Forskolin-Induced Swelling of Airway Organoids Derived from Healthy and Cystic Fibrosis Donors
Cystic fibrosis (CF) is a genetic disease caused by defects in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride anion channel expressed in multiple epithelial tissues. Defects in CFTR cause aberrant ion transport and mucus build-up in multiple organs, leading to difficulty breathing, recurring lung infections, and digestive issues. Recently, organoids have emerged as an excellent tool for studying CF, and hold great promise for drug discovery and efficacy testing due to their higher-throughput capabilities. Additionally, they have been shown to possess more physiologically relevant differentiated cell types compared to commonly used transformed cell lines, and have a similar cellular composition to that seen in mucociliary airway epithelium cultured at the air-liquid interface (ALI).
Forskolin is an activator of adenylyl cyclase that increases intracellular cyclic adenosine monophosphate (cAMP). When organoids in culture are exposed to forskolin, a signaling cascade will result in CFTR channel activation on the apical surface of the airway epithelium, allowing the efflux of chloride ions into the organoid lumen. As a result, water will follow into the lumen by osmosis, causing the organoid lumen to swell (Figure 1). Using intestinal organoids derived from colorectal biopsies, Dekkers and colleagues demonstrated that forskolin-induced swelling (FIS) of organoids is fully CFTR-dependent.1 In addition to forskolin, this method includes the use of amiloride, an inhibitor of ENaC, and genistein, a CFTR potentiator. These are used in tandem with forskolin to ensure the maximal degree of chloride efflux into the organoid lumen, and a minimal degree of sodium influx out of the organoid lumen.
Figure 1. Forskolin-Induced Swelling of Airway Organoids
Bright-field images of mature airway organoids derived in the PneumaCult™ Airway Organoid Kit display organoid swelling after 6-hour incubation in Forskolin-induced swelling conditions.
Since FIS assays can be used as a measure of CFTR function, 3D organotypic cultures provide a major advantage of throughput compared to existing electrophysiology assays using ALI cultures. Within several weeks, large data sets of CFTR function and response to selected drugs can be generated and assessed through the level of swelling using high-content microscopy imaging.2 Although the FIS assay is CFTR-dependent, it is important to note that the relationship between CFTR ion channel function is determined by an indirect outcome of water transport across the epithelium allowing the organoids to swell. In contrast, traditional Ussing chambers measure the transepithelial ion currents and represent a direct functional evaluation of CFTR channels, making them complementary after the pre-screen of CF drugs in high-throughput compatible FIS assays.3
The introduction of Highly Effective Modulator Therapies (HEMT) using Trikafta (VX-445, VX-661, and VX-770) has resulted in the improved rescue of the most common CFTR mutation. However, there remains a need to study rare mutations, which are currently excluded from CFTR modulator therapies, and to better predict the individual patient responses to CFTR drugs.
Below we describe a protocol for forskolin-induced swelling of Matrigel® embedded healthy and CF patient-derived airway organoids generated using PneumaCult™ Airway Organoid Kit, and treatment with VX-809. For complete culturing instructions, use this document in conjunction with the relevant Product Information Sheet.
Materials
- PneumaCult™ Airway Organoid Kit (Catalog #05060)
- NaCl
- KH₂PO₄
- NaHCO₃
- MgCl₂
- CaCl₂
- D-glucose
- Krebs-Ringer Solution, Bicarbonate-Buffered (Alfa Aesar Catalog #J67591)
Preparation of KBR Buffer and Glucose Solutions
- Prepare a 50X stock solution of NaCl by combining the following:
- 33.6 g NaCl in 100 mL dH₂O
- Prepare a 100X stock solution of KH₂PO₄ by combining the following:
- 3.81 g KH₂PO₄ in 100 mL dH₂O
- Prepare a 40X stock solution of NaHCO₃ by combining the following:
- 8.40 g NaHCO₃ in 100 mL dH₂O
- Prepare the KBR Buffer solution by combining the following:
- 900 mL dH₂O, 20 mL of the 50X NaCl stock solution
- 10 mL of the 100X KH₂PO₄ stock solution
- 25 mL of the 40X NaHCO₃ stock solution
- Top up the mixture to 1000 mL with dH₂O
- While stirring with a magnetic stir bar, adjust the pH of the mixture to 7.4 using 0.12N HCl.
- While stirring, insert a tube attached to a gas cylinder containing a mixture of 95% O₂, 5% CO₂ gas into the buffer to introduce a steady stream of bubbles for 15 minutes.
- While continually bubbling, slowly add 240 mg MgCl₂ to the mixture.
- While continually bubbling, very slowly add 180 mg CaCl₂ to the mixture.
Note: Adding CaCl₂ too quickly will result in precipitation. It is recommended to add small amounts of CaCl₂ at a time to the buffer.
- Continue to bubble and stir the mixture for an additional 15 minutes. Store at 4°C for 1 - 2 months, or until visible precipitation can be observed.
- Prepare a 100X stock solution of Glucose by combining the following:
- 9.0 g D-glucose in 50 mL dH₂O. Filter sterilize through a 0.22 μm pore filter (e.g. EMD Millipore Steriflip-GP Sterile Centrifuge Tube Top Filter Unit Catalog #SCGP00525) and store at 4°C for 1 - 2 months, or until visible precipitation can be observed.
Preparation of Drug Stock Solutions
- Prepare a 1000X stock solution by making a 10 mM Forskolin solution in DMSO. Store at -20°C.
- Prepare a 5000X stock solution by making a 100 mM Amiloride solution in DMSO. Store at -20°C.
- Prepare a 4000X stock solution by making a 100 mM Genistein solution in DMSO. Store at -20°C.
- Prepare a 5000X stock solution by making a 15 mM VX-809 solution in DMSO. Store at -20°C.
CFTR Corrector Pre-Treatment
- To pre-treat organoids with CFTR-corrector drugs (e.g. VX-809), treatment replicates and vehicle control replicates should be established (Figure 2).
Figure 2. Proposed Setup of 24-Well Plate for CFTR Corrector Pre-Treatment
Organoids derived from CF-diseased airway epithelial cells can be treated with CFTR-corrector drugs prior to a FIS assay to assess the efficacy of said CFTR-corrector on the rescue of CFTR function compared to vehicle control. Use the proposed setup to generate three technical replicate organoid wells per swelling condition, per treatment.
- To treat six technical replicates of 24-well plate format airway organoid cultures with CFTR-corrector drug VX-809, add 1.2 μL 5000X VX-809 stock solution to 6mL of PneumaCult™ Airway Organoid Differentiation Medium (AODM). Remove culture medium from six replicate airway organoid cultures and replace with room temperature (15 - 25°C) 1 mL 1X VX-809-AODM solution per well.
- For vehicle control replicates, prepare DMSO-AODM solution by adding 1.2 μL DMSO to 6 mL PneumaCult™ AODM. Remove culture medium from six additional technical replicate airway organoid cultures and replace with room temperature 1 mL DMSO-AODM solution per well.
- Incubate at 37°C, 5% CO₂ for 24 hours.
Forskolin-Induced Swelling Assay
- Allow the KBR buffer to come to room temperature. Prepare a 1X KBR-Glucose solution by adding 0.5 mL of 100X Glucose stock to 49.5 mL of KBR Buffer. Prepare volumes as needed for 1 mL per 24-well addition.
- Filter-sterilize the KBR-Glucose solution through a 0.22 μm pore filter.
- Combine KBR-Glucose solution with 5000X Amiloride stock, 1000X Forskolin stock, and 4000X Genistein stock to prepare a 1X KBR-Glucose-Amiloride-Forskolin-Genistein solution. For example, to 6 mL of KBR-Glucose solution, add 1.2 μL 5000X Amiloride stock, 6 μL 1000X Forskolin stock, and 1.5 μL 4000X Genistein stock.
- The final concentration of Amiloride is 20 μM
- The final concentration of Forskolin is 10 μM
- The final concentration of Genistein is 25 μM
- Combine 6 mL KBR-Glucose solution with 8.7 μL DMSO to prepare a 1X KBR-Glucose-DMSO vehicle control solution.
Note: Use media preparations with drug additions immediately, do not store for future use.
- Capture images of fully differentiated airway organoid cultures derived using PneumaCult™ Airway Organoid Kit prior to the addition of DMSO or Forskolin-swelling solutions (Time 0 h).
- Remove PneumaCult™ AODM and replace with room temperature 1 mL 1X KBR-Glucose-Amiloride-Forskolin-Genistein solution per well to perform positive Forskolin-induced swelling. For vehicle controls, remove PneumaCult™ AODM and replace it with 1 mL 1X KBR-Glucose-DMSO solution per well.
- Incubate for 6 hours at 37°C, 5% CO₂.
- Capture bright-field images at 2X magnification (or magnification of user choice) of organoid cultures after 6 hours (Time 6 h).
Note: Earlier time points can be imaged and assessed to generate additional kinetics of organoid swelling, such as rate of swelling. It is suggested to incubate swelling conditions for 6 hours to achieve maximal organoid swelling, since organoid swelling appeared to plateau after 6 hours. Organoid swelling performance has not been measured for longer than an 8-hour incubation.
- If additional characterizations are required to describe the organoid culture, such as ICC or qPCR, remove KBR-Glucose solutions from swelling conditions and vehicle controls and replace them with fresh PneumaCult™ AODM. Incubate overnight at 37°C, 5% CO₂ before continued characterization.
- The degree of organoid swelling at 6 hours can be expressed as a percentage increase relative to time 0, as described in Figure 3.
Figure 3. Degree of Swelling of Organoids at 6 Hours Following Treatment With Amiloride, Forskolin, and Genistein
Degree of swelling as based on the percent increase in organoid surface area measurements relative to Time 0. From left to right, treatment induced an 80% (n = 1) increase in P0 organoids size compared to a 92% in P3 (mean, n = 2), 99 ± 26% in P4 (mean ± SD, n = 6), and 87% ± 18% in P5 (mean ± SD, n = 3) organoids derived from commercial cryopreserved donor controls, respectively. Data point shapes represent distinct donors.
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