Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

Summary

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

Abstract

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Introduction

Cystic Fibrosis (CF) is a fatal, autosomal recessive disease affecting over 70,000 people worldwide¹. This life-shortening genetic disease is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR)². CFTR is a member of the adenosine triphosphate-binding cassette family, and functions as an ion channel allowing movement of chloride and bicarbonate across the apical membranes of multiple polarized epithelia including the gastrointestinal tract, sweat gland, and respiratory tree, among others^3,4. As such, dysfunctional CFTR lea....

Protocol

HNE samples were procured from subjects recruited through the Cincinnati Children's Hospital Medical Center CF Research Center. All methods described here have been approved by the Institutional Review Board (IRB) of Cincinnati Children's Hospital Medical Center. Written consent was obtained from all subjects prior to testing.

1. Prepare Expansion Media and Antibiotic Media

1. Gather media components listed in Table 1. Thaw frozen materials at room temperature and make necessary stock solutions as indicated in Table 1 under "Expansion Media".
   NOTE: Use care when handling chol....

Results

HNEs should attach to the culture dish and form small islands of cells within 72 h of seeding; examples of good and poor island formation at one week are shown in Figure 1A and 1B, respectively. These islands should expand to cover the dish over the course of 15-30 days. Small or suboptimal samples may take longer, and often will not yield useful spheroids. Contamination with infectious agents is evidenced by deep yellow/cloudy media, failure.......

Discussion

This protocol describes the generation of patient-derived nasal cell spheroid cultures able to produce an individualized, specific model of CFTR function. There are several key steps in the process that should be closely attended to avoid difficulty. First is a good sample acquisition from the patient's nose. A good sample should have >50,000 cells, limited mucus/debris, and be ready for processing within 4 h (though success is also easily achievable with overnight shipping on ice). Practice acquiring samples with cur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Eppendorf Tube	USA Scientific	4036-3204
150 mL Filter Flask	Midsci	TP99150	To filter Media
15 mL Conical Tube	Midsci	TP91015
1 L Filter Flask	Midsci	TP99950	To filter Media
35 mm Glass-Bottom Dish	MatTek Corporation	P35G-0-20-C	Optional
3-Isobutyl-1-Methylxanthine (IBMX)	Fisher Scientific	AC228420010	Prepare a 100 mM stock solution of 22.0 mg in 1 mL of DMSO
50 mL Conical Tube	Midsci	TP91050
Accutase	Innovative Cell Technologies, Inc.	AT-104	Cell detachment solution
Adenine	Sigma-Aldrich	A2786-25G	See Table 1
Amphotericin B	Sigma-Aldrich	A9528-100MG	See Table 1
Bovine Brain Extract (9mg/mL)	Lonza	CC-4098	See Table 2
Ceftazidime hydrate	Sigma-Aldrich	C3809-1G	See Table 1
Cell Scrapers 20 cm	Midsci	TP99010
CFTR Inh172	Tocris Bioscience	3430	Prepare a 10 mM stock solution of 4.0 mg in 1 mL of DMSO
Cholera Toxin B (From Vibrio cholerae)	Sigma-Aldrich	C8052-.5MG	See Table 1
CYB-1	Medical Packaging Corporation	CYB-1	Cytology brush
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D5879-500ML
Dulbecco's Modified Eagle Media (DMEM)/F12 Hepes	Life Technologies	11330-057	Base Medium; See Tables 1 and 2
Epidermal Growth Factor (Recombinant Human Protein, Animal-Origin Free)	Thermo Fisher Scientific	PHG6045	See Table 1
Epinephrine	Sigma-Aldrich	E4250-1G	See Table 2
Ethanol	Fisher Scientific	2701
Ethanolamine	Sigma-Aldrich	E0135-500ML	16.6 mM solution; See Table 2
Ethylenediaminetetraacetic Acid (EDTA)	TCI America	E0084
Fetal Bovine Serum (high performance FBS)	Invitrogen	10082147	See Table 1
Forskolin	Sigma-Aldrich	F6886-50	Prepare a 10 mM stock solution of 4.1 mg in 1 mL of DMSO
Growth Factor-Reduced Matrigel	Corning, Inc.	356231	Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, LDEV-Free, 10 mL.
Hemacytometer	Hausser Scientific	1483
Human Collagen Solution, Type I (VitroCol; 3 mg/mL)	Advanced BioMatrix	5007-A	Collagen solution
HyClone (aka FetalClone II)	GE Healthcare	SH30066.03HI	See Table 2
Hydrocortisone	StemCell Technologies	07904	See Tables 1 and 2
Insulin, human recombinant, zinc solution	Life Technologies	12585014	4 mg/mL solution; see Table 2
IVF 4-Well Dish, Non-treated	NUNC (via Fisher Scientific)	12566350	4-well plate for spheroids; similar well size to a 24-well plate
MEF-CF1-IRR	Globalstem	GSC-6001G	Irradiated murine embryonic fibroblasts
Metamorph 7.7	Molecular Devices		Analysis Software; https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software for a quote
Olympus IX51 Inverted Microscope	Olympus Corporation	Discontinued	Imaging Microscope. Replacment: Olympus IX53, https://www.olympus-lifescience.com/pt/microscopes/inverted/ix53/  for a quote
Pen Strep	Life Technologies	15140122	See Table 2
Phsophoryletheanolamine	Sigma-Aldrich	P0503-5G	See Table 2
Retinoic Acid	Sigma-Aldrich	R2625-50MG	See Table 2
Rhinoprobe	Arlington Scientific, Inc.	96-0905	Nasal curette
Slidebook 5.5	3i, Intelligent Imaging Innovations	Discontinued	Imaging Software. Replacement: Slidebook 6, https://www.intelligent-imaging.com/slidebook for a quote
Sterile Phosphate Buffered Saline (PBS)	Thermo Fisher Scientific	20012050
Sterile Water	Sigma-Aldrich	W3500-6X500ML
Tissue Culture Dish 100	Techno Plastic Products	93100	Tissue culture dish for expansion
Tobramycin	Sigma-Aldrich	T4014-100MG	See Table 1
Transferrin (Human Transferrin 0.5 mL)	Lonza	CC-4205	See Table 2
Triiodothryonine	Sigma-Aldrich	T6397-1G	3,3′,5-Triiodo-L-thyronine sodium salt  [T3]; See Table 2
Trypsin from Porcine Pancreas	Sigma-Aldrich	T4799-10G
Ultroser-G	Crescent Chemical (via Fisher Scientific)	NC0393024	20 mL lypophilized powder; See Table 2
Vancomycin hydrochloride from Streptomyces orientalis	Sigma-Aldrich	V2002-5G	See Table 1
VX770	Selleck Chemicals	S1144	Prepare a 1 mM stock solution of 0.4 mg in 1 mL of DMSO
VX809	Selleck Chemicals	S1565	Purchase or prepare a 10 mM stock solution of 4.5 mg in 1 mL of DMSO
Y-27632 Dihydrochloride  ROCK inhibitor	Enzo LifeSciences	ALX-270-333-M025	See Table 1