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

Katelin M. Allan; Sharon L. Wong; Laura K. Fawcett; Alexander Capraro; Adam Jaffe; Cristan Herbert; Elvis Pandzic; Shafagh A. Waters

doi:10.3791/63090

JoVE Journal > Medicine

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Médecine

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

Published: November 10, 2021

doi:

10.3791/63090

Katelin M. Allan², Sharon L. Wong², Laura K. Fawcett^2,3, Alexander Capraro², Adam Jaffe^2,3, Cristan Herbert, Elvis Pandzic, Shafagh A. Waters^2,3

¹School of Women’s and Children’s Health, Faculty of Medicine and Health,University of New South Wales, ²Molecular and Integrative Cystic Fibrosis Research Centre (miCF RC), Faculty of Medicine and Health,University of New South Wales, ³Department of Respiratory Medicine,Sydney Children’s Hospital, ⁴Department of Pathology, School of Medical Sciences,University of New South Wales Australia, ⁵Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre,University of New South Wales

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 patients with the same CFTR mutations³. 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 CBF.

Nasal epithelial sheets isolated from nasal mucosal brushings are directly used for measurements of ciliary function for PCD diagnosis⁴. Yet, while there is no control over the size or quality of the nasal epithelial sheets obtained, CBF varies depending on whether it is measured on single cells or cell sheets and on epithelial sheet ciliated edges that are disrupted or undisrupted⁵. As such, secondary dyskinesias caused by damage to cells during the collection of nasal mucosal brushings may influence CBF. Primary cell culture of nasal epithelial cells and their differentiation at Air-Liquid Interface (ALI) or in three-dimensional basement membrane matrix into ciliated airway epithelial organoids give rise to cilia that are free from secondary dyskinesias⁴^,⁶^,⁷^,⁸. Airway epithelial cells differentiated at ALI (henceforth termed ALI models) have been deemed an important secondary diagnostic aid that replicate the ciliary beat patterns and frequency of ex vivo nasal mucosal brushings⁶ and enable analysis of ciliary ultrastructure, beat pattern, and beat frequency while retaining patient-specific defects⁹. Yet, discrepancies exist in the methodologies used to create these pseudostratified, mucociliary differentiated cell models. Different culture expansion or differentiation protocols could induce distinct epithelial phenotypes (ciliated or secretory)¹⁰ and result in significant differences in CBF¹¹. CBF has been quantified in nasal epithelial brushings⁴^,⁶^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶, airway epithelial organoids¹⁴^,¹⁷^,¹⁸ and ALI models³^,⁴^,⁶^,¹³^,¹⁹^,²⁰^,²¹. Yet, amongst these protocols, there are large variabilities, and often many parameters are not controlled for. For example, in some studies, CBF is imaged in situ while the cells of the ALI model remain on the permeable support insert³^,¹⁹^,²⁰^,²¹, yet others scrape the cells from the permeable support insert and image them suspended in media⁴^,⁶^,¹³.

Furthermore, the wider application of techniques that measure ciliary function is limited by the extreme susceptibility of ciliary function to changes in environmental factors. Environmental factors such as temperature²², humidity²³^,²⁴, and pH²⁵^,²⁶ influence ciliary function and must be regulated to quantify CBF accurately. The various physiological parameters used across different laboratories and how they influence CBF has been reviewed previously²⁷.

Various imaging technologies and approaches to CBF measurements are reported in the literature. For PCD diagnostics, video microscopy is used to measure ciliary function²⁸^,²⁹. Recently, a video analysis algorithm based on differential dynamic microscopy was used to quantify both CBF and cilia coordination in airway epithelial cell ALI models³^,³⁰. This method enables the characterization of ciliary beating in airway epithelial cells in a fast and fully automated manner, without the need to segment or select regions. Various methods for imaging and quantification of CBF may add to the differences reported in CBF in the literature (Supplementary File 1).

A protocol from culture to quantification to streamline existing methods, standardization of culture conditions, and image acquisition, performed in strict environmentally controlled conditions, would enable consistent, reproducible quantification of CBF within and between individuals.

This protocol provides a complete description of the collection of epithelial cells, expansion and differentiation culture conditions, and quantification of CBF in three different airway epithelial cell model systems of nasal origin: 1) native epithelial sheets, 2) ALI models imaged on permeable support inserts and 3) Extracellular Matrix (ECM)-embedded three-dimensional organoids (Figure 1). Nasal epithelial cells obtained from nasal inferior turbinate brushings are used as representatives of the airway epithelium since they are an effective surrogate for bronchial epithelial cells³¹ while overcoming the invasive procedure associated with collecting bronchial brushings. The Conditional Reprogramming Cell (CRC) method is used to expand primary airway epithelial cells for the creation of ALI models and three-dimensional organoids. Conditional reprogramming of airway epithelial cells to a stem cell-like state is induced by co-culture with growth-arrested fibroblast feeder cell system and Rho-associated kinase (ROCK) inhibitor³². Importantly, the CRC method increases population doubling in airway epithelial cells while retaining their tissue-specific differentiation potential³³^,³⁴. In all airway epithelial cell models, the ciliary function is captured in a temperature-controlled chamber using a high-speed video camera with standardized image acquisition settings. Custom-built scripts are employed for the quantification of CBF.

Figure 1: Schematic of workflow. Following brushing the participants' nasal inferior turbinate, airway epithelial cells are utilized in one of two ways. Either airway epithelial sheets are isolated, and cilia beat frequency is imaged immediately, or airway epithelial cells are expanded via the conditional reprogramming cell method. CRC-expanded airway epithelial cells are differentiated to establish airway epithelial cells at an air-liquid interface or airway epithelial organoid cultures. Imaging of ciliary beat frequency is acquired using a live-cell imaging microscope with a heating and humidity environmental chamber and a fast frame rate (>100Hz) scientific camera. Data analysis is performed using custom-built scripts. Please click here to view a larger version of this figure.

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 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…

Divulgations

The authors have nothing to disclose.

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 Hospital Foundation. The authors would like to acknowledge Luminesce Alliance – Innovation for Children's Health for its contribution and support. Luminesce Alliance – Innovation for Children's Health is a not-for-profit cooperative joint venture between the Sydney Children's Hospitals Network, the Children's Medical Research Institute, and the Children's Cancer Institute. It has been established with the support of the NSW Government to coordinate and integrate pediatric research. Luminesce Alliance is also affiliated with the University of Sydney and the University of New South Wales Sydney. KMA is supported by an Australian Government Research Training Program Scholarship. LKF is supported by the Rotary Club of Sydney Cove/Sydney Children's Hospital Foundation and UNSW University postgraduate award scholarships.

Materials

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

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Allan, K. M., Wong, S. L., Fawcett, L. K., Capraro, A., Jaffe, A., Herbert, C., Pandzic, E., Waters, S. A. Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency. J. Vis. Exp. (177), e63090, doi:10.3791/63090 (2021).