Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Loretta Müller; Luisa E. Brighton; Johnny L. Carson; William A. Fischer II; Ilona Jaspers

doi:10.3791/50646

Research Article

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

DOI:

10.3791/50646

⸱

October 8th, 2013

Loretta Müller¹ , Luisa E. Brighton¹ , Johnny L. Carson¹^,² , William A. Fischer II¹^,³ , Ilona Jaspers¹^,²^,⁴

¹Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, ²Department of Pediatrics, The University of North Carolina at Chapel Hill, ³Pulmonary Diseases and Critical Care, The University of North Carolina at Chapel Hill, ⁴Curriculum in Toxicology, The University of North Carolina at Chapel Hill

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens.

Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc.

In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Goal of the technique

Epithelial cells (ECs) in the airways are among the first sites to be directly exposed to inhaled environmental stressors, such as pathogens, allergens or air pollutants. ECs are more than a structural barrier: They act as a switchboard to initiate and regulate the respiratory immune response^1-3 via the release of soluble mediators^4-6 and the expression of ligands/receptors on their surfac ^7-9. While immortalized cell lines can be used as models to study respiratory ECs in vitro, they fail to differentiate into heterogeneous cell populations composed of polarized ciliated and mucu....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Prepare Plastic Ware: Coat Plate

Add 500 μl PureCol (1:100 diluted with sterile water) to each well of a 12-well plate.
Incubate at 37 °C in an incubator for 30 min, remove the PureCol and rinse with HBSS right before the cells are added to the plate (see step 3.1 below).

2. Obtaining and Pretreating Biopsy of Human NECs

Nasal scrape biopsy
1. Obtain superficial ECs lining the nasal turbinates under direct vision through a 9 mm reusable polypropylene nasal speculum (Model 22009) on an operating otoscope with speculum (Model 21700). This device provides optimal visualization of the ....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

NECs cultured following the here described protocol re-differentiate into a heterogeneous layer of ECs composed of ciliated and non-ciliated cells, representing the in vivo situation (Figure 1). Some of the differentiated NECs are mucus producing (cells staining in blue using AB/PAS, Figure 1B). Even though the here presented protocol is optimized, differentiation can vary with regards to the distribution of the overall cell populations (i.e. different ratios of ciliate.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Respiratory ECs provide not only a physical barrier to protect the human body from exogenous stimuli²⁰, but also act as a switchboard to initiate and regulate respiratory immune defense responses^1-3 via secretion of soluble mediators or direct receptor-ligand cell-cell interactions. In order to further study the role of ECs in the immune response, to examine potential differences among ECs from diseased populations, or to study the effects of exogenous stressors on ECs, it is important to recapitula.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors would like to thank Lisa Dailey for the helpful inputs.

This work was supported by grants from the National Institutes of Health (ES013611 and HL095163), the Flight Attendant Medical Research Institute (FAMRI), and the Environmental Protection Agency (CR83346301) and declares no conflict of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Although the research described in this article has been funded in part by the U.S. Environmental Protection Agency through cooperative agreement CR83346301 with the Center for ....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article

Name

Company

Catalog Number

Comments

Nasal speculum

Welch Allyn

Model 22009

9 mm, reusable polyproylene

Operating otoscope

Welch Allyn

Model 21700

Rhino probe cell cuvette

Arlington Scientific

SY-96-092

bend the head of the cuvette a little more

12-well culture plates

Corning

3512

Transwell membrane cell culture inserts

Corning

3460

Tissue culture flask

Corning

430639 and 430641

T25 and T75 vented flask

RPMI 1640 media (with L-Glutamine)

Gibco

11875

Bronchial/Tracheal Epithelial Cell Growth Medium

Cell applications

511-500

BEGM Bronchial Epithelial Cell Growth Medium

Lonza

CC-3170

This comes as a kit of one bottle of medium and vials with supplements.

Sodium Bicarbonate 7.5% solution

Gibco

25080

Use as it is.

NuSerum

BD Biosciences

355104

Use as it is.

BAMBANKER freezing media

BAMBANKER

302-14681

Use as it is.

CoolCell

Biocision

HTBCS-136

(CryoPrep alcohol-free cell freezing container)

PureCol

AdvancedBioMatrix

5005-B

dilute 1:100 with sterile water (should be roughly 0.03 mg/ml)use 500 μl/well of a 12-well plate

HBSS with Ca² and Mg²⁺

Gibco

14025

DNAse 1

Sigma

DN25

Dissolve at 1.5 mg/ml, which is a 100x solution, add 10 μl to each ml of media

Trypsin, 0.25%, 1x, with phenol red

Gibco

25200-056

Use at it is

Soy Bean Trypsin Inhibitor (SBTI)

Sigma

T6522

Use dissolved in HBSS at a concentration of 1 mg/ml

Retinoic acid

Sigma

R7632

Make it up 100 μM stock solution (see below), dilute to 10 μM with absolute alcohol, take 5 μl of this and add it to 1 ml BEGM media (our working solution).

All-trans Retinol has a coefficient of extinction of 52,480 in ethanol at 325 nm. Dissolve 25 mg in 50 ml absolute ethanol. Serial dilutions need to be made, dilute 50 ml of stock in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, and again take 50 ml of this, dilute in 450 ml absolute Ethanol, take 100 ml of this and dilute in 900 ml absolute ethanol and read in the spectrophotometer at 325 nm, this number is then multiplied by its’ dilution factor, then divided by the extinction coefficient of 52,480, this will give a number in mM. Make a 100 mM stock (store at -20 °C) and from this stock a 10 mM aliquot is made, the daily retinoic acid needed would be 5 ml of this 10 mM aliquot in 995 ml ALI media. Example: 325nm reading is 1.15. 1.15 x 10,000 (dilution factor) / 52,480 = 0.219 = 219 mM, make a 100 mM stock from this, in absolute ethanol.

DMEM high glucose (1x), liquid, with L-glutamine and sodium pyruvate

Gibco

11995

Bovine Pituitary Extract

Gibco

13028-014

Used for BEGM ALI media.

Nystatin

Sigma

N4014-50 mg

Make up a solution of 3.3 mg/ml.

Bovine Serum Albumin

Fisher Scientific

BP1600-100

Make up a solution of 10 mg/ml solution in milliQ water.

PneumaCult-ALI media

StemCell

5001

This comes as a kit of one bottle of medium and vials with supplements. For details see below.

Hydrocortisone

StemCell

7904

Dissolve 2.4 mg hydrocortisone in 1 ml ddH₂O (used to make the 200x Hydrocortisone stock solution)

Heparin Sodium Salt in PBS (2 mg/ml)

StemCell

7980

Use at it is.

Filter flasks (500 ml)

Corning

431097

0.22 μm pore size

Filter tubes (50 ml)

Millipore

SCGP00525

Steriflip, 0.22 μm pore size

Pipette tips - ART Barrier Tips

Molecular Bioproducts

2139RI, 2069RI, 2179RI

Conical tubes, sterile, 15 ml and 50 ml

ddH₂O, absolute ethanol, ice

CO₂ incubator

refrigerated centrifuge

biological safety cabinet

pipettes (p2, p200, p1000), 9 in glass pipettes

gloves

lab coat with tight cuffs

Media and solutions used:	Recipe	Used for
General remark: always warm the media to room temperature
BEGM media	500 ml BEGM Cell Application media with one aliquot of retinoic acid	(mix them half-half and filter )
BEGM media	500 ml BEGM Lonza media with one aliquot of single quot supplements	digestion of the biopsy
BEGM+ media	50 ml BEGM media	maintaining cells on plastic in the plates (passage0)
	10 μl retinoic acid (working solution)	seeding cells in the flask (passage1; partially)
		maintain the cells in the flask
BEGM++ media	50 ml Lonza’s BEGM media with supplements	seeding fresh cells in 12-well plate on plastic
	1 ml Sodium BiCarb	maintaining cells in the plates on plastic (passage0; only partially)
	2.5 ml NuSerum	seeding cells in the flask (passage1; partially)
BEGM ALI media	250 ml DMEM-H	Maintaining cells on the transwell before they go ALI
	250 ml BEGM including supplements
	2 ml Bovine Pituitary Extract (12.5 mg/ml solution)
	1 ml Nystatin (3.3 mg/ml solution)
	75 μl BSA (10 mg/ml solution)
200X Hydrocortisone Stock Solution (96 μl/ml solution)	200 μl dissolved hydrocortisone	As a supplement to the PneumaCult-ALI Complete Base Medium for the last day in the flask and when cells are on inserts
	4250 μl dissolved sodium chloride
	540 μl ddH₂O
	10 μl absolute ethanol
	Filter solution through 0.22 μm filter, aliquot solution and store at -20 °C, avoid additional freeze/thaw cycles
PneumaCult-ALI Complete Base Media	450 ml PneumaCult-ALI Basal Medium	Base medium for all three PneumaCult-ALI media
	50 ml PneumaCult 10x Supplement	The PneumaCult media is light sensitive, thus always keep it in the dark
	(this is stable for 2 weeks at 2-8 °C or for up to 6 months at -20 °C)
PneumaCult-ALI Maintenance Medium	Per 1 ml of PneumaCult-ALI Complete Base Medium add:	ALI culture on inserts
	10 μl PneumaCult 100x Maintenance Supplement
	2 μl Heparin (of a 2 mg/ml stock solution)
	5 μl Hydrocortisone (of a 96 μl/ml stock solution)

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Swamy, M., Jamora, C., Havran, W., Hayday, A. Epithelial decision makers: in search of the 'epimmunome. Nat. Immunol. 11, 656-665 (2010).
Vareille, M., Kieninger, E., Edwards, M. R., Regamey, N. The airway epithelium: soldier in the fight against respiratory viruses. C....

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Request permission to reuse the text or figures of this JoVE article

Request Permission

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In This Article

Summary

Abstract

Introduction

Protocol

Results

Discussion

Disclosures

Acknowledgements

Materials

References

Reprints and Permissions

Tags