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Below we describe a protocol for forskolin-induced swelling of human intestinal organoids derived from healthy individuals and cultured in IntestiCult™ Organoid Growth Medium (Human) (Catalog #06010) or IntestiCult™ Plus Organoid Growth Medium (Catalog #100-1677). For complete culturing instructions, use this document in conjunction with the relevant Product Information Sheet (Document or ).

Introduction

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that leads to defective chloride ion transport across epithelial tissues. These mutations result in the accumulation of thick mucus in multiple organs, including the lungs and intestine. The CFTR channel is therefore a key target for therapeutic development and CFTR-modulator drug discovery. Human intestinal organoids provide a physiologically relevant 3D cell culture system for studying CFTR function in vitro since they can be efficiently established from intestinal biopsies and expanded and maintained in long-term in vitro culture.¹ These 3D intestinal organoids maintain the genotype and phenotype of the parental tissue and retain the expected CFTR function when cultured in vitro, making them a powerful translational model for CFTR functional assays and phenotypic screening.

Exposing intestinal organoids to forskolin causes the cells to rapidly increase their levels of cyclic adenosine monophosphate (cAMP), resulting in the opening of the CFTR channel.^1,2 As chloride ions move through the channel and into the lumen, the organoids increase in size due to water following by osmosis. This organoid swelling can be quantified as a measurement of CFTR activity. Because the swelling is dependent on functional CFTR, wild type organoids swell normally, while those carrying a CFTR mutation or exposed to a CFTR inhibitor exhibit diminished or absent swelling in the presence of forskolin.

The forskolin-induced swelling (FIS) assay serves as a functional readout of CFTR channel activity in a native epithelial context, can be used to benchmark normal CFTR function, and serves as a reference for evaluating the effects of CFTR modulators, inhibitors, or other compounds targeting epithelial ion transport. By providing a reproducible, quantitative measure of CFTR-dependent fluid secretion in human-derived tissue, this organoid-based assay offers a robust platform for drug screening, mechanism-of-action studies, and preclinical evaluation of potential therapies for cystic fibrosis and related epithelial disorders.

Protocol

1. Establish organoid cultures as described in the Product Information Sheet (Document or ). Grow organoids for 3 - 5 days after passage in IntestiCult™ Organoid Growth Medium (Human) (Catalog #06010) or for 6 - 7 days after passage in IntestiCult™ Plus Organoid Growth Medium (100-1677) prior to assessing forskolin-induced swelling.
2. Remove media from each well and replace with an equal volume of Krebs-Ringer Bicarbonate (KBR) buffer. Allow the buffer to equilibrate with the Corning^®Matrigel^® domes for 30 minutes at 37°C.
   NOTE: Forskolin-induced swelling can be performed and assayed in the culture medium; however, swelling is more pronounced if using KBR buffer.
3. OPTIONAL: If using CFTR inhibitors as an additional control (e.g. CFTRINH172 or GlyH-101), add the appropriate amount of inhibitor to each well as necessary (50 μM for CFTRINH172 or GlyH‐101) and incubate for 3 hours at 37°C. If inhibitors are not being used, proceed directly to step 4.
4. Capture time = 0 images before proceeding as organoid swelling may begin immediately after adding forskolin.
5. Remove the buffer from all wells being assayed and replace with fresh buffer, inhibitors (if applicable), and forskolin to a final concentration of 10 μM forskolin.
6. Image each well at regular intervals until the swelling in uninhibited wells ceases (usually 80 - 120 minutes).
7. To quantify forskolin-induced swelling, brightfield images at each time point are analyzed using ImageJ for average organoid area compared to the time = 0 images.