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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/Strep.......

Representative 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.......

Acknowledgements

We thank the study participants and their families for their contributions. We appreciate the assistance from Sydney Children's Hospitals (SCH) Randwick respiratory department in the organization and collection of patient biospecimens - special thanks to Dr. John Widger, Dr. Yvonne Belessis, Leanne Plush, Amanda Thompson, and Rhonda Bell. We acknowledge the assistance of Iveta Slapetova and Renee Whan from the Katharina Gaus Light Microscopy Facility within the Mark Wainwright Analytical Centre at UNSW Sydney. This work is supported by National Health and Medical Research Council (NHMRC) Australia (GNT1188987), CF Foundation Australia, and Sydney Children's Ho....

Materials

NameCompanyCatalog NumberComments
AdenineSigma-AldrichA278610 mg/mL
Advanced DMEM/F-12Thermo Fisher Scientific12634-010
Alanyl-glutamineSigma-AldrichG8541200 mM
Andor Zyla 4.2 sCMOSOxford InstrumentsFast frame rate (>100 Hz) scientific camera
Bottle-top vacuum filter systemSigma-AldrichCLS431098
Ceftazidime hydrateSigma-AldrichA698750 mg/mL
Cell Culture MicroscopeOlympusCKX53
CFI S Plan Fluor ELWD 20XCNikon Instruments Inc.MRH08230Long working distance objective lens. NA0.45 WD 8.2-6.9
Cholera toxinSigma-AldrichC8052-1MG200 µg/mL
Corning Gel Strainer 40 UMSigma-AldrichCLS431750Pore size 40 μm
Corning Matrigel Matrix (Phenol red-free)Corning356231Extracellular matrix (ECM)
Corning bottle-top vacuum filter systemSigma-AldrichCLS431098
Corning CoolCell LX Cell Freezing ContainerSigma-AldrichCLS432002
Corning Transwell polyester membrane cell culture insertsSigma-AldrichCLS3470Permeable support inserts. 6.5 mm Transwell with 0.4 μm pore polyester membrane insert.
Countess Cell Counting Chamber SlidesThermo Fisher ScientificC10228
Countess II Automated Cell CounterThermoFisher ScientificAMQAX1000Automated cell counter
Cytology brushesMcFarlane Medical33009
DMEM/F12-HamThermo Fisher Scientific11330032
DMEM/F12-HamThermo Fisher Scientific11330032
DMEM-High GlucoseThermo Fisher Scientific11965-092
Dulbecco′s Phosphate Buffered Saline (PBS)Sigma-AldrichD8537
Eclipse Ti2-ENikonLive-cell imaging microscope.
Fetal Bovine Serum, certified, heat inactivated, United StatesThermo Fisher Scientific10082147
Fungizone (Amphotericin B)Thermo Fisher Scientific15290018250 µg/mL
Gentamicin solutionSigma-AldrichG139750 mg/mL
Graphpad PrismGraphpadScientific analysis software
Greiner Cryo.s vialsSigma-AldrichV3135Cryogenic vials
HEPES solutionSigma-AldrichH08871 M
HI-FBSThermo Fisher Scientific10082-147
HydrocortisoneSigma-AldrichH08883.6 mg/mL
Incubator NL Ti2 BLACK 2000PeConMicroscope environmental chamber. Allows warm air incubation and local CO2 and O2 gassing
InsulinSigma-AldrichI26432 mg/mL
Lab Armor 74220 706 Waterless Bead Bath 6LJohn Morris Group74220 706Bead bath
Lab Armor BeadsThermo Fisher ScientificA1254302Thermal beads
MATLABMathWorksComputing software
Microsoft ExcelMicroscoftSpreadsheet software
NIH/3T3American Type Culture CollectionCRL-1658Irradiated NIH-3T3 mouse embryonic feeder cells
NIS-Elements ARNikon Instruments Inc.Image acquisition software
Penicillin-StreptomycinSigma-AldrichP433310,000 units penicillin and 10 mg streptomycin/mL
Dulbecco′s Phosphate Buffered Saline (PBS)Sigma-AldrichD8537
PneumaCult Airway Organoid KitStemCell Technologies5060Airway Organoid Kit
PneumaCult-ALI MediumStemCell Technologies5001
PneumaCult-Ex Plus MediumStemCell Technologies5040
PureCol-SAdvanced BioMatrix5015Type I Collagen solution
ReagentPack Subculture ReagentsLonzaCC-5034
rhEGF (Epidermal Growth Factor, human)Sigma-AldrichE964425 µg/mL
Y-27632 2HCl (ROCK inhibitor)SelleckchemS104910 mM
TobramycinSigma-AldrichT4014100 mg/mL
Trypan blue solutionSigma-AldrichT81540.4%
UNO Stage Top IncubatorOkolabMicroscope incubator. Allows temperature, humidity and CO2 conditioning

References

  1. Barbato, A., et al. Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. European Respiratory Journal. 34 (6), 1264-1276 (2009).
  2. Cutting, G. R.

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