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 ....

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 (Blu....

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

List of materials used in this article

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