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. 2022 May 27;3(2):101419.
doi: 10.1016/j.xpro.2022.101419. eCollection 2022 Jun 17.

Isolation, cultivation, and application of primary respiratory epithelial cells obtained by nasal brushing, polyp samples, or lung explants

Anita Golec  1 , Iwona Pranke  1 , Paolo Scudieri  2   3 , Kate Hayes  4   5 , Elise Dreano  1 , Fiona Dunlevy  5 , Aurelie Hatton  1 , Damian G Downey  6 , Luis Galietta  7   8 , Isabelle Sermet  9   10
Affiliations

Isolation, cultivation, and application of primary respiratory epithelial cells obtained by nasal brushing, polyp samples, or lung explants

Anita Golec et al. STAR Protoc. .

Abstract

Here, we present a standardized protocol for isolation, maintenance, and polarization of the respiratory epithelial primary cells from patient samples acquired from nasal brushing, polyp specimens, or lung explants. This protocol generates a clearly defined polarized layer of epithelial cells on filters, with a good number of ciliated cells and a thin layer of mucus. We detail the steps for samples prepared from patients with cystic fibrosis as well as from subjects without cystic fibrosis.

Keywords: Cell Biology; Cell culture; Cell isolation; Clinical Protocol.

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Conflict of interest statement

Damian Downey has received consultancy fees and/or grant support from Vertex pharmaceuticals, Proteostasis Therapeutics, Gilead, Chiesi, and Insmed. He is Director of the European CF Society – Clinical Trials Network and a member of the UK CF Trust Research Scientific Oversight Board of the Clinical Trials Accelerator Platform. Isabelle Sermet has received consultancy fees and/or grant support from Vertex Therapeutics, Eloxx, and PTC Therapeutics and is a Board Member of the European Cystic Fibrosis Society. Luis Galietta is a member of the Advisory Board of Enterprise Therapeutics.

Figures

None
Graphical abstract
Figure 1
Figure 1
Two types of brushes for nostril sampling (A) Brush for <5 years of age. (B) Brush for >5 years of age.
Figure 2
Figure 2
Cotton meshes, politzer tweezers, and brush for sampling procedure
Figure 3
Figure 3
Identification of the inferior turbinate
Figure 4
Figure 4
Soaking of cotton mesh in xylocaine 5% with naphazoline solution
Figure 5
Figure 5
Preparation of cotton meshes for insertion into nostrils
Figure 6
Figure 6
Placement of cotton mesh and nasal brush (A and B) Introducing (A) the cotton mesh into the nostril and (B) introducing the nasal brush into the nostril.
Figure 7
Figure 7
Locating the inferior turbinate: 1st and 2nd mesh localization and area to brush
Figure 8
Figure 8
Equipment and lung explant preparation
Figure 9
Figure 9
Lung explant
Figure 10
Figure 10
Recovery of cells from the lumen of the explant
Figure 11
Figure 11
Example of the pellet to which 5 mL of medium should be added
Figure 12
Figure 12
Dissociation of the airway epithelial cells from the polyp
Figure 13
Figure 13
Cell expansion (A) Cell suspension. (B) Cells after 1 day in culture. (C) Cells after 3 days in culture (60% of confluence).
Figure 14
Figure 14
Counting cells
Figure 15
Figure 15
Measurement of the transepithelial electrical resistance (TEER)
Figure 16
Figure 16
Suspension filter with layer of cells and visible mucus and layer of epithelium after 10 days in the air-liquid culture
Figure 17
Figure 17
Ciliated cells after approximately 2 weeks at the ALI
See this image and copyright information in PMC

References

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    1. Stokes A.B., Kieninger E., Schögler A., Kopf B.S., Casaulta C., Geiser T., Regamey N., Alves M.P. Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects. Exp. Lung Res. 2014;40:327–332. doi: 10.3109/01902148.2014.925987. - DOI - PubMed