #14-008). Informed consent for tissue collection, generation, storage, and use of the organoids was obtained from the patients at Wilhelmina Children's Hospital (WKZ)-UMCU.

Equipment	Consumable	Tools
Laminar flow hood	15 and 50 ml conical tubes	Zeiss LSM 800 – Zen 2 (Blue edition) software for measuring images
CO2 incubator	Microfuge tubes	Microsoft Excel program
Cell culture Microscope (light/optical microscope)	0.22 µm filters
Centrifuge	Serological pipettes
Rolling Shaker	Micropipette filter tips
4 °C room or 4 °C fridge for incubator	Cryovials
CoolCell Cell Freezing Container
Serological Pipettor
Micropippette (1,000, 200 and 20 µl)
Viaflo Repeat pipette
(Live cell) Confocal Microscope
Computer
Liquid nitrogen tank
Multichannel (200 µl)

1. Preparing Reagents for Culture

NOTE: All steps are performed inside a biosafety cabinet and following standard guidelines for working with cell cultures. In order to facilitate the formation of nice drops of basement membrane matrix, maintain a pre-warmed stock of 96-, 24-, and 6-well plates in the incubator at 37 °C.

1. Basal Medium Preparation
   NOTE: Basal medium (BM) refers to Advanced Dulbecco's Modified Eagle Medium with Ham's Nutrient Mixture F-12 (Ad-DF) supplemented with 4-(2-hydroxyethil)-1piperazineethanesulfonic acid (HEPES), glutamine, and penicillin/streptomycin (Pen/Strep).
   1. In a 500 ml Ad-DF medium bottle, add 5 ml of 200 mM glutamine, 5 ml of 1 M HEPES, and 5 ml of pen/strep solutions (10,000 U/ml, 10,000 µg/ml).
   2. Store BM in the refrigerator at 4 °C for at least 4 weeks.
2. Wnt-3A-conditioned Medium Preparation
   NOTE: Wnt-3A-conditioned medium is made in-house using the cell line L-Wnt-3A according to the manufacturer's instructions.
   1. For harvesting the Wnt-3A-conditioned medium, collect the medium exposed to the cells for one week and spin it down for 5 min at 450 x g to remove floating cells.
   2. Filter the Wnt-3A-conditioned medium through a 0.22 µm filter and divide it into 40 ml aliquots in 50 ml conical tubes. Store them at 4 °C for at least 4 months without a loss in activity.
   3. Test the activity of the Wnt-3A-conditioned medium in a TOP/FOP assay using human embryonic kidney (HEK)-293 cells transfected with TOP- and FOP-luciferase and TK-Renilla and measured with a Renilla-luciferase assay kit according to the manufacturer's instructions.
      NOTE: TOP-luciferase is a reporter plasmid that contains wild-type TCF binding regions. If the Wnt-3A-conditioned medium is active, the canonical Wnt signaling is activated. Beta-catenin translocates into the nucleus to associate with TCF/LEF transcription factors and activates transcription of the luciferase reporter, inducing an increase in relative luciferase activity when the substrate is added. The FOP-luciferase is used as a negative control because the TCF binding regions upstream of the luciferase gene are mutated.
3. Colon Medium Preparation
   NOTE: Prepare and dilute all growth factors and reagents according to the manufacturers' recommendations. Use small-size aliquots an avoid freeze-thaw cycles. Functional growth factors are essential for the results.
   1. Prepare colon medium (CM) by supplementing BM with 1x B27, 1.25 mM N-Acetylcysteine, 50 ng/ml hEGF, 5 nM gastrin, 10 mM nicotinamide, 300 ng/ml hRspo3, 100 ng/ml hNoggin, 500 µM A83-01, 10 µM SB202190, and 100 µg/ml of an antimicrobial for primary cells.
   2. Divide the CM into aliquots and freeze them at -20 °C for up to 4 months. Thaw the aliquots to prepare full colon medium (FCM) by adding 50% Wnt-3A-conditoned medium to the CM. Store FCM up to 7 days at 4 °C without a loss in activity.
   3. For the establishment of an organoid culture from rectal biopsies, supplement FCM with 50 µg/ml vancomycin, 50 µg/ml gentamycin, and 10 µM rho-associated coiled-coil-forming protein serine/threonine kinase inhibitor (RhoKi). Use this medium, called isolation medium (IM), only for the first two to three days in culture.
4. EDTA Stock Solution Preparation
   1. Prepare 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8, in ultrapure H₂O, sterilized with a 0.22 µm filter.
5. Manipulation of the Basement Membrane Matrix
   1. Prepare the basement membrane matrix (BMM) according to the manufacturer's recommendation.
   2. Thaw BMM overnight on ice.
   3. When transferring the BMM from the bottle to a 15 ml conical tube, use a 5 ml pipette and a 15 ml conical tube pre-cooled at -20 °C.
   4. Once thawed, store the BMM in a refrigerator at 4 °C and incubate on ice for at least 30-60 min before use.
      NOTE: In order to obtain the best results, the BMM must be cold and properly mixed before embedding crypts or organoids.

2. Establishing Colon Organoids from a CF Patient Biopsy

NOTE: After tissue collection, it is important to maintain the sample on ice in saline, Dulbecco's Phosphate-Buffered Saline without Ca²⁺ and Mg²⁺ (DPBS), or BM. Rapid processing of the biopsies is recommended, but it has proven possible to establish organoid cultures from biopsies stored on ice for up to 7 days.

1. Let the biopsies settle at the bottom of a 15 ml conical tube and remove the supernatant.
2. Add 10 ml of DPBS to the biopsies and pipette up and down 10-20 times using a 10 ml pipette.
   NOTE: The pipette needs to be pre-wetted with BM before pipetting the biopsy.
3. Let the biopsies settle at the bottom and remove the supernatant.
4. Repeat steps 2.2 and 2.3 4-5 times until the supernatant is clear.
5. Add 10 ml of DBPS and 200 µl EDTA (0.5 M) to the biopsies and place the tube on a rocking tube platform for 60-120 min at 4 °C.
   NOTE: Incubation time may differ per patient. If crypts are released, DPBS becomes cloudy. EDTA incubation can be finalized when crypts can be observed under a microscope.
6. Allow the crypts to settle. Discard the supernatant.
7. Take up 2 ml of BM and add it to a new 15 ml conical tube. Shake this new tube manually in such a way that the inside is covered with BM.
8. Using a pipette pre-wetted with BM, add 2 ml of DPBS to the tube containing the biopsies and pipette up and down 10-20 times.
9. Allow the biopsies to settle and transfer the DPBS with the crypts to the new tube containing the 2 ml of BM that was prepared in step 2.7.
10. Repeat steps 2.8 and 2.9 until no more crypts are released.
11. Spin the crypts down at 130 x g for 5 min at 8 °C.
12. In the meantime, transfer IM to the safety cabinet at room temperature (RT).
13. Discard the supernatant and add 10 ml of BM to the crypt pellet and repeat step 2.11.
14. Resuspend the pellet in 1 ml of BM. Take 5-10 µl and count the number of crypts by eye under a microscope.
15. Spin the crypts down at 130 x g for 5 min at 8 °C.
16. Remove the supernatant and resuspend the pellet in 55% BMM (v BMM to v IM).
    NOTE: The crypts are resuspended in the corresponding volume of 55% BMM at 1 crypt per µl. If there are not enough crypts, the minimum volume of BMM is 40 µl.
17. For seeding, pipette 35 µl per well (in a 24-well plate). Divide the 35 µl of BMM into 3-5 drops separately in order to improve the diffusion of growth factors into the BMM. Do not create bubbles.
18. Place and leave the plate upside down in the incubator at 37 °C for at least 20 min for the BMM to solidify.
19. Add 500 µl of IM per well and keep it in the incubator at 37 °C and 5% CO₂.
20. Refresh the medium every 2-3 days. Remove the old medium by pipetting out with a P1000 pipette, leaving the BMM drops intact. Carefully add FCM by pipetting it in the side of the well, not directly on the BMM drops.
    NOTE: If no crypts are released in the isolation protocol, centrifuge the supernatant, wash the pellet 2-3 times with BM, and resuspend it in BMM. Take the leftover tissue and cut it into little pieces with a razor. Collect these into a 15 ml conical tube, centrifuge them, and resuspend them in the same BMM. If any epithelial stem cells are present, these will also generate organoids.

3. Passaging of Colon Organoids for Maintenance, Freezing, and Forskolin-induced Swelling Assay (FIS)

NOTE: Every organoid culture has its own doubling time. Normally, colorectal CF organoids can be expanded 1:3-1:5 times every 7-10 days. It is a good sign if budding structures are observed. Colorectal CF organoids are less cystic (Figure 2A-2C) than colorectal normal organoids (Figure 2D). For establishment and maintenance, organoids are cultured in 24-well plates; for freezing, in 6-well plates; and for the FIS assay, in 96-well pates.

1. Passaging of Organoids
   1. Keep the BMM on ice for at least 30-60 min before using it.
   2. Keep the FCM at RT for at least 1 hr before using it.
   3. Label one 15 ml conical tube with the sample name and another tube as "washed."
   4. Carefully aspirate the medium from the wells using a P1000 pipette without disturbing the BMM drops.
   5. Add 1 ml of BM to 1 well and break up the BMM drops by using a P1000 pipette. Transfer this 1 ml into the 15 ml conical tube with the sample name.
   6. Wash the well with another 1 ml of BM and transfer it to the same 15 ml tube.
   7. Repeat steps 3.1.5 and 3.1.6 with 5 more wells (up to 6 wells of a 24-well plate will be washed in one 15 ml conical tube).
   8. Fill the tube up to 12 ml with BM. Pipette up and down using a pre-wetted 5 ml pipette.
   9. Spin at 85 x g for 5 min at 8 °C.
   10. Discard the supernatant and add 1 ml of BM to the pellet. With the same P1000 pipette tip, take up a P10 tip without a filter, and pipette up and down 20 times. Discard both P1000 and P10 tips.
   11. Add 4 ml of BM with a 5 ml pipette and hold the tube tilted at about 70° from the vertical to one side. Pre-wet a new P1000 tip and mix vigorously 2-3 times with the P1000 (1 ml volume).
   12. Count to 10 while remaining in the tilted position, collect the uppermost layer carefully with the P1000 pipette, and transfer 4 x 1 ml into the tube labelled as "washed."
   13. Observe the organoids settling to the bottom in the tilted tube; undisrupted organoids and bigger chunks will sink to the bottom. Centrifuge the 15 ml "washed" tube for 5 min at 85 x g and 8 °C.
   14. Discard the supernatant and add the required amount of medium and BMM to the organoid pellet to a final concentration of 55% BMM. Mix by pipetting up and down without creating bubbles (Figure 3B).
       NOTE: A good density is achieved by seeding 25-30 organoids in 10 µl of BMM.
   15. Follow steps 2.17-2.19.
2. Passaging for Freezing
   1. Keep the BM in ice for at least 30-60 min before use. Keep the FCM at RT for at least 1 hr before use.
   2. Prepare FCM supplemented with RhoKi (Step 1.3.3).
   3. Take trypsin from the refrigerator and leave it at RT for at least 30 min before use.
   4. Follow steps 3.1.5-3.1.10.
   5. Remove as much supernatant as possible, add 4 ml of trypsin, and vortex for 30 sec.
   6. Put the tube in a warm water bath at 37 °C for 1 min and vortex vigorously for 30 sec.
   7. Inspect the solution in the tube. If intact organoids are still visible under the microscope, repeat step 3.2.7.
   8. When the organoids are disrupted, add 8 ml of BM to neutralize the trypsin and pipette 10 times.
   9. Spin for 3 min at 450 x g at 8 °C.
   10. Discard the supernatant and add the required amount of medium and BMM to the organoids to reach a 55% BMM solution. Mix by pipetting up and down without creating bubbles.
       NOTE: For freezing, organoids must be seeded in a 1:1 ratio after trypsinization.
   11. Seed 250 µl in a single well of a pre-warmed 6-well tissue culture plate, producing tiny, separated droplets of ~10 µl. Place the plate upside down in the incubator at 37 °C and leave the BMM to solidify for 20-30 min.
   12. Add 2.5 ml of fresh FCM + RhoKi in each 6-well plate well and transfer the plate to the incubator (Figure 3C).
3. Passaging Organoids for the FIS Assay
   NOTE: Depending on the number and size of the organoids, between 4 and 6 wells of a 24-well plate are enough for seeding 27 wells of a 96-well plate.
   1. Process the organoids as described from steps 3.1-3.1.13.
   2. Resuspend the pellet in 120 µl of 50% BMM.
   3. Confirm the number of organoids (30-50) in a 3 µl BMM drop under the microscope.
   4. Plate the organoids using a single drop of 3 µl placed in the middle of each well of a 96-well pate.
   5. Place the plate in the 37 °C incubator for 15 min for the BMM to solidify; further steps are described in step 6.1.1.

4. Freezing Colon CF Organoids

NOTE: Organoids are ready to be frozen down 1-2 days after trypsinization and culturing, so organoids will still be small, which increases the efficiency of survival after thawing.

1. Add 1 ml of BM and break up the BMM drops using a P1000 pipette. Transfer to a 15 ml conical tube.
2. Wash the well with another 1 ml of BM and transfer to the same 15 ml tube.
3. Repeat steps 4.1 and 4.2 with all wells.
   NOTE: To avoid excess BMM, 3 wells of a 6-well plate are pooled in one 15 ml tube.
4. Fill up the 15 ml tube containing the organoids with 12 ml of cold BM and pipette up and down with a 5 ml pipette. Leave the tube on ice for 5 min.
5. Spin for 3 min at 450 x g and 8 °C and remove the supernatant.
6. Dissolve the pellet of organoids with freezing medium and pipette up and down to properly resuspend the organoids.
   NOTE: 500 µl of freezing medium is used for each 100 µl of BMM.
7. Using a 5 ml pipet, transfer 0.5 ml of organoids resuspended in freezing medium to sterile cryogenic vials.
8. Transfer the vials to a cell freezing container and put it at -80 °C.
9. After 24 hr, transfer the vials for storage in liquid nitrogen.

5. Establishing Cultures from Frozen Organoids

NOTE: Thaw the BMM on ice and keep it on ice. Let the BM reach RT, and warm a 10 ml aliquot to 37 °C before starting the procedure of thawing one cryovial.

1. Thaw the vial rapidly by agitation in a 37 °C water bath until there is still a little frozen material.
2. Transfer the thawed organoids to a 15 ml conical tube using a P1000 pipette.
3. Immediately after, add 1 ml of warm BM drop by drop while shaking the bottom of the tube. Once the 1 ml of medium is added, mix carefully by pipetting up and down a few times to dilute the freezing medium.
4. Slowly (drop by drop) add 9 ml of warm BM to the conical tube containing the organoids. Invert a few times.
5. Spin the cell suspension for 3 min at 85 x g and 8 °C.
6. Discard the supernatant carefully without disturbing the pellet. Resuspend the organoid in 90 µl of FCM supplemented with RhoKi (step 1.3.3). Then, add 110 µl of BMM.
7. Add 35 µl to each well of a pre-warmed 24-well plate, making tiny, separated droplets.
8. Transfer the plate to the 37 °C incubator, leaving the plate upside down for 20-30 min.
9. Add 500 µl of FCM with RhoKi to each well and transfer the plate back to the incubator (Figure 4A-4C).
10. Once organoids have recovered properly from thawing, dilute in more BMM so each 10 µl of BMM contains 25-30 organoids. This procedure can be done 2-3 days after thawing (Figure 4D-4G) and ensures the proper growth of the organoid.

6. Forskolin-induced Swelling Assay (FIS)

NOTE: Fsk titration allows for the measurement of CFTR residual function. For CFTR modulation, organoids are exposed to a given corrector (e.g., VX-809) and/or potentiator (e.g., VX-770), depending on genotype. Usually, VX-809 is added 18-24 h before the measurement, while VX-770 and Fsk are added right before starting the measurements. Be aware that some modulators (correctors/potentiators) can bind to the plastic surface of the assay plate.

1. Plating for the Assay
   NOTE: VX-809 stocks are aliquoted at 20 mM and stored at -80 °C. When thawing, leave at RT and protect from light.
   1. Process the organoids as described in section 3.3.
   2. If testing the VX-809 corrector, prepare a dose-response curve in triplicates with the following concentrations: 0.0003, 0.003, 0.03, 0.3, 3.0, and 30 µM. Prepare the dilutions in FCM.
   3. To the 96-well plate, add either 100 µl of FCM with the respective VX-809 concentration or 100 µl of FCM only and incubate the plate.
2. Measuring the Assay
   NOTE: VX-770 stocks are aliquoted at 20 mM, while Fsk is at 10 mM; both are stored at -80 °C. When thawing, leave at room temperature and protect from light.
   1. Take a vial of calcein and DMSO and leave at RT for 15 min.
   2. Add 5.1 µl of DMSO to the vial where the calcein is provided as a powder, if unopened. Otherwise, use an already resuspended calcein vial containing 2.5 µl of calcein.
   3. Add 2.5 µl of calcein to 580 µl of BM in a 1.5 ml tube and label it.
   4. Add 10 µl of this dye solution (BM + calcein) to each well using a repeat pipette.
   5. Resuspend once with a multichannel to ensure that the dye is mixed well.
   6. Incubate the plate in a 37 °C incubator for 30 min.
   7. During the calcein incubation, start preparing the Fsk and VX-770 solutions, if needed.
   8. If using a VX-770 potentiator, prepare a dose-response curve in triplicate with the following concentrations: 0.0003, 0.003, 0.03, 0.3, 3.0, and 30 µM. In case of an Fsk titration, the concentrations are: 0, 0.008, 0.02, 0.05, 0.12, 0.32, 0.8, 2.0, and 5.0 µM. Prepare the dilutions in BM.
      NOTE: For Fsk, VX-770, or any other drug added on the second day, the dilutions must be prepared at 2x final concentration, since 100 µl of Fsk/drug titration solution will be added to each well, which already contain 100 µl of FCM.
   9. Transfer the Fsk and VX-770 solutions, a p200 pipette, and tips to the microscope room.
   10. For imaging the FIS assay, use a live cell imaging confocal microscope equipped with an automated stage, a heated chamber, and CO₂ flow.
       NOTE: The following steps refer to a specific microscope. Different setups should apply to other microscopes following manufacturer's instructions.
   11. Put the 96-well plate in the plate holder.
   12. Enter the confocal settings for imaging.
       1. Preset the live imaging tool to 37 °C and 5% CO₂ and let the chamber pre-incubate for a minimum of 30 min prior to measurement.
       2. Use the "Smart setup" option to select the Alexa fluor-488 track.
       3. Set the 5X lens and scan area to 0.6X to zoom out and capture the entire well.
       4. Adapt the laser power and detector sensitivity to enable the optimal detection of calcein-green labeled organoids over the background. The pinhole can be increased to 130 µm and the image averaging can be set at 2.
       5. Set the bit depth at 8 and the frame size to 512 x 512.
       6. Use the time series option to set the measurement time, frequency, and intervals.
          NOTE: Suggested for regular measurements: 1 hr with 10 min interval: cycles = 7 (Cycle 1 is T = 0). For measurements of reduced swelling, organoids can be imaged for up to 3 hr.
       7. Use the tile option to manually determine the individual well positions.
   13. Add 100 µl of the Fsk and/or VX-770 solutions to the corresponding wells, following the same order as the measurement. Start adding the solutions in the first imaged well and continue with the imaging order.
   14. Start the measurement.
   15. After the experiment is finished, save the file.
3. Analysis of FIS Assay Data
   1. Create an "organoid analysis" macro for the data analysis of the FIS assay using the analysis tool in an image analysis software that recognizes all structures imaged through the Alexa-488 track and fills the identified structures to calculate the increase of total organoid area over the different time points.
   2. Open the data file with the acquired images in the image analysis program.
   3. Select the macro for organoid analysis.
   4. Set the threshold to balance the signal-to-noise ratio in one well at time point 1 and ensure that all organoid structures are recognized and filled.
      1. Check in 4-6 wells to see if all structures are also recognized at time point 7. Slightly modify the threshold to ensure that the background signals at time point 7 are not recognized as structures (the specific threshold may change somewhat between experiments).
   5. Set the criteria of minimal area size of recognized objects to 1,000 µm².
   6. Press "analyze" to start. The analysis takes approximately 3 min, depending on the software used.
   7. When the software is finished, go to "create table" and select the following data: well numbers (table should increase in this order), time points, and total area per well.
   8. Save file as an .xlm to export to a spreadsheet program.
   9. In this program, calculate the relative increase in area per well by setting the area of time point 1 at 100%. Calculate the area under the curve (AUC) from time points 1-7 per condition; the lower limit of the area (Y-value) is set at 100%. Fsk or drug titrations (X-axis) are plotted against the AUC (Y-axis).

Results

Figure 1A shows a representative fresh isolation of crypts embedded on BMM. The crypts are from a colorectal biopsy of a CF subject. Usually, an organoid is generated from each crypt (Figure 1A-C). Due to the dysfunction of the CFTR, most of the colon CF organoids are not cystic, but rather are compact and with projections and buddings (Figure 2A-2B). However, some CF organoid cultures, especially with hi...

Discussion

Here, we provide a complete protocol for the generation, expansion, freezing, and thawing of human colorectal organoids. While we have established human organoid cultures some time ago¹⁷, it has sometimes proven difficult to establish the technology in other labs without hands-on training. We anticipate that these protocols will replace such training.

Wnt-3A-conditioned medium is one of the most crucial reagents in order to succeed in establishing and maintaining a long...

Materials

Name	Company	Catalog Number	Comments
Advanced Dulbecco’s Modified Eagles Medium with Nutrient Mixture F-12 Hams (Ad-DF) 500 ml	Thermo Fisher Scientific:  Invitrogen	#12634	stored at 4 °C
GlutaMax	Thermo Fisher Scientific:  Invitrogen	#35050	stored at 4 °C
Hepes	Thermo Fisher Scientific:  Invitrogen	# 15630-056	stored at 4 °C
Penicillin/Streptomycin	Thermo Fisher Scientific:  Invitrogen	#15140-122	stored at -20 °C
96 well culture plate	Cellstar	#655180
24 well culture plate	Cellstar	#662160
6 well culture plate	Cellstar	#657160
Dulbecco's Phosphate Buffered Saline (-) CaCl2 (-) MgCl2) (DPBS)	Life Technologies: Gibco	#14190-094	stored at 4 °C
Dulbecco’s Modified Eagles Medium  (DMEM) 500 ml	Thermo Fisher Scientific:  Invitrogen	#31966-021	For Wnt-3A Conditioned Medium Production. Stored at 4 °C
Fetal Bovine Serum (FBS)	Bovogen	#SFBS LOT#11113	For Wnt-3A Conditioned Medium Production. Stored at -20 °C
L Wnt3A cell line	ATCC	#CRL-2647	For Wnt-3A Conditioend Medium Production.
TOP/FOP plasmids	Millipore	#17-285	For measuring Wnt activity
pTK-Renilla	Promega	#E2241	For measuring Wnt activity
HEK-293	ATCC	#CRL-1573	For measuring Wnt activity
Dual-Luciferase Reporter Assay System	Promega	#E1910	For measuring Wnt activity
Zeocin	Thermo Fisher Scientific:  Invitrogen	#R250-01	For Wnt-3A Cell line selection
B27 supplement	Thermo Fisher Scientific:  Invitrogen	#17504-044	stored at -20 °C
N-Acetylcysteine	Sigma Aldrich	#A9165-5G	stored at -20 °C
Nicotinamide	Sigma Aldrich	#N0636	stored at -20 °C
Human Epithelial Growth Factor (hEGF)	PrepoTech	#AF-100-15	stored at -20 °C
Gastrin	Sigma Aldrich	#G9145	stored at -20 °C
TGFb type I Receptor inhibitor (A83-01)	Tocris	#2939	stored at -20 °C
Y-27632 dihydrochloride (RhoKi)	Selleckchem	#S1049	stored at -20 °C
p38 MAPK inhibitor (p38i) (SB202190)	Sigma Aldrich	#S7067	stored at -20 °C
Primocin	InvivoGen	#ant-pm-1	stored at -20 °C
Human Noggin (hNoggin)	PrepoTech	#120-10C	stored at -20 °C
Human R-spondin 3 (hRspo-3)	R&D Systems	#3500-RS/CF	stored at -20 °C
Vancomycin	Sigma Aldrich	#861987- 250mg	stored at -20 °C
Gentamycin	Life Technologies: Gibco	#15710-049	stored at -20 °C
Ethylenediamine tetraacetic acid (EDTA)	Sigma Aldrich	#431788	Stored at 4 °C
Matrigel	Corning	#354230	stored at -80 °C
TryplE Express	Life Technologies: Gibco	#12605-010	for trypsinizing organoids for freezing
Recovery Cell Culture Freezing Medium	Life Technologies: Gibco	#12648010	for freezing
Calcein	Life Technologies: Gibco	#C3100MP	stored at -20 °C
Forskolin	R&D Systems	#1099-50 mg	stored at -80 °C
Lumacaftor (VX-809)	Selleckchem	#s1565	stored at -80 °C
Ivacaftor (VX-770)	Selleckchem	#s1144	stored at -80 °C
Name of Reagents/Material	Solvent	Stock Concentration	Final Concentration
GlutaMax		200 mM	2 mM
Hepes		1 M	10 mM
Penicillin/Streptomycin		10K U/ml 10K µg/ml	100 U/ml 100 µg/ml
Zeocin		100 mg/ml	125 µg/ml
B27 supplement		100x	1x
N-Acetylcysteine	MiliQ H2O	500 mM
Nicotinamide	DPBS	1 M	10 mM
Human Epithelial Growth Factor (hEGF)	DPBS 0.1% BSA	0.5 mg/ml	50 ng/ml
Gastrin	DPBS	100 µM	10 nM
TGFb type I Receptor inhibitor (A83-01)	DMSO	5 mM	500 nM
Y-27632 dihydrochloride (RhoKi)	DMSO	10 mM	10 µM
p38 MAPK inhibitor (p38i) (SB202190)	DMSO	30 mM	10 µM
Primocin		50 mg/ml	100 µg/ml
Human Noggin (hNoggin)	DPBS 0.1%BSA	100 µg/ml	100 ng/ml
Human R-spondin 3 (hRspo-3)		varies per lot	300 ng/ml
Vancomycin		10 mg/ml	50 µg/ml
Gentamycin		10 mg/ml	50 µg/ml
Ethylenediamine tetraacetic acid (EDTA)	MiliQ H2O	0.5 M	2 mM
Calcein	DMSO	10 µg/ml	3.3 ng/ml
Forskolin	DMSO	10 mM	variable
Lumacaftor (VX-809)	DMSO	20 mM	variable
Ivacaftor (VX-770)	DMSO	20 mM	variable