Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Summary

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Abstract

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

Introduction

Measurements of Cilia Beat Frequency (CBF) and pattern have been established as diagnostic tools for respiratory diseases such as Primary Ciliary Dyskinesia (PCD)¹. In Cystic Fibrosis (CF), dysfunction of the CF Transmembrane conductance Regulator (CFTR) chloride channel causes dehydration of the airway surface liquid and impaired mucociliary clearance². Ciliary function has been investigated in vitro in primary airway cell models as an indicator of CFTR channel activity³. However, considerable patient-to-patient variability exists in CBF in response to CFTR-modulating drugs, even for pat....

Protocol

Study approval was received from the Sydney Children's Hospital Network Ethics Review Board (HREC/16/SCHN/120). Written consent was obtained from all participants (or participants' guardian) prior to the collection of biospecimens.

1. Preparations for establishing airway epithelial cell models

1. Prepare nasal cell collection media by combining 80% Dulbecco's Modified Eagle Medium and 20% Fetal Bovine Serum. Supplement with 1 µL/mL of Penicillin/Streptomycin. Store at 4 °C for up to 3 months.
2. Coat the flasks or permeable support inserts with Collagen solution on a per-need basis foll....

Results

To demonstrate the efficiency of this protocol in quantifying CBF, the results of CBF measured in airway epithelial cell ALI models derived from three participants with CF and three healthy control participants are presented. On Day 14 of culture differentiation, beating cilia were present (Figure 6). From Day 14 to 21 of culture differentiation, a statistically significant (P < 0.0345) increase in CBF was observed within both cohorts. On Day 21 of culture differentiation, the mean CBF f.......

Discussion

There are multiple factors that could obscure the quantification of CBF in nasal epithelial sheets. Epithelial sheets should be imaged within 3-9 hrs of sample collection since the ciliary function is most stable during this time³⁷. Less red blood cells and debris are most optimal for imaging since these interfere with data acquisition. When selecting an ROI for imaging, it is important to select an epithelial sheet that edge has not been damaged or disrupted during the collection of the sample, a.......

Materials

Name	Company	Catalog Number	Comments
Adenine	Sigma-Aldrich	A2786	10 mg/mL
Advanced DMEM/F-12	Thermo Fisher Scientific	12634-010
Alanyl-glutamine	Sigma-Aldrich	G8541	200 mM
Andor Zyla 4.2 sCMOS	Oxford Instruments		Fast frame rate (>100 Hz) scientific camera
Bottle-top vacuum filter system	Sigma-Aldrich	CLS431098
Ceftazidime hydrate	Sigma-Aldrich	A6987	50 mg/mL
Cell Culture Microscope	Olympus	CKX53
CFI S Plan Fluor ELWD 20XC	Nikon Instruments Inc.	MRH08230	Long working distance objective lens. NA0.45 WD 8.2-6.9
Cholera toxin	Sigma-Aldrich	C8052-1MG	200 µg/mL
Corning Gel Strainer 40 UM	Sigma-Aldrich	CLS431750	Pore size 40 μm
Corning Matrigel Matrix (Phenol red-free)	Corning	356231	Extracellular matrix (ECM)
Corning bottle-top vacuum filter system	Sigma-Aldrich	CLS431098
Corning CoolCell LX Cell Freezing Container	Sigma-Aldrich	CLS432002
Corning Transwell polyester membrane cell culture inserts	Sigma-Aldrich	CLS3470	Permeable support inserts. 6.5 mm Transwell with 0.4 μm pore polyester membrane insert.
Countess Cell Counting Chamber Slides	Thermo Fisher Scientific	C10228
Countess II Automated Cell Counter	ThermoFisher Scientific	AMQAX1000	Automated cell counter
Cytology brushes	McFarlane Medical	33009
DMEM/F12-Ham	Thermo Fisher Scientific	11330032
DMEM/F12-Ham	Thermo Fisher Scientific	11330032
DMEM-High Glucose	Thermo Fisher Scientific	11965-092
Dulbecco′s Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537
Eclipse Ti2-E	Nikon		Live-cell imaging microscope.
Fetal Bovine Serum, certified, heat inactivated, United States	Thermo Fisher Scientific	10082147
Fungizone (Amphotericin B)	Thermo Fisher Scientific	15290018	250 µg/mL
Gentamicin solution	Sigma-Aldrich	G1397	50 mg/mL
Graphpad Prism	Graphpad		Scientific analysis software
Greiner Cryo.s vials	Sigma-Aldrich	V3135	Cryogenic vials
HEPES solution	Sigma-Aldrich	H0887	1 M
HI-FBS	Thermo Fisher Scientific	10082-147
Hydrocortisone	Sigma-Aldrich	H0888	3.6 mg/mL
Incubator NL Ti2 BLACK 2000	PeCon		Microscope environmental chamber. Allows warm air incubation and local CO2 and O2 gassing
Insulin	Sigma-Aldrich	I2643	2 mg/mL
Lab Armor 74220 706 Waterless Bead Bath 6L	John Morris Group	74220 706	Bead bath
Lab Armor Beads	Thermo Fisher Scientific	A1254302	Thermal beads
MATLAB	MathWorks		Computing software
Microsoft Excel	Microscoft		Spreadsheet software
NIH/3T3	American Type Culture Collection	CRL-1658	Irradiated NIH-3T3 mouse embryonic feeder cells
NIS-Elements AR	Nikon Instruments Inc.		Image acquisition software
Penicillin-Streptomycin	Sigma-Aldrich	P4333	10,000 units penicillin and 10 mg streptomycin/mL
Dulbecco′s Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537
PneumaCult Airway Organoid Kit	StemCell Technologies	5060	Airway Organoid Kit
PneumaCult-ALI Medium	StemCell Technologies	5001
PneumaCult-Ex Plus Medium	StemCell Technologies	5040
PureCol-S	Advanced BioMatrix	5015	Type I Collagen solution
ReagentPack Subculture Reagents	Lonza	CC-5034
rhEGF (Epidermal Growth Factor, human)	Sigma-Aldrich	E9644	25 µg/mL
Y-27632 2HCl (ROCK inhibitor)	Selleckchem	S1049	10 mM
Tobramycin	Sigma-Aldrich	T4014	100 mg/mL
Trypan blue solution	Sigma-Aldrich	T8154	0.4%
UNO Stage Top Incubator	Okolab		Microscope incubator. Allows temperature, humidity and CO2 conditioning